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Pepperkok TeamPublications
Plasma membrane translocation of REDD1 governed by GPCRs contributes to mTORC1 activation.
Michel, G., Matthes, H.W., Hachet-Haas, M., El Baghdadi, K., de Mey, J., Pepperkok, R., Simpson, J.C., Galzi, J.L. & Lecat, S.
J Cell Sci. 2014 Feb 15;127(Pt 4):773-87. doi: 10.1242/jcs.136432. Epub 2013 Dec11.
The mTORC1 kinase promotes cell growth in response to growth factors by activation of receptor tyrosine kinase. It is regulated by the cellular energy level and the availability of nutrients. mTORC1 activity is also inhibited by cellular stresses through overexpression of REDD1 (regulated in development and DNA damage responses). We report the identification of REDD1 in a fluorescent live-imaging screen aimed at discovering new proteins implicated in G-protein-coupled receptor signaling, based on translocation criteria. Using a sensitive and quantitative plasma membrane localization assay based on bioluminescent resonance energy transfer, we further show that a panel of endogenously expressed GPCRs, through a Ca(2+)/calmodulin pathway, triggers plasma membrane translocation of REDD1 but not of its homolog REDD2. REDD1 and REDD2 share a conserved mTORC1-inhibitory motif characterized at the functional and structural level and differ most in their N-termini. We show that the N-terminus of REDD1 and its mTORC1-inhibitory motif participate in the GPCR-evoked dynamic interaction of REDD1 with the plasma membrane. We further identify REDD1 as a novel effector in GPCR signaling. We show that fast activation of mTORC1 by GPCRs correlates with fast and maximal translocation of REDD1 to the plasma membrane. Overexpression of functional REDD1 leads to a reduction of mTORC1 activation by GPCRs. By contrast, depletion of endogenous REDD1 protein unleashes mTORC1 activity. Thus, translocation to the plasma membrane appears to be an inactivation mechanism of REDD1 by GPCRs, which probably act by sequestering its functional mTORC1-inhibitory motif that is necessary for plasma membrane targeting.
Europe PMC
PIP(3) induces the recycling of receptor tyrosine kinases.
Laketa, V., Zarbakhsh, S., Traynor-Kaplan, A., Macnamara, A., Subramanian, D., Putyrski, M., Mueller, R., Nadler, A., Mentel, M., Saez-Rodriguez, J., Pepperkok, R. & Schultz, C.
Sci Signal. 2014 Jan 14;7(308):ra5. doi: 10.1126/scisignal.2004532.
Down-regulation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) is achieved by endocytosis of the receptor followed by degradation or recycling. We demonstrated that in the absence of ligand, increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations induced clathrin- and dynamin-mediated endocytosis of EGFR but not that of transferrin or G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. Endocytosis of the receptor in response to binding of EGF resulted in a decrease in the abundance of the EGFR, but PIP3-induced internalization decreased receptor ubiquitination and phosphorylation and resulted in recycling of the receptor to the plasma membrane. An RNA interference (RNAi) screen directed against lipid-binding domain-containing proteins identified polarity complex proteins, including PARD3 (partitioning defective 3), as essential for PIP3-induced receptor tyrosine kinase recycling. Thus, PIP3 and polarity complex proteins regulate receptor tyrosine kinase trafficking, which may enhance cellular responsiveness to growth factors.
Europe PMC
An RNAi screen identifies KIF15 as a novel regulator of the endocytic trafficking of integrin.
Eskova, A., Knapp, B., Matelska, D., Reusing, S., Arjonen, A., Lisauskas, T., Pepperkok, R., Russell, R., Eils, R., Ivaska, J., Kaderali, L., Erfle, H. & Starkuviene, V.
J Cell Sci. 2014 Jun 1;127(Pt 11):2433-47. doi: 10.1242/jcs.137281. Epub 2014 Mar21.
alpha2beta1 integrin is one of the most important collagen-binding receptors, and it has been implicated in numerous thrombotic and immune diseases. alpha2beta1 integrin is a potent tumour suppressor, and its downregulation is associated with increased metastasis and poor prognosis in breast cancer. Currently, very little is known about the mechanism that regulates the cell-surface expression and trafficking of alpha2beta1 integrin. Here, using a quantitative fluorescence-microscopy-based RNAi assay, we investigated the impact of 386 cytoskeleton-associated or -regulatory genes on alpha2 integrin endocytosis and found that 122 of these affected the intracellular accumulation of alpha2 integrin. Of these, 83 were found to be putative regulators of alpha2 integrin trafficking and/or expression, with no observed effect on the internalization of epidermal growth factor (EGF) or transferrin. Further interrogation and validation of the siRNA screen revealed a role for KIF15, a microtubule-based molecular motor, as a significant inhibitor of the endocytic trafficking of alpha2 integrin. Our data suggest a novel role for KIF15 in mediating plasma membrane localization of the alternative clathrin adaptor Dab2, thus impinging on pathways that regulate alpha2 integrin internalization.
Europe PMC
CNIH4 interacts with newly synthesized GPCR and controls their export from the endoplasmic reticulum.
Sauvageau, E., Rochdi, M.D., Oueslati, M., Hamdan, F.F., Percherancier, Y., Simpson, J.C., Pepperkok, R. & Bouvier, M.
Traffic. 2014 Apr;15(4):383-400. doi: 10.1111/tra.12148. Epub 2014 Feb 6.
The molecular mechanisms regulating G protein-coupled receptors (GPCRs) trafficking from their site of synthesis in the endoplasmic reticulum (ER) to their site of function (the cell surface) remain poorly characterized. Using a bioluminescence resonance energy transfer-based proteomic screen, we identified a novel GPCR-interacting protein; the human cornichon homologue 4 (CNIH4). This previously uncharacterized protein is localized in the early secretory pathway where it interacts with members of the 3 family of GPCRs. Both overexpression and knockdown expression of CNIH4 caused the intracellular retention of GPCRs, indicating that this ER-resident protein plays an important role in GPCR export. Overexpression of CNIH4 at low levels rescued the maturation and cell surface expression of an intracellularly retained mutant form of the beta2-adrenergic receptor, further demonstrating a positive role of CNIH4 in GPCR trafficking. Taken with the co-immunoprecipitation of CNIH4 with Sec23 and Sec24, components of the COPII coat complex responsible for ER export, these data suggest that CNIH4 acts as a cargo-sorting receptor, recruiting GPCRs into COPII vesicles.
Europe PMC
Adaptive fluorescence microscopy by online feedback image analysis.
Tischer, C., Hilsenstein, V., Hanson, K. & Pepperkok, R.
Methods Cell Biol. 2014;123C:489-503. doi: 10.1016/B978-0-12-420138-5.00026-4.
Obtaining sufficient statistics in quantitative fluorescence microscopy is often hampered by the tedious and time-consuming task of manually locating comparable specimen and repeatedly launching the same acquisition protocol. Recent advances in combining fluorescence microscopy with online image analysis tackle this problem by fully integrating the task of identifying and locating the specimen of interest in an automated acquisition workflow. Here, we describe the general requirements and specific microscope control and image analysis software solutions for implementing such automated online feedback microscopy. We demonstrate the power of the method by two selected applications addressing high-throughput 3D imaging of sparsely parasite-infected tissue culture cells and automated fluorescence recovery after photobleaching experiments to quantify the turnover of vesicular coat proteins at ER exit sites.
Europe PMC
Imaging ER-to-Golgi transport: towards a systems view.
Verissimo, F. & Pepperkok, R.
J Cell Sci. 2013 Nov 15;126(Pt 22):5091-100. doi: 10.1242/jcs.121061.
Proteins synthesised at the endoplasmic reticulum (ER) have to undergo a number of consecutive and coordinated steps to reach the Golgi complex. To understand the dynamic complexity of ER-to-Golgi transport at the structural and molecular level, light microscopy approaches are fundamental tools that allow in vivo observations of protein dynamics and interactions of fluorescent proteins in living cells. Imaging protein and organelle dynamics close to the ultra-structural level became possible by combining light microscopy with electron microscopy analyses or super-resolution light microscopy methods. Besides, increasing evidence suggests that the early secretory pathway is tightly connected to other cellular processes, such as signal transduction, and quantitative information at the systems level is fundamental to achieve a comprehensive molecular understanding of these connections. High-throughput microscopy in fixed and living cells in combination with systematic perturbation of gene expression by, e.g. RNA interference, will open new avenues to gain such an understanding of the early secretory pathway at the systems level. In this Commentary, we first outline examples that revealed the dynamic organisation of ER-to-Golgi transport in living cells. Next, we discuss the use of advanced imaging methods in studying ER-to-Golgi transport and, finally, delineate the efforts in understanding ER-to-Golgi transport at the systems level.
Europe PMC
PtdIns(3)P-bound UVRAG coordinates Golgi-ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex.
He, S., Ni, D., Ma, B., Lee, J.H., Zhang, T., Ghozalli, I., Pirooz, S.D., Zhao, Z., Bharatham, N., Li, B., Oh, S., Lee, W.H., Takahashi, Y., Wang, H.G., Minassian, A., Feng, P., Deretic, V., Pepperkok, R., Tagaya, M., Yoon, H.S. & Liang, C.
Nat Cell Biol. 2013 Oct;15(10):1206-19. doi: 10.1038/ncb2848. Epub 2013 Sep 22.
Endoplasmic reticulum (ER)-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and disease. Here, we identify UVRAG, a beclin-1-binding autophagic factor, as a phosphatidylinositol-3-phosphate (PtdIns(3)P)-binding protein that depends on PtdIns(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-beclin-1-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis.
Europe PMC
High-Content siRNA Screen Reveals Global ENaC Regulators and Potential Cystic Fibrosis Therapy Targets.
Almaca, J., Faria, D., Sousa, M., Uliyakina, I., Conrad, C., Sirianant, L., Clarke, L.A., Martins, J.P., Santos, M., Heriche, J.K., Huber, W., Schreiber, R., Pepperkok, R., Kunzelmann, K. & Amaral, M.D.
Cell. 2013 Sep 12;154(6):1390-400. doi: 10.1016/j.cell.2013.08.045.
Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKiota, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na(+) and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.
Europe PMC
Characterization of drug-induced transcriptional modules: towards drug repositioning and functional understanding.
Iskar, M., Zeller, G., Blattmann, P., Campillos, M., Kuhn, M., Kaminska, K.H., Runz, H., Gavin, A.C., Pepperkok, R., van Noort, V. & Bork, P.
Mol Syst Biol. 2013;9:662. doi: 10.1038/msb.2013.20.
In pharmacology, it is crucial to understand the complex biological responses that drugs elicit in the human organism and how well they can be inferred from model organisms. We therefore identified a large set of drug-induced transcriptional modules from genome-wide microarray data of drug-treated human cell lines and rat liver, and first characterized their conservation. Over 70% of these modules were common for multiple cell lines and 15% were conserved between the human in vitro and the rat in vivo system. We then illustrate the utility of conserved and cell-type-specific drug-induced modules by predicting and experimentally validating (i) gene functions, e.g., 10 novel regulators of cellular cholesterol homeostasis and (ii) new mechanisms of action for existing drugs, thereby providing a starting point for drug repositioning, e.g., novel cell cycle inhibitors and new modulators of alpha-adrenergic receptor, peroxisome proliferator-activated receptor and estrogen receptor. Taken together, the identified modules reveal the conservation of transcriptional responses towards drugs across cell types and organisms, and improve our understanding of both the molecular basis of drug action and human biology.
Europe PMC
Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells.
Stadler, C., Rexhepaj, E., Singan, V.R., Murphy, R.F., Pepperkok, R., Uhlen, M., Simpson, J.C. & Lundberg, E.
Nat Methods. 2013 Apr;10(4):315-23. doi: 10.1038/nmeth.2377. Epub 2013 Feb 24.
Imaging techniques such as immunofluorescence (IF) and the expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Here we report a systematic analysis of >500 human proteins comparing the localizations obtained in live versus fixed cells using FPs and IF, respectively. We identify systematic discrepancies between IF and FPs as well as between FP tagging at the N and C termini. The analysis shows that for 80% of the proteins, IF and FPs yield the same subcellular distribution, and the locations of 250 previously unlocalized proteins were determined by the overlap between the two methods. Approximately 60% of proteins localize to multiple organelles for both methods, indicating a complex subcellular protein organization. These results show that both IF and FP tagging are reliable techniques and demonstrate the usefulness of an integrative approach for a complete investigation of the subcellular human proteome.
Europe PMC
RNAi-Based Functional Profiling of Loci from Blood Lipid Genome-Wide Association Studies Identifies Genes with Cholesterol-Regulatory Function.
Blattmann, P., Schuberth, C., Pepperkok, R. & Runz, H.
PLoS Genet. 2013 Feb;9(2):e1003338. doi: 10.1371/journal.pgen.1003338. Epub 2013Feb 28.
Genome-wide association studies (GWAS) are powerful tools to unravel genomic loci associated with common traits and complex human disease. However, GWAS only rarely reveal information on the exact genetic elements and pathogenic events underlying an association. In order to extract functional information from genomic data, strategies for systematic follow-up studies on a phenotypic level are required. Here we address these limitations by applying RNA interference (RNAi) to analyze 133 candidate genes within 56 loci identified by GWAS as associated with blood lipid levels, coronary artery disease, and/or myocardial infarction for a function in regulating cholesterol levels in cells. Knockdown of a surprisingly high number (41%) of trait-associated genes affected low-density lipoprotein (LDL) internalization and/or cellular levels of free cholesterol. Our data further show that individual GWAS loci may contain more than one gene with cholesterol-regulatory functions. Using a set of secondary assays we demonstrate for a number of genes without previously known lipid-regulatory roles (e.g. CXCL12, FAM174A, PAFAH1B1, SEZ6L, TBL2, WDR12) that knockdown correlates with altered LDL-receptor levels and/or that overexpression as GFP-tagged fusion proteins inversely modifies cellular cholesterol levels. By providing strong evidence for disease-relevant functions of lipid trait-associated genes, our study demonstrates that quantitative, cell-based RNAi is a scalable strategy for a systematic, unbiased detection of functional effectors within GWAS loci.
Europe PMC
Cell-to-cell communication: current views and future perspectives.
Gerdes, H.H. & Pepperkok, R.
Cell Tissue Res. 2013 Apr;352(1):1-3. doi: 10.1007/s00441-013-1590-1. Epub 2013Feb 26. Europe PMC
Golgi depletion from living cells with laser nanosurgery.
Ronchi, P. & Pepperkok, R.
Methods Cell Biol. 2013;118:311-24. doi: 10.1016/B978-0-12-417164-0.00019-7.
How Golgi biogenesis occurs in mammalian cells is a controversial problem. Can the Golgi complex (GC) form de novo from ER membranes or does it require a template? The method described in this chapter uses growth of cells on micropatterns to displace the GC from its juxtanuclear position and laser nanosurgery to subsequently deplete it from living cells. Golgi-depleted karyoplasts can be followed by time-lapse microscopy to address if and how the GC can be de novo synthesized from ER membranes. Furthermore, the study of different processes in the absence of the GC can shed light on the role of this organelle in the intracellular signaling and homeostasis.
Europe PMC
Chemical chaperones improve protein secretion and rescue mutant factor VIII in mice with hemophilia A.
Roth, S.D., Schuttrumpf, J., Milanov, P., Abriss, D., Ungerer, C., Quade-Lyssy, P., Simpson, J.C., Pepperkok, R., Seifried, E. & Tonn, T.
PLoS One. 2012;7(9):e44505. doi: 10.1371/journal.pone.0044505. Epub 2012 Sep 4.
Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.
Europe PMC
Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway.
Simpson, J.C., Joggerst, B., Laketa, V., Verissimo, F., Cetin, C., Erfle, H., Bexiga, M.G., Singan, V.R., Heriche, J.K., Neumann, B., Mateos, A., Blake, J., Bechtel, S., Benes, V., Wiemann, S., Ellenberg, J. & Pepperkok, R.
Nat Cell Biol. 2012 Jun 3;14(7):764-74. doi: 10.1038/ncb2510.
The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.
Europe PMC
Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1.
Moudry, P., Lukas, C., Macurek, L., Neumann, B., Heriche, J.K., Pepperkok, R., Ellenberg, J., Hodny, Z., Lukas, J. & Bartek, J.
Cell Death Differ. 2012 May;19(5):798-807. doi: 10.1038/cdd.2011.150. Epub 2011Nov 11.
53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-beta interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-beta pathway and implicate this mechanism in the maintenance of genome integrity.
Europe PMC
Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy.
Stadler, C., Hjelmare, M., Neumann, B., Jonasson, K., Pepperkok, R., Uhlen, M. & Lundberg, E.
J Proteomics. 2012 Apr 3;75(7):2236-51. doi: 10.1016/j.jprot.2012.01.030. Epub2012 Feb 15.
We have developed a platform for validation of antibody binding and protein subcellular localization data obtained from immunofluorescence using siRNA technology combined with automated confocal microscopy and image analysis. By combining the siRNA technology with automated sample preparation, automated imaging and quantitative image analysis, a high-throughput assay has been set-up to enable confirmation of accurate protein binding and localization in a systematic manner. Here, we describe the analysis and validation of the subcellular location of 65 human proteins, targeted by 75 antibodies and silenced by 130 siRNAs. A large fraction of (80%) the subcellular locations, including locations of several previously uncharacterized proteins, could be confirmed by the significant down-regulation of the antibody signal after the siRNA silencing. A quantitative analysis was set-up using automated image analysis to facilitate studies of targets found in more than one compartment. The results obtained using the platform demonstrate that siRNA silencing in combination with quantitative image analysis of antibody signals in different compartments of the cells is an attractive approach for ensuring accurate protein localization as well as antibody binding using immunofluorescence. With a large fraction of the human proteome still unexplored, we suggest this approach to be of great importance under the continued work of mapping the human proteome on a subcellular level.
Europe PMC
A new approach to manipulate the fate of single neural stem cells in tissue.
Taverna, E., Haffner, C., Pepperkok, R. & Huttner, W.B.
Nat Neurosci. 2012;15(2):329-37. doi: 10.1038/nn.3008.
A challenge in the field of neural stem cell biology is the mechanistic dissection of single stem cell behavior in tissue. Although such behavior can be tracked by sophisticated imaging techniques, current methods of genetic manipulation do not allow researchers to change the level of a defined gene product on a truly acute time scale and are limited to very few genes at a time. To overcome these limitations, we established microinjection of neuroepithelial/radial glial cells (apical progenitors) in organotypic slice culture of embryonic mouse brain. Microinjected apical progenitors showed cell cycle parameters that were indistinguishable to apical progenitors in utero, underwent self-renewing divisions and generated neurons. Microinjection of single genes, recombinant proteins or complex mixtures of RNA was found to elicit acute and defined changes in apical progenitor behavior and progeny fate. Thus, apical progenitor microinjection provides a new approach to acutely manipulating single neural stem and progenitor cells in tissue.
Europe PMC
Quantifying the influence of yellow fluorescent protein photoconversion on acceptor photobleaching-based fluorescence resonance energy transfer measurements.
Seitz, A., Terjung, S., Zimmermann, T. & Pepperkok, R.
J Biomed Opt. 2012 Jan;17(1):011010. doi: 10.1117/1.JBO.17.1.011010.
Fluorescence resonance energy transfer (FRET) efficiency measurements based on acceptor photobleaching of yellow fluorescent protein (YFP) are affected by the fact that bleaching of YFP produces a fluorescent species that is detectable in cyan fluorescent protein (CFP) image channels. The presented quantitative measurement of this conversion makes it possible to correct the obtained FRET signal to increase the accuracy of intensity based CFP/YFP FRET measurements. The described method can additionally be used to compare samples with very different fluorescence levels.
Europe PMC
Molecular recognition of a single sphingolipid species by a protein's transmembrane domain.
Contreras, F.X., Ernst, A.M., Haberkant, P., Bjorkholm, P., Lindahl, E., Gonen, B., Tischer, C., Elofsson, A., von Heijne, G., Thiele, C., Pepperkok, R., Wieland, F. & Brugger, B.
Nature. 2012 Jan 9;481(7382):525-9. doi: 10.1038/nature10742.
Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.
Europe PMC
At the cutting edge: applications and perspectives of laser nanosurgery in cell biology.
Ronchi, P., Terjung, S. & Pepperkok, R.
Biol Chem. 2012 Apr;393(4):235-48. doi: 10.1515/hsz-2011-0237.
Laser-mediated nanosurgery has become popular in the last decade because of the previously unexplored possibility of ablating biological material inside living cells with sub-micrometer precision. A number of publications have shown the potential applications of this technique, ranging from the dissection of sub-cellular structures to surgical ablations of whole cells or tissues in model systems such as Drosophila melanogaster or Danio rerio . In parallel, the recent development of micropatterning techniques has given cell biologists the possibility to shape cells and reproducibly organize the intracellular space. The integration of these two techniques has only recently started yet their combination has proven to be very interesting. The aim of this review is to present recent applications of laser nanosurgery in cell biology and to discuss the possible developments of this approach, particularly in combination with micropattern-mediated endomembrane organization.
Europe PMC
Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases.
Ritzerfeld, J., Remmele, S., Wang, T., Temmerman, K., Brugger, B., Wegehingel, S., Tournaviti, S., Strating, J.R., Wieland, F.T., Neumann, B., Ellenberg, J., Lawerenz, C., Hesser, J., Erfle, H., Pepperkok, R. & Nickel, W.
Genome Res. 2011 Nov;21(11):1955-68. doi: 10.1101/gr.116087.110. Epub 2011 Jul27.
SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes.
Europe PMC
Quantitative Proteomics and Dynamic Imaging of the Nucleolus Reveal Distinct Responses to UV and Ionizing Radiation.
Moore, H.M., Bai, B., Boisvert, F.M., Latonen, L., Rantanen, V., Simpson, J.C., Pepperkok, R., Lamond, A.I. & Laiho, M.
Mol Cell Proteomics. 2011 Oct;10(10):M111.009241. Epub 2011 Jul 21.
The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.
Europe PMC
A novel laser nanosurgery approach supports de novo Golgi biogenesis in mammalian cells.
Tangemo, C., Ronchi, P., Colombelli, J., Haselmann, U., Simpson, J.C., Antony, C., Stelzer, E.H., Pepperkok, R. & Reynaud, E.G.
J Cell Sci. 2011 Mar 15;124(Pt 6):978-87. doi: 10.1242/jcs.079640.
The Golgi complex has a central role in the secretory pathway of all higher organisms. To explain the synthesis of its unique stacked structure in mammalian cells, two major models have been proposed. One suggests that it is synthesized de novo from the endoplasmic reticulum. The second model postulates a pre-existing Golgi template that serves as a scaffold for its biogenesis. To test these hypotheses directly, we have developed an approach in which we deplete the Golgi complex from living cells by laser nanosurgery, and subsequently analyze the 'Golgi-depleted' karyoplast using time-lapse and electron microscopy. We show that biosynthetic transport is blocked after Golgi depletion, but is restored 12 hours later. This recovery of secretory transport coincides with an ordered assembly of stacked Golgi structures, and we also observe the appearance of matrix proteins before that of Golgi enzymes. Functional experiments using RNA interference-mediated knockdown of GM130 further demonstrate the importance of the matrix during Golgi biogenesis. By contrast, the centrosome, which can also be removed by laser nanosurgery and is not reformed within the considered time frame, is not required for this process. Altogether, our data provide evidence that de novo Golgi biogenesis can occur in mammalian cells.
Europe PMC
Micropilot: automation of fluorescence microscopy-based imaging for systems biology.
Conrad, C., Wunsche, A., Tan, T.H., Bulkescher, J., Sieckmann, F., Verissimo, F., Edelstein, A., Walter, T., Liebel, U., Pepperkok, R. & Ellenberg, J.
Nat Methods. 2011 Mar;8(3):246-9. doi: 10.1038/nmeth.1558. Epub 2011 Jan 23.
Quantitative microscopy relies on imaging of large cell numbers but is often hampered by time-consuming manual selection of specific cells. The 'Micropilot' software automatically detects cells of interest and launches complex imaging experiments including three-dimensional multicolor time-lapse or fluorescence recovery after photobleaching in live cells. In three independent experimental setups this allowed us to statistically analyze biological processes in detail and is thus a powerful tool for systems biology.
Europe PMC
Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.
Reiss, S., Rebhan, I., Backes, P., Romero-Brey, I., Erfle, H., Matula, P., Kaderali, L., Poenisch, M., Blankenburg, H., Hiet, M.S., Longerich, T., Diehl, S., Ramirez, F., Balla, T., Rohr, K., Kaul, A., Buhler, S., Pepperkok, R., Lengauer, T., Albrecht, M., Eils, R., Schirmacher, P., Lohmann, V. & Bartenschlager, R.
Cell Host Microbe. 2011 Jan 20;9(1):32-45. doi: 10.1016/j.chom.2010.12.002.
Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIalpha), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIalpha product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIalpha was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIalpha and stimulate its kinase activity. The absence of PI4KIIIalpha activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.
Europe PMC
Functional genomics assays to study CFTR traffic and ENaC function.
Almaca, J., Dahimene, S., Appel, N., Conrad, C., Kunzelmann, K., Pepperkok, R. & Amaral, M.D.
Methods Mol Biol. 2011;742:249-64. doi: 10.1007/978-1-61779-120-8_15.
As several genomes have been sequenced, post-genomic approaches like transcriptomics and proteomics, identifying gene products differentially expressed in association with a given pathology, have held promise both of understanding the pathways associated with the respective disease and as a fast track to therapy. Notwithstanding, these approaches cannot distinguish genes and proteins with mere secondary pathological association from those primarily involved in the basic defect(s). New global strategies and tools identifying gene products responsible for the basic cellular defect(s) in CF pathophysiology currently being performed are presented here. These include high-content screens to determine proteins affecting function and trafficking of CFTR and ENaC.
Europe PMC
High-throughput microscopy using live mammalian cells.
Terjung, S., Walter, T., Seitz, A., Neumann, B., Pepperkok, R. & Ellenberg, J.
Cold Spring Harb Protoc. 2010 Aug 1;2010(7):pdb.top84. doi: 10.1101/pdb.top84. Europe PMC
In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.
Grecco, H.E., Roda-Navarro, P., Girod, A., Hou, J., Frahm, T., Truxius, D.C., Pepperkok, R., Squire, A. & Bastiaens, P.I.
Nat Methods. 2010 Jun;7(6):467-72. doi: 10.1038/nmeth.1458. Epub 2010 May 9.
Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ. FLIM on cell arrays that express fluorescent protein fusions can quantify tyrosine phosphorylation patterns in large networks in individual cells. We identified tyrosine kinase substrates by imaging their phosphorylation levels after inhibition of protein tyrosine phosphatases. Analysis of the correlation between protein phosphorylation and expression levels at single cell resolution allowed us to identify positive feedback motifs. Using FLIM on cell arrays (CA-FLIM), we uncovered components that transduce signals from epidermal growth factor receptor.
Europe PMC
Tec-kinase-mediated phosphorylation of fibroblast growth factor 2 is essential for unconventional secretion.
Ebert, A.D., Laussmann, M., Wegehingel, S., Kaderali, L., Erfle, H., Reichert, J., Lechner, J., Beer, H.D., Pepperkok, R. & Nickel, W.
Traffic. 2010 Jun;11(6):813-26. doi: 10.1111/j.1600-0854.2010.01059.x. Epub 2010Mar 10.
Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi-independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5-biphosphate (PI(4,5)P(2)) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post-translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.
Europe PMC
Activation of membrane-permeant caged PtdIns(3)P induces endosomal fusion in cells.
Subramanian, D.*, Laketa, V.*, Muller, R., Tischer, C., Zarbakhsh, S., Pepperkok, R. & Schultz, C. * - equally contributing first authors.
Nat Chem Biol. 2010 May;6(5):324-6. Epub 2010 Apr 4.
Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phospholipid residing on early endosomes, where it is proposed to be involved in endosomal fusion. We synthesized membrane-permeant derivatives of PtdIns(3)P, including a caged version that is to our knowledge the first photoactivatable phosphoinositide derivative developed so far. In living cells, photoactivation of caged PtdIns(3)P induced rapid endosomal fusion in an EEA1-dependent fashion, thus providing in vivo evidence that PtdIns(3)P is a sufficient signal for driving this process.
Europe PMC
Automatic identification and clustering of chromosome phenotypes in a genome wide RNAi screen by time-lapse imaging.
Walter, T., Held, M., Neumann, B., Heriche, J.K., Conrad, C., Pepperkok, R. & Ellenberg, J.
J Struct Biol. 2010 Apr;170(1):1-9. doi: 10.1016/j.jsb.2009.10.004. Epub 2009 Oct23.
High-throughput time-lapse microscopy is an excellent way of studying gene function by collecting time-resolved image data of the cellular responses to gene perturbations. With the increase in both data amount and complexity, computational methods capable of dealing with large image data sets are required. While image processing methods have been successfully applied to endpoint assays in the past, the analysis of complex time-resolved read-outs was so far still too immature to be applied on a large-scale. Here, we present a complete computational processing pipeline for such screens. By automatic image processing and machine learning, a quantitative description of phenotypic dynamics is obtained from the raw bitmaps. In order to visualize the resulting phenotypes in their temporal context, we introduce Event Order Maps allowing a concise representation of the major tendencies of causes and consequences of phenotypic classes. In order to cluster the phenotypic kinetics, we propose a novel technique based on trajectory representation of multidimensional time series. We demonstrate the use of these methods applying them on a genome wide RNAi screen by time-lapse microscopy.
Europe PMC
Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.
Neumann, B., Walter, T., Heriche, J.K., Bulkescher, J., Erfle, H., Conrad, C., Rogers, P., Poser, I., Held, M., Liebel, U., Cetin, C., Sieckmann, F., Pau, G., Kabbe, R., Wünsche, A., Satagopam, V., Schmitz, M.H., Chapuis, C., Gerlich, D.W., Schneider, R., Eils, R., Huber, W., Peters, J.M., Hyman, A.A., Durbin, R., Pepperkok, R. & Ellenberg, J.
Nature. 2010 Apr 1;464(7289):721-7.
Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.
Europe PMC
From experimental setup to bioinformatics: An RNAi screening platform to identify host factors involved in HIV-1 replication.
Borner, K., Hermle, J., Sommer, C., Brown, N.P., Knapp, B., Glass, B., Kunkel, J., Torralba, G., Reymann, J., Beil, N., Beneke, J., Pepperkok, R., Schneider, R., Ludwig, T., Hausmann, M., Hamprecht, F., Erfle, H., Kaderali, L., Krausslich, H.G. & Lehmann, M.J.
Biotechnol J. 2009 Dec 10;5(1):39-49.
RNA interference (RNAi) has emerged as a powerful technique for studying loss-of-function phenotypes by specific down-regulation of gene expression, allowing the investigation of virus-host interactions by large-scale high-throughput RNAi screens. Here we present a robust and sensitive small interfering RNA screening platform consisting of an experimental setup, single-cell image and statistical analysis as well as bioinformatics. The workflow has been established to elucidate host gene functions exploited by viruses, monitoring both suppression and enhancement of viral replication simultaneously by fluorescence microscopy. The platform comprises a two-stage procedure in which potential host factors are first identified in a primary screen and afterwards re-tested in a validation screen to confirm true positive hits. Subsequent bioinformatics allows the identification of cellular genes participating in metabolic pathways and cellular networks utilised by viruses for efficient infection. Our workflow has been used to investigate host factor usage by the human immunodeficiency virus-1 (HIV-1), but can also be adapted to other viruses. Importantly, we expect that the description of the platform will guide further screening approaches for virus-host interactions. The ViroQuant-CellNetworks RNAi Screening core facility is an integral part of the recently founded BioQuant centre for systems biology at the University of Heidelberg and will provide service to external users in the near future.
Europe PMC
Membrane-permeant phosphoinositide derivatives as modulators of growth factor signaling and neurite outgrowth.
Laketa, V., Zarbakhsh, S., Morbier, E., Subramanian, D., Dinkel, C., Brumbaugh, J., Zimmermann, P., Pepperkok, R. & Schultz, C.
Chem Biol. 2009 Nov 25;16(11):1190-6.
Phosphoinositides are important signaling molecules that govern a large number of cellular processes such as proliferation, differentiation, membrane remodeling, and survival. Here we introduce a fully synthetic membrane-permeant derivative of a novel, easily accessible, and very potent phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] mimic: phosphatidylinositol 3,4,5,6-tetrakisphosphate [PtdIns(3,4,5,6)P(4)]. The membrane-permeant PtdIns(3,4,5,6)P(4) derivative activated pathways downstream of phosphatidylinositol 3-kinase (PI3K), including protein kinase B, p70S6K, mitogen-activated protein kinase, and protein kinase C, more potently than similar membrane-permeant PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) derivatives in the absence of receptor stimulation. In addition, we demonstrate that treatment of PC12 cells with the membrane-permeant PtdIns(3,4)P(2), PtdIns(3,4,5)P(3), and PtdIns(3,4,5,6)P(4) derivatives increases the number of neurites per cell in the presence of NGF. This work establishes membrane-permeant phosphoinositides as powerful tools to study PI3K signaling and directly demonstrates that 3-phosphorylated phosphoinositides are instrumental for neurite initiation.
Europe PMC
Factor VIII-eGFP fusion proteins with preserved functional activity for the analysis of the early secretory pathway of factor VIII.
Heinz, S., Schuttrumpf, J., Simpson, J.C., Pepperkok, R., Nicolaes, G.A., Abriss, D., Milanov, P., Roth, S., Seifried, E. & Tonn, T.
Thromb Haemost. 2009 Nov;102(5):925-35.
Considering the difficulty in detecting factor (F)VIII in vivo, fluorescently labelled FVIII protein provides a tool to analyse the intracellular localisation, bio distribution, and pharmacokinetics of the protein in living organisms. Here, we report the use of FVIII full length and B-domain deleted proteins, fused to enhanced green fluorescent protein (eGFP) at the C-terminus of the coagulation protein via a nine amino acid spanning linker. Comparison of the FVIII-eGFP fusion proteins to their unlabelled counterparts showed no impairment with respect to recombinant expression levels, intracellular processing, specific coagulant activity and decay at physiological temperature. Confocal live cell imaging demonstrated ER-Golgi-transport of B-domain deleted FVIII-eGFP in vesicular tubular carriers. Using temperature blocks and release experiments, imaging of FVIII-eGFP fusion proteins enabled for the first time the visualisation of the early secretory pathway of B-domain deleted FVIII in living cells and in particular highlighted the apparent deficit of active transport carriers, an observation consistent with the low rates of FVIII secretion seen in recombinant expression systems.
Europe PMC
DetecTiff: a novel image analysis routine for high-content screening microscopy.
Gilbert, D.F., Meinhof, T., Pepperkok, R. & Runz, H.
J Biomol Screen. 2009 Sep;14(8):944-55. doi: 10.1177/1087057109339523. Epub 2009Jul 29.
In this article, the authors describe the image analysis software DetecTiff, which allows fully automated object recognition and quantification from digital images. The core module of the LabView-based routine is an algorithm for structure recognition that employs intensity thresholding and size-dependent particle filtering from microscopic images in an iterative manner. Detected structures are converted into templates, which are used for quantitative image analysis. DetecTiff enables processing of multiple detection channels and provides functions for template organization and fast interpretation of acquired data. The authors demonstrate the applicability of DetecTiff for automated analysis of cellular uptake of fluorescence-labeled low-density lipoproteins as well as diverse other image data sets from a variety of biomedical applications. Moreover, the performance of DetecTiff is compared with preexisting image analysis tools. The results show that DetecTiff can be applied with high consistency for automated quantitative analysis of image data (e.g., from large-scale functional RNAi screening projects).
Europe PMC
Reversible phosphorylation as a molecular switch to regulate plasma membrane targeting of acylated SH4 domain proteins.
Tournaviti, S., Pietro, E.S., Terjung, S., Schafmeier, T., Wegehingel, S., Ritzerfeld, J., Schulz, J., Smith, D.F., Pepperkok, R. & Nickel, W.
Traffic. 2009 Aug;10(8):1047-60. Epub 2009 May 12.
Acylated SH4 domains represent N-terminal targeting signals that anchor peripheral membrane proteins such as Src kinases in the inner leaflet of plasma membranes. Here we provide evidence for a novel regulatory mechanism that may control the levels of SH4 proteins being associated with plasma membranes. Using a fusion protein of the SH4 domain of Leishmania HASPB and GFP as a model system, we demonstrate that threonine 6 is a substrate for phosphorylation. Substitution of threonine 6 by glutamate (to mimic a phosphothreonine residue) resulted in a dramatic redistribution from plasma membranes to intracellular sites with a particular accumulation in a perinuclear region. As shown by both pharmacological inhibition and RNAi-mediated down-regulation of the threonine/ serine-specific phosphatases PP1 and PP2A, recycling back to the plasma membrane required dephosphorylation of threonine 6. We provide evidence that a cycle of phosphorylation and dephosphorylation may also be involved in intracellular targeting of other SH4 proteins such as the Src kinase Yes.
Europe PMC
Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II.
Schwarz, K., Iolascon, A., Verissimo, F., Trede, N.S., Horsley, W., Chen, W., Paw, B.H., Hopfner, K.P., Holzmann, K., Russo, R., Esposito, M.R., Spano, D., De Falco, L., Heinrich, K., Joggerst, B., Rojewski, M.T., Perrotta, S., Denecke, J., Pannicke, U., Delaunay, J., Pepperkok, R. & Heimpel, H.
Nat Genet. 2009 Aug;41(8):936-40. Epub 2009 Jun 28.
Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.
Europe PMC
ojoplano-mediated basal constriction is essential for optic cup morphogenesis.
Martinez-Morales, J.R., Rembold, M., Greger, K., Simpson, J.C., Brown, K.E., Quiring, R., Pepperkok, R., Martin-Bermudo, M.D., Himmelbauer, H. & Wittbrodt, J.
Development. 2009 Jul;136(13):2165-75.
Although the vertebrate retina is a well-studied paradigm for organogenesis, the morphogenetic mechanisms that carve the architecture of the vertebrate optic cup remain largely unknown. Understanding how the hemispheric shape of an eye is formed requires addressing the fundamental problem of how individual cell behaviour is coordinated to direct epithelial morphogenesis. Here, we analyze the role of ojoplano (opo), an uncharacterized gene whose human ortholog is associated with orofacial clefting syndrome, in the morphogenesis of epithelial tissues. Most notably, when opo is mutated in medaka fish, optic cup folding is impaired. We characterize optic cup morphogenesis in vivo and determine at the cellular level how opo affects this process. opo encodes a developmentally regulated transmembrane protein that localizes to compartments of the secretory pathway and to basal end-feet of the neuroepithelial precursors. We show that Opo regulates the polarized localization of focal adhesion components to the basal cell surface. Furthermore, tissue-specific interference with integrin-adhesive function impairs optic cup folding, resembling the ocular phenotype observed in opo mutants. We propose a model of retinal morphogenesis whereby opo-mediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.
Europe PMC
Identification of cholesterol-regulating genes by targeted RNAi screening.
Bartz, F., Kern, L., Erz, D., Zhu, M., Gilbert, D., Meinhof, T., Wirkner, U., Erfle, H., Muckenthaler, M., Pepperkok, R. & Runz, H.
Cell Metab. 2009 Jul;10(1):63-75.
Elevated plasma cholesterol levels are considered responsible for excess cardiovascular morbidity and mortality. Cholesterol in plasma is tightly controlled by cholesterol within cells. Here, we developed and applied an integrative functional genomics strategy that allows systematic identification of regulators of cellular cholesterol levels. Candidate genes were identified by genome-wide gene-expression profiling of sterol-depleted cells and systematic literature queries. The role of these genes in cholesterol regulation was then tested by targeted siRNA knockdown experiments quantifying cellular cholesterol levels and the efficiency of low-density lipoprotein (LDL) uptake. With this strategy, 20 genes were identified as functional regulators of cellular cholesterol homeostasis. Of these, we describe TMEM97 as SREBP target gene that under sterol-depleted conditions localizes to endo-/lysosomal compartments and binds to LDL cholesterol transport-regulating protein Niemann-Pick C1 (NPC1). Taken together, TMEM97 and other factors described here are promising to yield further insights into how cells control cholesterol levels.
Europe PMC
RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins.
Doil, C., Mailand, N., Bekker-Jensen, S., Menard, P., Larsen, D.H., Pepperkok, R., Ellenberg, J., Panier, S., Durocher, D., Bartek, J., Lukas, J. & Lukas, C.
Cell. 2009 Feb 6;136(3):435-46.
DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.
Europe PMC
Rab18 and Rab43 have key roles in ER-Golgi trafficking.
Dejgaard, S.Y., Murshid, A., Erman, A., Kizilay, O., Verbich, D., Lodge, R., Dejgaard, K., Ly-Hartig, T.B., Pepperkok, R., Simpson, J.C. & Presley, J.F.
J Cell Sci. 2008 Aug 15;121(Pt 16):2768-81. doi: 10.1242/jcs.021808. Epub 2008Jul 29.
Rabs and Arfs/Arls are Ras-related small GTPases of particular relevance to membrane trafficking. It is thought that these proteins regulate specific pathways through interactions with coat, motor, tether and SNARE proteins. We screened a comprehensive list of Arf/Arl/Rab proteins, previously identified on purified Golgi membranes by a proteomics approach (37 in total), for Golgi or intra-Golgi localization, dominant-negative and overexpression phenotypes. Further analysis of two of these proteins, Rab18 and Rab43, strongly indicated roles in ER-Golgi trafficking. Rab43-T32N redistributed Golgi elements to ER exit sites without blocking trafficking of the secretory marker VSVG-GFP from ER to cell surface. Wild-type Rab43 redistributes the p150(Glued) subunit of dynactin, consistent with a specific role in regulating association of pre-Golgi intermediates with microtubules. Overexpression of wild-type GFP-Rab18 or incubation with any of three siRNAs directed against Rab18 severely disrupts the Golgi complex and reduces secretion of VSVG. Rab18 mutants specifically enhance retrograde Golgi-ER transport of the COPI-independent cargo beta-1,4-galactosyltransferase (Galtase)-YFP but not the COPI-dependent cargo p58-YFP from the Golgi to ER in a photobleach assay. Rab18-S22N also potentiated brefeldin-A-induced ER-Golgi fusion. This study is the first comprehensive application of large-scale proteomics to the cell biology of small GTPases of the secretory pathway.
Europe PMC
Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates.
Erfle, H., Neumann, B., Rogers, P., Bulkescher, J., Ellenberg, J. & Pepperkok, R.
J Biomol Screen. 2008 Aug;13(7):575-80. Epub 2008 Jul 3.
Solid-phase reverse transfection on cell microarrays is a high-throughput method for the parallel transfection of mammalian cells. However, the cells transfected in this way have been restricted so far to microscopy-based analyses. Analysis methods such as reverse transcriptase-polymerase chain reaction (RT-PCR) and access to higher cell numbers for statistical reasons in microscopy-based assays are not possible with solid-phase reverse transfection on cell microarrays. We have developed a quick and reliable protocol for automated solid-phase reverse transfection of human cells with siRNAs in multiwell plates complementing solid-phase reverse transfection on cell microarrays. The method retains all advantages of solid-phase reverse transfection such as long-term storage capacity after fabrication, reduced cytotoxicity, and reduced cost per screen compared with liquid-phase transfection in multiwell plates. The protocol has been tested for the RNAi-mediated knockdown of several genes in different cell lines including U20S, RPE1, A549, and HeLa cells. We show that even 3 months after production of the "ready to transfect" multiwell plates, there is no reduction in their transfection efficiency as assessed by RT-PCR and nuclear phenotyping by fluorescence microscopy. We conclude that solid-phase reverse transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays.
Europe PMC
In migrating cells, the Golgi complex and the position of the centrosome depend on geometrical constraints of the substratum.
Pouthas, F., Girard, P., Lecaudey, V., Ly, T.B., Gilmour, D., Boulin, C., Pepperkok, R. & Reynaud, E.G.
J Cell Sci. 2008 Jul 15;121(Pt 14):2406-14. Epub 2008 Jun 24.
Although cells migrate in a constrained 3D environment in vivo, in-vitro studies have mainly focused on the analysis of cells moving on 2D substrates. Under such conditions, the Golgi complex is always located towards the leading edge of the cell, suggesting that it is involved in the directional movement. However, several lines of evidence indicate that this location can vary depending on the cell type, the environment or the developmental processes. We have used micro contact printing (microCP) to study the migration of cells that have a geometrically constrained shape within a polarized phenotype. Cells migrating on micropatterned lines of fibronectin are polarized and migrate in the same direction. Under such conditions, the Golgi complex and the centrosome are located behind the nucleus. In addition, the Golgi complex is often displaced several micrometres away from the nucleus. Finally, we used the zebrafish lateral line primordium as an in-vivo model of cells migrating in a constrained environment and observe a similar localization of both the Golgi and the centrosome in the leading cells. We propose that the positioning of the Golgi complex and the centrosome depends on the geometrical constraints applied to the cell rather than on a precise migratory function in the leading region.
Europe PMC
Human Proteinpedia enables sharing of human protein data.
Mathivanan, S., Ahmed, M., Ahn, N.G., Alexandre, H., Amanchy, R., Andrews, P.C., Bader, J.S., Balgley, B.M., Bantscheff, M., Bennett, K.L., Bjorling, E., Blagoev, B., Bose, R., Brahmachari, S.K., Burlingame, A.S., Bustelo, X.R., Cagney, G., Cantin, G.T., Cardasis, H.L., Celis, J.E., Chaerkady, R., Chu, F., Cole, P.A., Costello, C.E., Cotter, R.J., Crockett, D., DeLany, J.P., De Marzo, A.M., DeSouza, L.V., Deutsch, E.W., Dransfield, E., Drewes, G., Droit, A., Dunn, M.J., Elenitoba-Johnson, K., Ewing, R.M., Van Eyk, J., Faca, V., Falkner, J., Fang, X., Fenselau, C., Figeys, D., Gagne, P., Gelfi, C., Gevaert, K., Gimble, J.M., Gnad, F., Goel, R., Gromov, P., Hanash, S.M., Hancock, W.S., Harsha, H.C., Hart, G., Hays, F., He, F., Hebbar, P., Helsens, K., Hermeking, H., Hide, W., Hjerno, K., Hochstrasser, D.F., Hofmann, O., Horn, D.M., Hruban, R.H., Ibarrola, N., James, P., Jensen, O.N., Jensen, P.H., Jung, P., Kandasamy, K., Kheterpal, I., Kikuno, R.F., Korf, U., Korner, R., Kuster, B., Kwon, M.S., Lee, H.J., Lee, Y.J., Lefevre, M., Lehvaslaiho, M., Lescuyer, P., Levander, F., Lim, M.S., Lobke, C., Loo, J.A., Mann, M., Martens, L., Martinez-Heredia, J., McComb, M., McRedmond, J., Mehrle, A., Menon, R., Miller, C.A., Mischak, H., Mohan, S.S., Mohmood, R., Molina, H., Moran, M.F., Morgan, J.D., Moritz, R., Morzel, M., Muddiman, D.C., Nalli, A., Navarro, J.D., Neubert, T.A., Ohara, O., Oliva, R., Omenn, G.S., Oyama, M., Paik, Y.K., Pennington, K., Pepperkok, R., Periaswamy, B., Petricoin, E.F., Poirier, G.G., Prasad, T.S., Purvine, S.O., Rahiman, B.A., Ramachandran, P., Ramachandra, Y.L., Rice, R.H., Rick, J., Ronnholm, R.H., Salonen, J., Sanchez, J.C., Sayd, T., Seshi, B., Shankari, K., Sheng, S.J., Shetty, V., Shivakumar, K., Simpson, R.J., Sirdeshmukh, R., Siu, K.W., Smith, J.C., Smith, R.D., States, D.J., Sugano, S., Sullivan, M., Superti-Furga, G., Takatalo, M., Thongboonkerd, V., Trinidad, J.C., Uhlen, M., Vandekerckhove, J., Vasilescu, J., Veenstra, T.D., Vidal-Taboada, J.M., Vihinen, M., Wait, R., Wang, X., Wiemann, S., Wu, B., Xu, T., Yates, J.R., Zhong, J., Zhou, M., Zhu, Y., Zurbig, P. & Pandey, A.
Nat Biotechnol. 2008 Feb;26(2):164-7. doi: 10.1038/nbt0208-164. Europe PMC
ER-to-Golgi transport.
Verissimo, F. & Pepperkok, R..
In Mironov, A.A. & Pavelka, M. (eds.) The Golgi Apparatus: State of the art 110 years after Camillo Golgi's discovery (2008), Springer, pp. 333-341.
Biosynthetic cargo enters the secretory pathway at the endoplasmic reticu-lum (ER) where it is properly folded before it becomes sorted and concentrated into transport carriers that move towards the Golgi complex. The basic protein machinery involved in bidirectional transport between the ER and the Golgi complex has long been identified and is fairly well characterized (Lee et al. 2004). The vesicular coat complex COPII mediates ER exit, while retrograde transport from the Golgi complex to the ER involves the function of the vesicular coat complex COPI. In the following paragraphs we focus on our current knowledge on the spatial and temporal organization of ER-to-Golgi transport at the molecular level.
Integrating systems biology with clinical research.
Pepperkok, R. & Wiemann, S.
Genome Biol. 2008;9(7):314. Epub 2008 Jul 1.
A report on the conference 'Systems Genomics 2008', Heidelberg, Germany, 2-3 May 2008.
Europe PMC
A correlative light and electron microscopy method based on laser micropatterning and etching.
Colombelli, J., Tangemo, C., Haselman, U., Antony, C., Stelzer, E.H., Pepperkok, R. & Reynaud, E.G.
Methods Mol Biol. 2008;457:203-13.
Correlative microscopy is a hybrid method that allows the localization of events observed under visible, ultraviolet, or infrared light, at molecular and submolecular levels, combining two microscopy techniques. However, the main limitation of correlative microscopy is to develop a labeling technique that can be easily used first in light and then in electron microscopy. Laser etching is a well-established method to create precisely designed shapes or volumes in various materials including glass. We have applied this technique to develop a new correlative light and electron microscopy method and to apply it in our study of the Golgi apparatus. The location of the cell of interest is laser-inscribed into the glass allowing a simple follow-up in light and fluorescence microscopy. Furthermore, the glass surface is laser-etched and upon fixation and flat embedding, the inverse ridge can be localized as well as the cell of interest, which is then processed for electron microscopy.
Europe PMC
The potential of high-content high-throughput microscopy in drug discovery.
Starkuviene, V. & Pepperkok, R.
Br J Pharmacol. 2007 Sep;152(1):62-71. Epub 2007 Jul 2.
Fluorescence microscopy is a powerful method to study protein function in its natural habitat, the living cell. With the availability of the green fluorescent protein and its spectral variants, almost any gene of interest can be fluorescently labelled in living cells opening the possibility to study protein localization, dynamics and interactions. The emergence of automated cellular systems allows rapid visualization of large groups of cells and phenotypic analysis in a quantitative manner. Here, we discuss recent advances in high-content high-throughput microscopy and its potential application to several steps of the drug discovery process.
Europe PMC
Differential requirements for ts-O45-G and procollagen biosynthetic transport.
Starkuviene, V. & Pepperkok, R.
Traffic. 2007 Aug;8(8):1035-51. Epub 2007 Jun 5.
Emerging experimental evidence favours the existence of cargo sorting occurring upon the endoplasmic reticulum (ER) exit. Recent studies revealed that, in contrast to the conventional secretory marker ts-O45-G, procollagen (PC I) exits the ER at sites not coated with coat protein II and is transported to the Golgi complex in carriers devoid of coat protein I. Here, we investigated whether PC I trafficking requires a different molecular machinery in comparison with the ts-O45-G. By combining colocalization of the cargoes with endogenous markers, downregulation of transport machinery by RNA interference and knock-ins by complementary DNA over-expression, we provide strong evidence that PC I and ts-O45-G have common but also different molecular requirements during pre- and post-Golgi trafficking events.
Europe PMC
Transfected cell microarrays: an efficient tool for high-throughput functional analysis.
Starkuviene, V., Pepperkok, R. & Erfle, H.
Expert Rev Proteomics. 2007 Aug;4(4):479-89.
Transfected cell microarrays are considered to be a breakthrough methodology for high-throughput and high-content functional genomics. Here, recent advances in the cell microarray field are reviewed, along with its potential to increase the speed of determining gene function. These advances, combined with an increasing number and diversity of gene perturbing systems, such as RNAi and ectopic gene expression, provide tools for expanding our understanding of biology at the systems level.
Europe PMC
An RNAi screening platform to identify secretion machinery in mammalian cells.
Simpson, J.C., Cetin, C., Erfle, H., Joggerst, B., Liebel, U., Ellenberg, J. & Pepperkok, R.
J Biotechnol. 2007 Apr 30;129(2):352-65. Epub 2007 Jan 14.
Integrative approaches to study protein function in a cellular context are a vital aspect of understanding human disease. Genome sequencing projects provide the basic catalogue of information with which to unravel gene function, but more systematic applications of this resource are now necessary. Here, we describe and test a platform with which it is possible to rapidly use RNA interference in cultured mammalian cells to probe for proteins involved in constitutive protein secretion. Synthetic small interfering RNA molecules are arrayed in chambered slides, then incubated with cells and an assay for secretion performed. Automated microscopy is used to acquire images from the experiments, and automated single-cell analysis rapidly provides reliable quantitative data. In test arrays of 92 siRNA spots targeting 37 prospective membrane traffic proteins, our approach identifies 7 of these as being important for the correct delivery of a secretion marker to the cell surface. Correlating these findings with other screens and bioinformatic information makes these candidates highly likely to be novel membrane traffic machinery components.
Europe PMC
Maturation of the mammalian secretome.
Simpson, J.C., Mateos, A. & Pepperkok, R.
Genome Biol. 2007 Apr 30;8(4):211.
ABSTRACT: A recent use of quantitative proteomics to determine the constituents of the endoplasmic reticulum and Golgi complex is discussed in the light of other available methodologies for cataloging the proteins associated with the mammalian secretory pathway.
Europe PMC
High-content microscopy identifies new neurite outgrowth regulators.
Laketa, V., Simpson, J.C., Bechtel, S., Wiemann, S. & Pepperkok, R.
Mol Biol Cell. 2007 Jan;18(1):242-52. Epub 2006 Nov 8.
Neurons, with their long axons and elaborate dendritic arbour, establish the complex circuitry that is essential for the proper functioning of the nervous system. Whereas a catalogue of structural, molecular, and functional differences between axons and dendrites is accumulating, the mechanisms involved in early events of neuronal differentiation, such as neurite initiation and elongation, are less well understood, mainly because the key molecules involved remain elusive. Here we describe the establishment and application of a microscopy-based approach designed to identify novel proteins involved in neurite initiation and/or elongation. We identified 21 proteins that affected neurite outgrowth when ectopically expressed in cells. Complementary time-lapse microscopy allowed us to discriminate between early and late effector proteins. Localization experiments with GFP-tagged proteins in fixed and living cells revealed a further 14 proteins that associated with neurite tips either early or late during neurite outgrowth. Coexpression experiments of the new effector proteins provide a first glimpse on a possible functional relationship of these proteins during neurite outgrowth. Altogether, we demonstrate the potential of the systematic microscope-based screening approaches described here to tackle the complex biological process of neurite outgrowth regulation.
Europe PMC
Birth and life of tissue macrophages and their migration in embryogenesis and inflammation in medaka.
Grabher, C., Cliffe, A., Miura, K., Pepperkok, R., Rorth, P. & Wittbrodt, J.
J Leukoc Biol. 2007 Jan;81(1):263-71.
Macrophages detecting and migrating toward sites of injury and infection represent one of the first steps in an immune response. Here we directly image macrophage birth and migration in vivo in transgenic medaka fish. Macrophages are born as frequently dividing, immotile cells with spherical morphology that differentiate into flat, highly motile cells. They retain mitotic activity while spreading over the entire body. Cells follow restricted paths not only in directed migration, but also during patrolling. Along those paths the macrophages rapidly patrol the tissue and respond to wounding and bacterial infection from long distances. Upon injury they increase their speed and migratory persistence. Specifically targeting PI3-kinase isoforms efficiently blocks the wounding response and results in a distinct inhibition of cell motility and chemotaxis. Our study provides in situ insights into the properties of immature and migratory macrophages and presents a unique model to further test modulating compounds in vivo.
Europe PMC
Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.
von Brunn, A., Teepe, C., Simpson, J.C., Pepperkok, R., Friedel, C.C., Zimmer, R., Roberts, R., Baric, R. & Haas, J.
PLoS ONE. 2007 May 23;2(5):e459.
The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.
Europe PMC
Membrane dynamics in the early secretory pathway.
Robinson, D.G., Herranz, M.C., Bubeck, J., Pepperkok, R. & Ritzenthaler, C.
Critical Reviews in Plant Sciences 2007 26(4) 199-225
All eukaryotes possess a secretory pathway, and the major molecular players involved in secretion are well conserved. However, the morphological manifestation of this pathway at the level of the participant organelles shows great divergences between yeasts, mammals and plants. The unique features of the early secretory pathway in plants-a polydisperse mobile Golgi apparatus and the lack of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus-suggests the participation of many plant-specific molecules in the maintenance and regulation of protein trafficking. The advent of live cell imaging fluorescently-tagged proteins and the increased usage of cryotechniques in electron microscopy has led to dramatic advances in our understanding of the early secretory pathway of plants. In contrast, contradictions have sometimes emerged and interpretations for the same observations have not necessarily reached a consensus. In this review we have attempted to provide the reader with a critical, yet balanced overview of this rapidly expanding research area. Wherever possible we have contrasted a particular event or parameter with the corresponding situation in yeast or mammalian cells. We have also taken the opportunity to suggest suitable experimentation in newly emerging sectors.
Reverse transfection on cell arrays for high content screening microscopy.
Erfle, H., Neumann, B., Liebel, U., Rogers, P., Held, M., Walter, T., Ellenberg, J. & Pepperkok, R.
Nat Protoc. 2007;2(2):392-9.
Here, we describe a robust protocol for the reverse transfection of cells on small interfering (siRNA) arrays, which, in combination with multi-channel immunofluorescence or time-lapse microscopy, is suitable for genome-wide RNA interference (RNAi) screens in intact human cells. The automatic production of 48 'transfection ready' siRNA arrays, each containing 384 samples, takes in total 7 h. Pre-fabricated siRNA arrays can be used without loss of transfection efficiency at least up to 15 months after printing. Different human cell lines that have been successfully transfected using the protocol are presented here. The present protocol has been applied to two genome-wide siRNA screens addressing mitosis and constitutive protein secretion.
Europe PMC
A role for kinesin-2 in COPI-dependent recycling between the ER and the Golgi complex.
Stauber, T., Simpson, J.C., Pepperkok, R. & Vernos, I.
Curr Biol. 2006 Nov 21;16(22):2245-51.
Transport carriers operating between early compartments in the mammalian secretory pathway have to travel long distances in the cell by mostly relying on the microtubule network and its associated motor proteins. Although anterograde transport from the endoplasmic reticulum (ER) to the Golgi complex is mediated by cytoplasmic dynein, the identity of the motor(s) mediating transport in the retrograde direction is presently unclear. Some studies have suggested that the heterotrimeric kinesin-2 complex plays a role in transport between the ER and the Golgi. Here, we have examined kinesin-2 function by using an RNA-interference approach to downregulate the expression of KAP3, the nonmotor subunit of kinesin-2, in HeLa cells. KAP3 silencing results in the fragmentation of the Golgi apparatus and a change in the steady-state localization of the KDEL-receptor (KDEL-R). Using specific transport assays, we show that the rate of anterograde secretory traffic is unaffected in these cells but that KDEL-R-dependent retrograde transport is strongly abrogated. Our data strongly support a role for kinesin-2 in the KDEL-R-/COPI-dependent retrograde transport pathway from the Golgi complex to the ER.
Europe PMC
High-throughput fluorescence microscopy for systems biology.
Pepperkok, R. & Ellenberg, J.
Nat Rev Mol Cell Biol. 2006 Sep;7(9):690-6. Epub 2006 Jul 19.
In this post-genomic era, we need to define gene function on a genome-wide scale for model organisms and humans. The fundamental unit of biological processes is the cell. Among the most powerful tools to assay such processes in the physiological context of intact living cells are fluorescence microscopy and related imaging techniques. To enable these techniques to be applied to functional genomics experiments, fluorescence microscopy is making the transition to a quantitative and high-throughput technology.
Europe PMC
Detection and quantification of protein-microtubules interactions using green fluorescent protein photoconversion.
Brunet, S., Zimmermann, T., Reynaud, E.G., Vernos, I., Karsenti, E. & Pepperkok, R.
Traffic. 2006 Sep;7(9):1283-9.
We present an in vitro system to analyze quantitatively the interactions of green fluorescent protein (GFP)-tagged recombinant proteins with microtubules. This method relies on photoconversion of GFP and time-lapse microscopy. Specific interactions can be detected and binding kinetics can be determined rapidly and accurately. This method provides an alternative to classical in vitro microtubule-binding assays to analyze microtubule-associated proteins binding to microtubules. It has the potential to be extended to study interactions of proteins or multi-protein complexes with different biopolymers like actin microfilaments or organelle membranes.
Europe PMC
Protein kinase WNK3 increases cell survival in a caspase-3-dependent pathway.
Verissimo, F., Silva, E., Morris, J.D., Pepperkok, R. & Jordan, P.
Oncogene. 2006 Jul 13;25(30):4172-82. Epub 2006 Feb 27.
The subfamily of WNK (with no K= lysine) protein kinases has four human members and germline mutations in the WNK1 and WNK4 genes were recently found to cause pseudohypoaldosteronism type II, a familial hypertension disease. Here, we describe cloning and functional analysis of a further WNK member, human WNK3. Endogenous WNK3 protein is an active protein kinase when immunoprecipitated from cells and its overexpression increases the survival of HeLa cells by delaying the onset of apoptosis. Suppression of endogenous WNK3 protein by RNA interference accelerates the apoptotic response and promotes the activation of caspase-3. The mechanism of WNK3 action involves interaction with procaspase-3 and heat-shock protein 70. These results demonstrate a role for WNK3 in promoting cell survival and suggest a mechanism at the level of procaspase-3 activation.
Europe PMC
Sterols regulate ER-export dynamics of secretory cargo protein ts-O45-G.
Runz, H., Miura, K., Weiss, M. & Pepperkok, R.
EMBO J. 2006 Jul 12;25(13):2953-65. Epub 2006 Jun 22.
Alterations in endoplasmic reticulum (ER) cholesterol are fundamental for a variety of cellular processes such as the regulation of lipid homeostasis or efficient protein degradation. We show that reduced levels of cellular sterols cause a delayed ER-to-Golgi transport of the secretory cargo membrane protein ts-O45-G and a relocation to the ER of an endogenous protein cycling between the ER and the Golgi complex. Transport inhibition is characterized by a delay in the accumulation of ts-O45-G in ER-exit sites (ERES) and correlates with a reduced mobility of ts-O45-G within ER membranes. A simple mathematical model describing the kinetics of ER-exit predicts that reduced cargo loading to ERES and not the reduced mobility of ts-O45-G accounts for the delayed ER-exit and arrival at the Golgi. Consistent with this, membrane turnover of the COPII component Sec23p is delayed in sterol-depleted cells. Altogether, our results demonstrate the importance of sterol levels in COPII mediated ER-export.
Europe PMC
Laser nanosurgery in cell biology
Colombelli, J., Pepperkok, R., Stelzer, E.H.K. & Reynaud, E.G.
Med Sci (Paris). 2006 Jul-Aug;22(6-7):651-8.
Since their first use in the early 60's, pulsed lasers have become increasingly popular for their ability to ablate biological tissue. Short laser pulses allow high precision surgery for biological and medical applications with minimal invasiveness. Performing highly targeted manipulation and ablation allows experiments impossible so far in development biology, cellular biology or even assisted reproductive technologies and laser surgery has been increasingly used over the last five years to answer key questions in Biology. Recently, picosecond UV and femtosecond IR laser pulses have been used to cleave microtubules and to severe actin stress fibers in vivo with a spatial precision in the submicrometer range to study their dynamics without affecting cell viability. We review recent findings on the underlying principles of pulsed laser nanosurgery mechanisms showing how the use of ultra short laser pulses increases precision and non-invasiveness of laser surgery. We show how the understanding of the surgical process allows one to distinguish between single cell ablation in living organisms or intracellular nanosurgery in living cells and we review recent applications to the study of forces and the quantification of cytoskeleton dynamics. double dagger.
Europe PMC
High-throughput RNAi screening by time-lapse imaging of live human cells.
Neumann, B., Held, M., Liebel, U., Erfle, H., Rogers, P., Pepperkok, R. & Ellenberg, J.
Nat Methods. 2006 May;3(5):385-90.
RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.
Europe PMC
Calcium-dependent regulation of NF-(kappa)B activation in cystic fibrosis airway epithelial cells.
Tabary, O., Boncoeur, E., de Martin, R., Pepperkok, R., Clement, A., Schultz, C. & Jacquot, J.
Cell Signal. 2006 May;18(5):652-60. Epub 2005 Aug 9.
Dysregulation of nuclear factor kappa B (NF-(kappa)B) and increased Ca(2+) signals have been reported in airway epithelial cells of patients with cystic fibrosis (CF). The hypothesis that Ca(2+) signaling may regulate NF-(kappa)B activation was tested in a CF bronchial epithelial cell line (IB3-1, CFTR genotype DeltaF508/W1282X) and compared to the CFTR-corrected epithelial cell line S9 using fluorescence microscopy to visualized in situ NF-(kappa)B activation at the single cell level. Upon stimulation with IL-1beta,we observed a slow but prolonged [Ca(2+)](i) increase (up to 10 min) in IB3-1 cells compared to S9 cells. The IL-1beta-induced [Ca(2+)](i) response was accompanied by an activation of NF-(kappa)B in IB3-1 but not in S9 cells. Pretreatment of IB3-1 cells with the ER Ca(2+) pump inhibitor thapsigargin inhibited the IL-1beta-induced [Ca(2+)](i) response. Treatment with either the calcium chelator BAPTA or an inhibitor of I(kappa)Balpha phosphorylation (digitoxin) led to a drastic [Ca(2+)](i) decrease accompanied by an inhibition of NF-(kappa)B activation of IL-1beta-stimulated IB3-1 cells in comparison to untreated cells. In IB3-1 cells cultured at low temperature (26 degrees C) for 16 h, the IL-1beta-induced [Ca(2+)](i) response was inhibited and no significant NF-(kappa)B activation was observed. To our knowledge, this is the first report of visualization of the Ca(2+)-mediated activation of NF-(kappa)B in individual living airway epithelial cells. Our results support the concept that [Ca(2+)](i) is a key regulator of NF-(kappa)B activation in CF airway epithelial cells.
Europe PMC
Biogenesis of tubular ER-to-Golgi transport intermediates.
Simpson, J.C., Nilsson, T. & Pepperkok, R.
Mol Biol Cell. 2006 Feb;17(2):723-37. Epub 2005 Nov 28.
Tubular transport intermediates (TTIs) have been described as one class of transport carriers in endoplasmic reticulum (ER)-to-Golgi transport. In contrast to vesicle budding and fusion, little is known about the molecular regulation of TTI synthesis, transport and fusion with target membranes. Here we have used in vivo imaging of various kinds of GFP-tagged proteins to start to address these questions. We demonstrate that under steady-state conditions TTIs represent approximately 20% of all moving transport carriers. They increase in number and length when more transport cargo becomes available at the donor membrane, which we induced by either temperature-related transport blocks or increased expression of the respective GFP-tagged transport markers. The formation and motility of TTIs is strongly dependent on the presence of intact microtubules. Microinjection of GTPgammaS increases the frequency of TTI synthesis and the length of these carriers. When Rab proteins are removed from membranes by microinjection of recombinant Rab-GDI, the synthesis of TTIs is completely blocked. Microinjection of the cytoplasmic tails of the p23 and p24 membrane proteins also abolishes formation of p24-containing TTIs. Our data suggest that TTIs are ER-to-Golgi transport intermediates that form preferentially when transport-competent cargo exists in excess at the donor membrane. We propose a model where the interaction of the cytoplasmic tails of membrane proteins with microtubules are key determinants for TTI synthesis and may also serve as a so far unappreciated model for aspects of transport carrier formation.
Europe PMC
Secretory cargo regulates the turnover of COPII subunits at single ER exit sites.
Forster, R., Weiss, M., Zimmermann, T., Reynaud, E.G., Verissimo, F., Stephens, D.J. & Pepperkok, R.
Curr Biol. 2006 Jan 24;16(2):173-9.
The COPII coat complex mediates the formation of transport carriers at specialized sites of the endoplasmic reticulum (ERES). It consists of the Sar1p GTPase and the Sec23/24p and the Sec13/31p subcomplexes . Both stimulate the GTPase activity of Sar1p , which itself triggers coat disassembly. This built-in GAP activity makes the COPII complex in principle unstable and raises the question of how sufficient stability required for cargo capture and carrier formation is achieved. To address this, we analyzed COPII turnover at single ERES in living cells. The half times for Sar1p, Sec23p, and Sec24p turnover are 1.1, 3.7, and 3.9 s, respectively. Decreasing the amount of transport-competent cargo in the endoplasmic reticulum accelerates turnover of the Sec23/24p and slows down that of Sar1p. A mathematical model of COPII membrane turnover that reproduces the experimental in vivo FRAP kinetics and is consistent with existing in vitro data predicts that Sec23/24p remains membrane associated even after GTP hydrolysis by Sar1p for a duration that is strongly increased by the presence of cargo. We conclude that secretory cargo retains the COPII complex on membranes, after Sar1p release has occurred, and prevents premature disassembly of COPII during cargo sorting and transport carrier formation.
Europe PMC
Systematic Subcellular Localization of Novel Proteins.
Simpson, J.C. & Pepperkok, R.
In Cell Biology. A Laboratory Handbook (3rd ed.), 2006, vol. 3, pp. 121?127.
This chapter describes the basic methodology used to systematically determine the subcellular localization of novel human proteins, as they have been derived from past and current cDNA sequencing projects worldwide. Since, localization is wholly dependent upon targeting sequences within the protein of interest, tagging of a protein with green fluorescent protein (GFP) always carries the risk that these targeting sequences become masked, which will finally lead to a mislocalization of the tagged protein. The localization that best matches the bioinformatic data is then considered as the correct one. Finally, data are verified by colocalization of the GFP fusion proteins with established endogenous organelle-specific markers. The localization that best matches the bioinformatic data is then considered as the correct one. Finally, data are verified by colocalization of the GFP fusion proteins with established endogenous organelle-specific markers. In parallel cultures or following image acquisition from live cells, it is important to fix the cells and remove any soluble GFP signal that may be obscuring more subtle localization patterns.
The subcellular localization of the mammalian proteome comes a fraction closer.
Simpson, J.C. & Pepperkok, R.
Genome Biol. 2006;7(6):222.
Another step along the road towards determining the subcellular localization of a complete mammalian proteome has been taken with a study using cellular fractionation and protein correlation profiling to identify and localize organellar proteins. Here we discuss this new work in the context of other strategies for large-scale subcellular localization.
Europe PMC
The LIFEdb database in 2006.
Mehrle, A., Rosenfelder, H., Schupp, I., del Val, C., Arlt, D., Hahne, F., Bechtel, S., Simpson, J., Hofmann, O., Hide, W., Glatting, K.H., Huber, W., Pepperkok, R., Poustka, A. & Wiemann, S.
Nucleic Acids Res. 2006 Jan 1;34(Database issue):D415-8.
LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.
Europe PMC
Production of siRNA- and cDNA-Transfected Cell Arrays on Noncoated Chambered Coverglass for High-Content Screening Microscopy in Living Cells.
Erfle, H. & Pepperkok, R.
Methods Mol Biol. 2006;360:155-62.
In this chapter, we provide a protocol for the production of transfected cell arrays in living mammalian cells on noncoated chambered coverglass for the systematic functional analyses of human genes by high-content screening microscopy. This method should facilitate drug target validation by small-interfering RNAs.
Europe PMC
In vivo selective cytoskeleton dynamics quantification in interphase cells induced by pulsed ultraviolet laser nanosurgery.
Colombelli, J., Reynaud, E.G., Rietdorf, J., Pepperkok, R. & Stelzer, E.H.K.
Traffic. 2005 Dec;6(12):1093-102.
We report on the manipulation of intracellular filaments using a nanosurgery system based on a subnanosecond pulsed UV laser optimized for the localized severing of biological polymers. By inducing artificial catastrophe of selected microtubules (MTs), we perform shrinkage-rate measurements in interphase Ptk-2 cells throughout the entire cell. We quantify the impact of two labeling methods and three fluorescent markers, showing a 25% faster depolymerization with Alexa-488 tubulin compared with Rhodamine and yellow fluorescent protein (YFP) tubulins and a 20% higher variability induced by microinjection compared with stable transfection. Using EB3-GFP as a tip marker, we establish a new protocol to measure shrinkage rate, growth rate and rescue frequency simultaneously with high temporal and spatial specificity in live cells. As our analysis shows, laser-induced MT dynamics are physiologically relevant. The high statistical efficiency that the method offers in terms of numbers of measured events and therefore reduced standard deviations represents an important quantitative improvement in the measurement of dynamic instability parameters in vivo. We extend the application of the method by demonstrating induced dynamic behavior of actin-stress fibers after severing. This new method enables the quantitative investigation of cytoskeleton dynamics in a local confinement.
Europe PMC
Human Lsg1 defines a family of essential GTPases that correlates with the evolution of compartmentalization.
Reynaud, E.G., Andrade, M.A., Bonneau, F., Ly, T.B., Knop, M., Scheffzek, K. & Pepperkok, R.
BMC Biol. 2005 Oct 7;3:21.
BACKGROUND: Compartmentalization is a key feature of eukaryotic cells, but its evolution remains poorly understood. GTPases are the oldest enzymes that use nucleotides as substrates and they participate in a wide range of cellular processes. Therefore, they are ideal tools for comparative genomic studies aimed at understanding how aspects of biological complexity such as cellular compartmentalization evolved. RESULTS: We describe the identification and characterization of a unique family of circularly permuted GTPases represented by the human orthologue of yeast Lsg1p. We placed the members of this family in the phylogenetic context of the YlqF Related GTPase (YRG) family, which are present in Eukarya, Bacteria and Archea and include the stem cell regulator Nucleostemin. To extend the computational analysis, we showed that hLsg1 is an essential GTPase predominantly located in the endoplasmic reticulum and, in some cells, in Cajal bodies in the nucleus. Comparison of localization and siRNA datasets suggests that all members of the family are essential GTPases that have increased in number as the compartmentalization of the eukaryotic cell and the ribosome biogenesis pathway have evolved. CONCLUSION: We propose a scenario, consistent with our data, for the evolution of this family: cytoplasmic components were first acquired, followed by nuclear components, and finally the mitochondrial and chloroplast elements were derived from different bacterial species, in parallel with the formation of the nucleolus and the specialization of nuclear components.
Europe PMC
IR micro spectroscopy of live cells.
Moss, D.A., Keese, M., Pepperkok, R.
Vibrational Spectroscopy 2005 JUL;38(1-2): 185-191.
The objective of the present study was to establish the conditions for infrared spectroscopy of single living cells in cell culture under aqueous media, using a high brilliance synchrotron light source to achieve acceptable S/N at the necessary high spatial resolution. This is experimentally challenging, but brings a number of significant advantages. The data presented show that the infrared spectra of single living cells can be monitored over several hours with high reproducibility, as well as confirming that acquisition of spectral data from single cells can contribute new insights that are not available from measurements of cell populations. (c) 2005 Elsevier B.V. All rights reserved.
Imaging epidermal growth factor receptor phosphorylation in human colorectal cancer cells and human tissues.
Keese, M., Magdeburg, R.J., Herzog, T., Hasenberg, T., Offterdinger, M., Pepperkok, R., Sturm, J.W. & Bastiaens, P.I.
J Biol Chem 2005 Jul 29;280(30):27826-31. Epub 2005 May 20.
In tumor cells, high phosphorylation levels of receptor tyrosine kinases may occur in the absence of exogenous ligands due to autocrine signaling or enhanced tyrosine kinase activity. Here we show that the phosphorylation state of the endogenous epidermal growth factor receptor (EGFR) can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies against the receptor and phosphotyrosine, respectively. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation with EGF or pervanadate, dependent on the level of basal EGFR phosphorylation in the respective cell lines. This basal activity correlated inversely with receptor expression. Using the acceptor photobleaching fluorescence resonance energy transfer imaging approach, a significantly higher phosphorylation state of EGFR was also found in resected human colorectal tumor samples as compared with adjacent healthy tissue. Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth.
Europe PMC
Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions.
Knauer, S.K., Moodt, S., Berg, T., Liebel, U., Pepperkok, R. & Stauber, R.H.
Traffic 2005 Jul;6(7):594-606.
Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.
Europe PMC
Dynamics of COPII vesicles and the Golgi apparatus in cultured Nicotiana tabacum BY-2 cells provides evidence for transient association of Golgi stacks with endoplasmic reticulum exit sites.
Yang, Y.D., Elamawi, R., Bubeck, J., Pepperkok, R., Ritzenthaler, C. & Robinson, D.G.
Plant Cell 2005 May;17(5):1513-31. Epub 2005 Apr 1.
Despite the ubiquitous presence of the COPI, COPII, and clathrin vesicle budding machineries in all eukaryotes, the organization of the secretory pathway in plants differs significantly from that in yeast and mammalian cells. Mobile Golgi stacks and the lack of both transitional endoplasmic reticulum (ER) and a distinct ER-to-Golgi intermediate compartment are the most prominent distinguishing morphological features of the early secretory pathway in plants. Although the formation of COPI vesicles at periphery of Golgi cisternae has been demonstrated in plants, exit from the ER has been difficult to visualize, and the spatial relationship of this event is now a matter of controversy. Using tobacco (Nicotiana tabacum) BY-2 cells, which represent a highly active secretory system, we have used two approaches to investigate the location and dynamics of COPII binding to the ER and the relationship of these ER exit sites (ERES) to the Golgi apparatus. On the one hand, we have identified endogenous COPII using affinity purified antisera generated against selected COPII-coat proteins (Sar1, Sec13, and Sec23); on the other hand, we have prepared a BY-2 cell line expressing Sec13:green fluorescent protein (GFP) to perform live cell imaging with red fluorescent protein-labeled ER or Golgi stacks. COPII binding to the ER in BY-2 cells is visualized as fluorescent punctate structures uniformly distributed over the surface of the ER, both after antibody staining as well as by Sec13:GFP expression. These structures are smaller and greatly outnumber the Golgi stacks. They are stationary, but have an extremely short half-life (<10 s). Without correlative imaging data on the export of membrane or lumenal ER cargo it was not possible to equate unequivocally these COPII binding loci with ERES. When a GDP-fixed Sar1 mutant is expressed, ER export is blocked and the visualization of COPII binding is perturbed. On the other hand, when secretion is inhibited by brefeldin A, COPII binding sites on the ER remain visible even after the Golgi apparatus has been lost. Live cell imaging in a confocal laser scanning microscope equipped with spinning disk optics allowed us to investigate the relationship between mobile Golgi stacks and COPII binding sites. As they move, Golgi stacks temporarily associated with COPII binding sites at their rims. Golgi stacks were visualized with their peripheries partially or fully occupied with COPII. In the latter case, Golgi stacks had the appearance of a COPII halo. Slow moving Golgi stacks tended to have more peripheral COPII than faster moving ones. However, some stationary Golgi stacks entirely lacking COPII were also observed. Our results indicate that, in a cell type with highly mobile Golgi stacks like tobacco BY-2, the Golgi apparatus is not continually linked to a single ERES. By contrast, Golgi stacks associate intermittently and sometimes concurrently with several ERES as they move.
Europe PMC
Gamma-BAR, a novel AP-1-interacting protein involved in post-Golgi trafficking.
Neubrand, V.E., Will, R.D., Mobius, W., Poustka, A., Wiemann, S., Schu, P., Dotti, C.G., Pepperkok, R. & Simpson, J.C.
EMBO J 2005 Mar 23;24(6):1122-33. Epub 2005 Mar 10.
A novel peripheral membrane protein (2c18) that interacts directly with the gamma 'ear' domain of the adaptor protein complex 1 (AP-1) in vitro and in vivo is described. Ultrastructural analysis demonstrates a colocalization of 2c18 and gamma1-adaptin at the trans-Golgi network (TGN) and on vesicular profiles. Overexpression of 2c18 increases the fraction of membrane-bound gamma1-adaptin and inhibits its release from membranes in response to brefeldin A. Knockdown of 2c18 reduces the steady-state levels of gamma1-adaptin on membranes. Overexpression or downregulation of 2c18 leads to an increased secretion of the lysosomal hydrolase cathepsin D, which is sorted by the mannose-6-phosphate receptor at the TGN, which itself involves AP-1 function for trafficking between the TGN and endosomes. This suggests that the direct interaction of 2c18 and gamma1-adaptin is crucial for membrane association and thus the function of the AP-1 complex in living cells. We propose to name this protein gamma-BAR.
Europe PMC
Live imaging of bidirectional traffic from the ERGIC.
Ben-Tekaya, H., Miura, K., Pepperkok, R. & Hauri, H.P.
J Cell Sci 2005 Jan 15;118(Pt 2):357-67. Epub 2005 Jan 04.
The endoplasmic reticulum-Golgi intermediate compartment (ERGIC) defined by the cycling lectin ERGIC-53 consists of tubulovesicular clusters, but it is unknown if these membranes are transport vehicles or stationary entities. Here, we show by live imaging that GFP-ERGIC-53 mainly localizes to long-lived stationary and some short-lived highly mobile elements. Unlike the anterograde marker VSV-G-GFP, GFP-ERGIC-53 does not vectorially move to the Golgi upon exit from the ERGIC, as assessed by a novel quantitative vector field method. Dual-color imaging of GFP-ERGIC-53 and a secretory protein (signal-sequence-tagged dsRed) reveals that the stationary elements are sites of repeated sorting of retrograde and anterograde cargo, and are interconnected by highly mobile elements. These results suggest that the ERGIC is stationary and not simply a collection of mobile carriers that mediate protein traffic from endoplasmic reticulum to Golgi.
Europe PMC
Regulation of microtubule-dependent recycling at the trans-Golgi network by Rab6A and Rab6A'.
Young, J., Stauber, T., del Nery, E., Vernos, I., Pepperkok, R. & Nilsson, T.
Mol Biol Cell 2005 Jan;16(1):162-77. Epub 2004 Oct 13.
The small GTPase rab6A but not the isoform rab6A' has previously been identified as a regulator of the COPI-independent recycling route that carries Golgi-resident proteins and certain toxins from the Golgi to the endoplasmic reticulum (ER). The isoform rab6A' has been implicated in Golgi-to-endosomal recycling. Because rab6A but not A', binds rabkinesin6, this motor protein is proposed to mediate COPI-independent recycling. We show here that both rab6A and rab6A' GTP-restricted mutants promote, with similar efficiency, a microtubule-dependent recycling of Golgi resident glycosylation enzymes upon overexpression. Moreover, we used small interfering RNA mediated down-regulation of rab6A and A' expression and found that reduced levels of rab6 perturbs organization of the Golgi apparatus and delays Golgi-to-ER recycling. Rab6-directed Golgi-to-ER recycling seems to require functional dynactin, as overexpression of p50/dynamitin, or a C-terminal fragment of Bicaudal-D, both known to interact with dynactin inhibit recycling. We further present evidence that rab6-mediated recycling seems to be initiated from the trans-Golgi network. Together, this suggests that a recycling pathway operates at the level of the trans-Golgi linking directly to the ER. This pathway would be the preferred route for both toxins and resident Golgi proteins.
Europe PMC
Coupling of ER exit to microtubules through direct interaction of COPII with dynactin.
Watson, P., Forster, R., Palmer, K.J., Pepperkok, R. & Stephens, D.J.
Nat Cell Biol 2005 Jan;7(1):48-55. Epub 2004 Dec 5.
Transport of proteins from the endoplasmic reticulum (ER) to the Golgi is mediated by the sequential action of two coat complexes: COPII concentrates cargo for secretion at ER export sites, then COPI is subsequently recruited to nascent carriers and retrieves recycling proteins back to the ER. These carriers then move towards the Golgi along microtubules, driven by the dynein/dynactin complexes. Here we show that the Sec23p component of the COPII complex directly interacts with the dynactin complex through the carboxy-terminal cargo-binding domain of p150(Glued). Functional assays, including measurements of the rate of recycling of COPII on the ER membrane and quantitative analyses of secretion, indicate that this interaction underlies functional coupling of ER export to microtubules. Together, our data suggest a mechanism by which membranes of the early secretory pathway can be linked to motors and microtubules for subsequent organization and movement to the Golgi apparatus.
Europe PMC
EAMNET - a resource and teaching framework for advanced light microscopy.
Terjung, S., Pepperkok, R., Ankerhold, R., Caiolfa, V.R., Castel, S., Majer, P., Geiger, B., Luini, A., Salamero, J., Shorte, S. & Small, J.V.
European Journal of Cell Biology 2005 84 108
Imaging platforms for measurement of membrane trafficking.
Pepperkok, R., Simpson, J.C., Rietdorf, J., Cetin, C., Liebel, U., Terjung, S. & Zimmermann, T.
Methods Enzymol. 2005;404:8-18.
In this chapter we describe automated imaging methods used to measure the transport of an established membrane transport marker from the endoplasmic reticulum to the plasma membrane. The method is fast and significantly robust to be applied in systematic studies on a large scale such as genome-wide screening projects. We further describe the use of software macros and plugins in Image J that allow the quantification of the kinetics of membrane transport intermediates in fluorescence microscopy time-lapse sequences.
Europe PMC
Bioinformatic "Harvester": a search engine for genome-wide human, mouse, and rat protein resources.
Liebel, U., Kindler, B. & Pepperkok, R.
Methods Enzymol. 2005;404:19-26.
Harvester is a meta search engine for gene and protein information. It searches 16 major databases and prediction servers and combines the results on pregenerated HTML pages. In this way Harvester can provide comprehensive gene-protein information from different servers in a convenient and fast manner. The Harvester search engine works similar to Google, offering genome-wide ranked results at very high speed. Here we describe how to use this bioinformatic tool along with selected examples.
Europe PMC
Arrays of transfected mammalian cells for high content screening microscopy.
Erfle, H. & Pepperkok, R.
Methods Enzymol. 2005;404:1-8.
In this chapter we describe protocols for reverse transfection to generate mammalian cell arrays for systematic gene knock-downs by RNAi or knock-ins by ectopic cDNA expression. The method is suitable for high content screening microscopy at a high spatial and temporal resolution allowing even time-lapse analysis of hundreds of samples in parallel.
Europe PMC
Novel cancer relevant cell cycle modulators identified in automated cell-based assays
Dorit Arlt, Huber, W., Mamatha Sauermann, Meher Majety, Florian Hahne, Pepperkok, R., Annemarie Poustka, and Stefan Wiemann
European Journal of Cell Biology, 84(Suppl. 55):30, 2005
Functional profiling: from microarrays via cell-based assays to novel tumor relevant modulators of the cell cycle.
Arlt, D., Huber, W., Liebel, U., Schmidt, C., Majety, M., Sauermann, M., Rosenfelder, H., Bechtel, S., Mehrle, A., Bannasch, D., Schupp, I., Seiler, M., Simpson, J.C., Hahne, F., Moosmayer, P., Ruschhaupt, M., Guilleaume, B., Wellenreuther, R., Pepperkok, R., Sultmann, H., Poustka, A. & Wiemann, S.
Cancer Res 2005 Sep 1;65(17):7733-42.
Cancer transcription microarray studies commonly deliver long lists of "candidate" genes that are putatively associated with the respective disease. For many of these genes, no functional information, even less their relevance in pathologic conditions, is established as they were identified in large-scale genomics approaches. Strategies and tools are thus needed to distinguish genes and proteins with mere tumor association from those causally related to cancer. Here, we describe a functional profiling approach, where we analyzed 103 previously uncharacterized genes in cancer relevant assays that probed their effects on DNA replication (cell proliferation). The genes had previously been identified as differentially expressed in genome-wide microarray studies of tumors. Using an automated high-throughput assay with single-cell resolution, we discovered seven activators and nine repressors of DNA replication. These were further characterized for effects on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling (G1-S transition) and anchorage-independent growth (tumorigenicity). One activator and one inhibitor protein of ERK1/2 activation and three repressors of anchorage-independent growth were identified. Data from tumor and functional profiling make these proteins novel prime candidates for further in-depth study of their roles in cancer development and progression. We have established a novel functional profiling strategy that links genomics to cell biology and showed its potential for discerning cancer relevant modulators of the cell cycle in the candidate lists from microarray studies.
Europe PMC
Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts.
Brunet, S., Sardon, T., Zimmerman, T., Wittmann, T., Pepperkok, R., Karsenti, E. & Vernos, I.
Mol Biol Cell 2004 Dec;15(12):5318-28. Epub .
TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.
Europe PMC
From ORFeome to biology: a functional genomics pipeline.
Wiemann, S., Arlt, D., Huber, W., Wellenreuther, R., Schleeger, S., Mehrle, A., Bechtel, S., Sauermann, M., Korf, U., Pepperkok, R., Sultmann, H. & Poustka, A.
Genome Res. 2004 Oct;14(10B):2136-44.
As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.
Europe PMC
High-content screening microscopy identifies novel proteins with a putative role in secretory membrane traffic.
Starkuviene, V., Liebel, U., Simpson, J.C., Erfle, H., Poustka, A., Wiemann, S. & Pepperkok, R.
Genome Res 2004 Oct;14(10A):1948-56.
Here we describe the establishment of microscope-based functional screening assays in intact cells that allow us to systematically identify new proteins involved in secretory membrane traffic, and proteins that can influence the integrity of the Golgi complex. We were able to identify 20 new proteins that affected either secretory transport, Golgi morphology, or both, when overexpressed in cells. Control experiments with human orthologs to yeast proteins with a role in membrane traffic, or already well characterized mammalian regulators of the secretory pathway, confirmed the specificity and significance of our results. Proteins localized to the Golgi complex or endoplasmic reticulum (ER) showed preferential interference in our assays. Bioinformatic analysis of the new proteins interfering with membrane traffic and/or Golgi integrity revealed broad functional variety, but demonstrated a bias towards proteins with predicted coiled-coil domains and repeat structures. Extending our approach to a much larger set of novel proteins in the future will be an important step toward a more comprehensive understanding of the molecular basis of the secretory pathway. It will also serve as an example for similar microscope-based screens addressing different biological questions.
Europe PMC
siRNA cell arrays for high-content screening microscopy.
Erfle, H., Simpson, J.C., Bastiaens, P.I. & Pepperkok, R.
Biotechniques 2004 Sep;37(3):454-8, 460, 462.
RNA interference (RNAi) is a recent advance that provides the possibility to reduce the expression of specific target genes in cultured mammalian cells with potential applications on a genome-wide scale. However, to achieve this, robust methodologies that allow automated and efficient delivery of small interfering RNAs (siRNAs) into living cultured cells and reliable quality control of siRNA function must be in place. Here we describe the production of cell arrays for reverse transfection of tissue culture cells with siRNA and plasmid DNA suitable for subsequent high-content screening microscopy applications. All the necessary transfection components are mixed prior to the robotic spotting on noncoated chambered coverglass tissue culture dishes, which are ideally suited for time-lapse microscopy applications in living cells. The addition of fibronectin to the spotting solution improves cell adherence. After cell seeding, no further cell culture manipulations, such as medium changes or the addition of 7 serum, are needed. Adaptation of the cell density improves autofocus performance for high-quality data acquisition and cell recognition. The co-transfection of a nonspecific fluorescently labeled DNA oligomer with the specific siRNA helps to mark each successfully transfected cell and cell cluster. We demonstrate such an siRNA cell array in a microscope-based functional assay in living cells to determine the effect of various siRNA oligonucleotides against endogenous targets on cellular secretion.
Europe PMC
'Harvester': a fast meta search engine of human protein resources.
Liebel, U., Kindler, B. & Pepperkok, R.
Bioinformatics 2004 Aug 12;20(12):1962-3. Epub 2004 Feb 26.
SUMMARY: We have developed a Web-based tool named 'Harvester' that bulk-collects bioinformatic data on human proteins from various databases and prediction servers. The information on every single protein is assembled on a single HTML page as a combination of database screen-shots and plain text. A full text meta search engine, similar to Google trade mark, allows screening of the whole genome proteome for current protein functions and predictions in a few seconds. With Harvester it is now possible to compare and check the quality of different database entries and prediction algorithms on a single page. A feedback forum allows users to comment on Harvester and to report database inconsistencies. AVAILABILITY: The service is freely available to the academic community at http://harvester.embl.de.
Europe PMC
Differential effects of a GTP-restricted mutant of Sar1p on segregation of cargo during export from the endoplasmic reticulum.
Stephens, D.J. & Pepperkok, R.
J Cell Sci 2004 Jul 15;117(Pt 16):3635-44.
Export of cargo from the endoplasmic reticulum (ER) is the first membrane trafficking step in the secretory pathway. To date, all cargo proteins appear to use a common set of machinery for the initial stages of export, namely the COPII coat complex. Recent data from both yeast and mammalian systems have emerged suggesting that specific cargoes could be sorted from one another at the point of exit from the endoplasmic reticulum or immediately afterwards. Here, we have examined the mechanisms used for export of different types of cargo molecule from the endoplasmic reticulum. All cargoes examined utilise the COPII machinery, but specific differences are seen in the accumulation of cargo into ER-derived pre-budding complexes following expression of a GTP-restricted mutant of the Sar1p GTPase. Glycosylphosphatidylinositol (GPI)-anchored GFP is seen to be restricted to the ER under these conditions whereas other cargoes, including ts045-G and lumFP accumulate in pre-budding complexes. Following exit, GPI-FP, lumFP and ts045-G-FP all travel to the Golgi in the same vesicular tubular clusters (VTCs). These data show a differential requirement for efficient GTP hydrolysis by the Sar1p GTPase in export of cargo from the ER.
Europe PMC
Confocal microscopy analysis of native, full length and B-domain deleted coagulation factor VIII trafficking in mammalian cells.
Becker, S., Simpson, J.C., Pepperkok, R., Heinz, S., Herder, C., Grez, M., Seifried, E. & Tonn, T.
Thromb Haemost 2004 Jul;92(1):23-35.
In mammalian cells, factor VIII (FVIII) secretion depends upon its interaction with chaperones of the endoplasmic reticulum (ER) and requires a unique ATP-dependent step to dissociate aggregates formed within the ER. To further elucidate mechanisms which might account for the inefficient secretion of recombinant FVIII (rFVIII), we have analyzed the pathways of recombinant full length (rFVIII-FL) and B-domain deleted (rFVIII Delta B) FVIII and compared these to the secretion route of native FVIII in primary hepatocytes. Using confocal laser scanning microscopy in combination with a pulse chase of a known secretion marker, we describe the trafficking route of FVIII, which upon release from the ER--where it colocalizes with calnexin--is transported to the Golgi complex in vesicular-tubular transport complexes (VTCs) which could be further identified as being COP I coated. However, a large portion of rFVIII is retained in the ER and additionally in structures which could not be assigned to the ER, Golgi complex or intermediate compartment. Moderate BiP transcription levels indicate that this observed retention of FVIII does not reflect cellular stress due to an overexpression of FVIII-protein in transduced cells. Moreover, a pulse of newly synthesized rFVIII protein is released within 4 hrs, indicating that once rFVIII is released from the ER there is no further limitation to its secretion. Our data provide new details about the secretory route of FVIII, which may ultimately help to identify factors currently limiting the efficient and physiological expression of FVIII in gene therapy and manufacture.
Europe PMC
Automatic identification of subcellular phenotypes on human cell arrays.
Conrad, C., Erfle, H., Warnat, P., Daigle, N., Lorch, T., Ellenberg, J., Pepperkok, R. & Eils, R.
Genome Res. 2004 Jun;14(6):1130-6.
Light microscopic analysis of cell morphology provides a high-content readout of cell function and protein localization. Cell arrays and microwell transfection assays on cultured cells have made cell phenotype analysis accessible to high-throughput experiments. Both the localization of each protein in the proteome and the effect of RNAi knock-down of individual genes on cell morphology can be assayed by manual inspection of microscopic images. However, the use of morphological readouts for functional genomics requires fast and automatic identification of complex cellular phenotypes. Here, we present a fully automated platform for high-throughput cell phenotype screening combining human live cell arrays, screening microscopy, and machine-learning-based classification methods. Efficiency of this platform is demonstrated by classification of eleven subcellular patterns marked by GFP-tagged proteins. Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells.
Europe PMC
LIFEdb: a database for functional genomics experiments integrating information from external sources, and serving as a sample tracking system.
Bannasch, D., Mehrle, A., Glatting, K.H., Pepperkok, R., Poustka, A. & Wiemann, S.
Nucleic Acids Res 2004 Jan 1;32 Database issue:D505-8.
We have implemented LIFEdb (http://www.dkfz.de/LIFEdb) to link information regarding novel human full-length cDNAs generated and sequenced by the German cDNA Consortium with functional information on the encoded proteins produced in functional genomics and proteomics approaches. The database also serves as a sample-tracking system to manage the process from cDNA to experimental read-out and data interpretation. A web interface enables the scientific community to explore and visualize features of the annotated cDNAs and ORFs combined with experimental results, and thus helps to unravel new features of proteins with as yet unknown functions.
Europe PMC
A microscope-based screening platform for large-scale functional protein analysis in intact cells.
Liebel, U., Starkuviene, V., Erfle, H., Simpson, J.C., Poustka, A., Wiemann, S. & Pepperkok, R.
FEBS Lett 2003 Nov 20;554(3):394-8.
A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects.
Europe PMC
CDNAs for functional genomics and proteomics: the German Consortium.
Wiemann, S., Mehrle, A., Bechtel, S., Wellenreuther, R., Pepperkok, R. & Poustka, A.
C R Biol. 2003 Oct-Nov;326(10-11):1003-9.
To functionally characterize numerous novel proteins encoded by cDNAs sequenced by the German Consortium, 800 were tagged with green fluorescent protein. The subcellular localizations of the fusion proteins were examined in living cells, enabling their classification in subcellular groups. Their activity in cell growth, cell death, and protein transport was screened in high throughput using robotic liquid handling and reading stations. The resulting information is integrated with functional genomics and proteomics data for further understanding of protein functions in the cellular context.
Europe PMC
Spectral imaging and its applications in live cell microscopy.
Zimmermann, T., Rietdorf, J. & Pepperkok, R.
FEBS Lett 2003 Jul 3;546(1):87-92.
In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct fluorescent molecules at the same time in the same sample. However, the spectral properties of the fluorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods. Spectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging. This method also offers advantages for fast multicolor time-lapse microscopy and fluorescence resonance energy transfer measurements in living samples. Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples.
Europe PMC
The German cDNA network: cDNAs, functional genomics and proteomics.
Wiemann, S., Bechtel, S., Bannasch, D., Pepperkok, R. & Poustka, A.
J Struct Funct Genomics 2003;4(2-3):87-96.
Among the greatest challenges facing biology today is the exploitation of huge amounts of genomic data, and their conversion into functional information about the proteins encoded. For example, the large-scale cDNA sequencing project of the German cDNA Consortium is providing vast numbers of open reading frames (ORFs) encoding novel proteins of completely unknown function. As a first step towards their characterization we have tagged over 500 of these with the green fluorescent protein (GFP), and examined the subcellular localizations of these fusion proteins in living cells. These data have allowed us to classify the proteins into subcellular groups which determines the next step towards a detailed functional characterization. To make further use of these GFP-tagged constructs, a series of functional assays have been designed and implemented to assess the effect of these novel proteins on processes such as cell growth, cell death, and protein transport. Functional assays with such a large set of molecules is only possible by automation. Therefore, we have developed, and adapted, functional assays for use by robotic liquid handling stations and reading stations. A transport assay allows to identify proteins which localize to distinct organelles of the secretory pathway and have the potential to be new regulators in protein transport, a proliferation assay helps identifying proteins that stimulate or repress mitosis. Further assays to monitor the effects of the proteins in apoptosis and signal transduction pathways are in progress. Integrating the functional information that is generated in the assays with data from expression profiling and further functional genomics and proteomics approaches, will ultimately allow us to identify functional networks of proteins in a morphological context, and will greatly contribute to our understanding of cell function.
Europe PMC
Localizing the proteome.
Simpson, J.C. & Pepperkok, R.
Genome Biol 2003;4(12):240. Epub 2003 Nov 18.
The subcellular localization of the entire proteome of an organism, the yeast Saccharomyces cerevisiae, has been revealed for the first time. Comparison with less comprehensive studies of mammalian cells provides insights into the localization of the mammalian proteome.
Europe PMC
Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair.
Zimmermann, T., Rietdorf, J., Girod, A., Georget, V. & Pepperkok, R.
FEBS Lett 2002 Nov 6;531(2):245-9.
Spectral variants of the green fluorescent protein (GFP) have been extensively used as reporters to image molecular interactions in living cells by fluorescence resonance energy transfer (FRET). However, those GFP variants which are the most efficient donor acceptor pairs for FRET measurements show a high degree of spectral overlap which has hampered in the past their use in FRET applications. Here we use spectral imaging and subsequent un-mixing to quantitatively separate highly overlapping donor and acceptor emissions in FRET measurements. We demonstrate the method in fixed and living cells using a novel GFP based FRET pair (GFP2-YFP (yellow)), which has an increased FRET efficiency compared to the most commonly used FRET pair consisting of cyan fluorescent protein and YFP. Moreover, GFP2 has its excitation maximum at 396 nm at which the YFP acceptor is excited only below the detection level and thus this FRET pair is ideal for applications involving sensitized emission.
Europe PMC
Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells.
Gruss, O.J., Wittmann, M., Yokoyama, H., Pepperkok, R., Kufer, T., Sillje, H., Karsenti, E., Mattaj, I.W. & Vernos, I.
Nat Cell Biol 2002 Nov;4(11):871-9.
In Xenopus laevis egg extracts, TPX2 is required for the Ran-GTP-dependent assembly of microtubules around chromosomes. Here we show that interfering with the function of the human homologue of TPX2 in HeLa cells causes defects in microtubule organization during mitosis. Suppressing the expression of human TPX2 by RNA interference leads to the formation of two microtubule asters that do not interact and do not form a spindle. Our results suggest that in vivo, even in the presence of duplicated centrosomes, spindle formation requires the function of TPX2 to generate a stable bipolar spindle with overlapping antiparallel microtubule arrays. This indicates that chromosome-induced microtubule production is a general requirement for the formation of functional spindles in animal cells.
Europe PMC
Inhibitors of COP-mediated transport and cholera toxin action inhibit simian virus 40 infection.
Richards, A.A., Stang, E., Pepperkok, R. & Parton, R.G.
Mol Biol Cell 2002 May;13(5):1750-64.
Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20 degrees C. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to betaCOP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection.
Europe PMC
Imaging of procollagen transport reveals COPI-dependent cargo sorting during ER-to-Golgi transport in mammalian cells.
Stephens, D.J. & Pepperkok, R.
J Cell Sci 2002 Mar 15;115(Pt 6):1149-60.
We have examined the ER-to-Golgi transport of procollagen, which, when assembled in the lumen of the ER, is thought to be physically too large to fit in classically described 60-80 nm COPI- and COPII-coated transport vesicles. We found that procollagen exits the ER via COPII- coated ER exit sites and is transported to the Golgi along microtubules in defined transport complexes. These procollagen-containing transport complexes are, however, distinct from those containing other cargo proteins like ERGIC-53 and ts-045-G. Furthermore, they do not label for the COPI coat complex in contrast to those containing ts-045-G. Inhibition of COPII or COPI function before addition of ascorbate, which is required for the folding of procollagen, inhibits export of procollagen from the ER. Inactivation of COPI coat function after addition of ascorbate results in the localisation of procollagen to transport complexes that now also contain ERGIC-53 and are inhibited in their transport to the Golgi complex. These data reveal the existence of an early COPI-dependent, pre-Golgi cargo sorting step in mammalian cells.
Europe PMC
Inhibition of intracellular cholesterol transport alters presenilin localization and amyloid precursor protein processing in neuronal cells.
Runz, H., Rietdorf, J., Tomic, I., de Bernard, M., Beyreuther, K., Pepperkok, R. & Hartmann, T.
J Neurosci 2002 Mar 1;22(5):1679-89.
Generation of amyloid-beta (Abeta) from the amyloid precursor protein (APP) requires proteolytic cleavage by two proteases, beta- and gamma-secretase. Several lines of evidence suggest a role for cholesterol on secretase activities, although the responsible cellular mechanisms remain unclear. Here we show that alterations in cholesterol transport from late endocytic organelles to the endoplasmic reticulum have important consequences for both APP processing and the localization of gamma-secretase-associated presenilins (PS). Exposure of neuronal cells to cholesterol transport-inhibiting agents resulted in a marked decrease in beta-cleavage of full-length APP. In contrast, gamma-secretase activity on APP C-terminal fragments was enhanced, increasing the production of both Abeta40 and Abeta42. Remarkably, retention of cholesterol in endosomal/lysosomal compartments induced PS1 and PS2 to accumulate in Rab7-positive vesicular organelles implicated in cholesterol sorting. Accumulation of PS in vesicular compartments was prominent in both Chinese hamster ovary cells deficient in Niemann-Pick C1 protein as well as in neuronal cells exposed to the cholesterol transport-inhibiting agent U18666A. Because Abeta42 also localized to PS1-containing vesicular compartments, organelles involved in cholesterol transport might represent an important site for gamma-secretase activity. Our results suggest that the subcellular distribution of cholesterol may be an important factor in how cholesterol alters Abeta production and the risk of Alzheimer's disease.
Europe PMC
A fully automated high content screening system for cell based genome analyses.
Liebel, U.& Pepperkok, R.
Transkript Laborwelt 2002 1 4-8
The Spir actin organizers are involved in vesicle transport processes.
Kerkhoff, E., Simpson, J.C., Leberfinger, C.B., Otto, I.M., Doerks, T., Bork, P., Rapp, U.R., Raabe, T. & Pepperkok, R.
Curr Biol 2001 Dec 11;11(24):1963-8.
The p150-Spir protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spir proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spir encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spir family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as "Spir-box." The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of p150-Spir and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spir protein colocalized specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spir localization pattern was dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibited the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein was arrested in membrane structures, largely colocalizing with the TGN marker TGN46. Our findings that the Spir actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport.
Europe PMC
The many ways to cross the plasma membrane.
Stephens, D.J. & Pepperkok, R.
Proc Natl Acad Sci U S A 2001 Apr 10;98(8):4295-8. Europe PMC
Illuminating the secretory pathway: when do we need vesicles?
Stephens, D.J. & Pepperkok, R.
J Cell Sci 2001 Mar;114(Pt 6):1053-9.
Recent studies using GFP-tagged markers and time-lapse microscopy have allowed direct visualisation of membrane traffic in the secretory pathway in living mammalian cells. This work shows that larger membrane structures, 300-500 nm in size, are the vehicles responsible for long distance, microtubule-dependent ER-to-Golgi and trans-Golgi to plasma membrane transport of secretory markers. At least two retrograde transport pathways from the Golgi to the ER exist, both of which are proposed to involve a further class of long, tubular membrane carrier that forms from the Golgi and fuses with the ER. Together, this has challenged established transport models, raising the question of whether larger pleiomorphic structures, rather than small 60-80 nm transport vesicles, mediate long-range transport between the ER and Golgi and between the Golgi and plasma membrane.
Europe PMC
Illuminating the human genome.
Simpson, J.C., Neubrand, V.E., Wiemann, S. & Pepperkok, R.
Histochem Cell Biol 2001 Jan;115(1):23-9.
The identification and analysis of novel genes and their encoded protein products remains a vigorous area of research in biology today. Worldwide genomic and cDNA sequencing projects are now identifying new molecules every day and the need for methodologies to functionally characterise these proteins has never been greater. The distinct compartmental arrangement of eukaryotic cells helps define the processes which occur within or in proximity to these membranes, and as such provides one means of inferring protein function. We describe here some of the methods recently reported in the literature, which use the subcellular localisation of proteins as a first step towards their further characterisation.
Europe PMC
Being in the right location at the right time.
Pepperkok, R., Simpson, J.C. & Wiemann, S.
Genome Biol 2001;2(9):REVIEWS1024.
Taking each coding sequence from the human genome in turn and identifying the subcellular localization of the corresponding protein would be a significant contribution to understanding the function of each of these genes and to deciphering functional networks. This article highlights current approaches aimed at achieving this goal.
Europe PMC
Observing proteins in their natural habitat: the living cell.
Bastiaens, P.I. & Pepperkok, R.
Trends Biochem Sci 2000 Dec 1;25(12):631-637
Fluorescence microscopy has played a tremendous role in uncovering the morphological features of cells and the expression pattern of proteins by immunofluorescence. Since the discovery of green-fluorescent proteins (GFPs), this technique has undergone a revival in the life sciences as the spatial distribution of ectopically expressed fusion proteins inside living cells can now be followed more easily. By further exploiting the photophysical properties of the emitted fluorescence with microspectroscopic methods, spatial information on the biochemical parameters of intracellular processes and reactions can be obtained. This possibility will not only play an important role in the understanding of biochemical reactions in signal processing and fidelity but also help to uncover the molecular mechanisms of organelle and cell morphogenesis.
Europe PMC
Breaking the COPI monopoly on Golgi recycling.
Storrie, B., Pepperkok, R. & Nilsson, T.
Trends Cell Biol. 2000 Sep;10(9):385-91.
The unexpected discovery of a transport pathway from the Golgi to the endoplasmic reticulum (ER) independent of COPI coat proteins sheds light on how Golgi resident enzymes and protein toxins gain access to the ER from as far as the trans Golgi network. This new pathway provides an explanation for how membrane is recycled to allow for an apparent concentration of anterograde cargo at distinct stages of the secretory pathway. As signal-mediated COPI-dependent recycling also involves the concentration of resident proteins into retrograde COPI vesicles, the main bulk of lipids must be recycled, possibly through a COPI-independent pathway.
Europe PMC
Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing.
Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R. & Wiemann, S.
EMBO rep 2000 Sep;1(3):287-92.
As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N- and C-terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of > 100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For > 80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.
Europe PMC
COPI-coated ER-to-Golgi transport complexes segregate from COPII in close proximity to ER exit sites.
Stephens, D.J., Lin-Marq, N., Pagano, A., Pepperkok, R. & Paccaud, J.P.
J Cell Sci 2000 Jun;113 ( Pt 12):2177-85.
Transport of proteins between the endoplasmic reticulum and Golgi apparatus is mediated by two distinct membrane coat complexes, COPI and COPII. Genetic, biochemical and morphological data have accumulated into a model which suggests a sequential mode of action with COPII mediating the selection of cargo and formation of transport vesicles at the ER membrane for ER-to-Golgi transport and COPI mediating recycling of the transport machinery from post-ER membranes. To test this transport model directly in vivo, and to study the precise temporal sequence of COPI and COPII action in ER-to-Golgi transport, we have used time lapse microscopy of living cells to visualise simultaneously the dynamics of COPII and COPI, as well as COPII and GFP tagged secretory markers in living cells. The majority of COPII labelling appears tightly associated with ER membranes that move only within a limited area (less than 2 microm). Secretory cargo segregates from these sites and is then transported to the Golgi apparatus without any apparent association with COPII. COPI-coated transport complexes are seen to form adjacent to the COPII sites on the ER before segregating and moving directionally towards the Golgi apparatus. COPII is not present on these transport complexes and remains associated with the ER. These data demonstrate for the first time directly in vivo that ER-to-Golgi transport is organised in two steps characterised by a sequential mode of action of COPII and COPI.
Europe PMC
Intracellular distribution of mammalian protein kinase A catalytic subunit altered by conserved Asn2 deamidation.
Pepperkok, R., Hotz-Wagenblatt, A., Konig, N., Girod, A., Bossemeyer, D. & Kinzel, V.
J Cell Biol 2000 Feb 21;148(4):715-26
The catalytic (C) subunit of protein kinase A functions both in the cytoplasm and the nucleus. A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH(2)-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A. Girod, A. Tholey, N. Konig, S. Thullner, V. Kinzel, and D. Bossemeyer. 1998. Protein Sci. 7:457-469). Because of the increase of electronegativity by generation of Asp2, it is reminiscent of a myristoyl-electrostatic switch. To compare the intracellular distribution of the enzymes, both forms of porcine or bovine heart enzyme were microinjected into the cytoplasm of mouse NIH 3T3 cells after conjugation with fluorescein, rhodamine, or in unlabeled form. The nuclear/cytoplasmic fluorescence ratio (N/C) was analyzed in the presence of cAMP (in the case of unlabeled enzyme by antibodies). Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form. Comparable data were obtained with a human cell line. The differential intracellular distribution of both enzyme forms is also reflected by functional data. It correlates with the degree of phosphorylation of the key serine in CREB family transcription factors in the nucleus. Microinjection of myristoylated recombinant bovine Calpha and the Asn2 deletion mutant of it yielded N/C ratios in the same range as encoded native enzymes. Thus, Asn2 seems to serve as a potential site for modulating electronegativity. The data indicate that the NH(2)-terminal domain of the PKA C-subunit contributes to the intracellular distribution of free enzyme, which can be altered by site-specific in vivo deamidation. The model character for other signaling proteins starting with myrGly-Asn is discussed.
Europe PMC
The p24 family member p23 is required for early embryonic development.
Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J.J., Solari, R.C. & Owen, M.J.
Curr Biol 2000 Jan 13;10(1):55-8
The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation. Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex. Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast. In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature). In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality. Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members. The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules. The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway.
Europe PMC
COPI vesicles accumulating in the presence of a GTP restricted Arf1 mutant are depleted of anterograde and retrograde cargo.
Pepperkok, R., Whitney, J.A., Gomez, M. & Kreis, T.E.
J Cell Sci 2000;113(Pt 1):135-144
Microinjection of the slowly hydrolyzable GTP analogue GTP(&ggr;)S or the ectopic expression of a GTP restricted mutant of the small GTPase arf1 (arf1[Q71L]) leads to the rapid accumulation of COPI coated vesicles and buds in living cells. This effect is blocked at 15 degrees C and by microinjection of antibodies against (&bgr;)-COP. Anterograde and retrograde membrane protein transport markers, which have been previously shown to be incorporated into COPI vesicles between the endoplasmic reticulum and Golgi complex, are depleted from the GTP(&ggr;)S or arf1[Q71L] induced COPI coated vesicles and buds. In contrast, in control cells 30 to 60% of the COPI carriers co-localize with these markers. These in vivo data corroborate recent in vitro work, suggesting that GTP(&ggr;)S and arf1[Q71L] interfere with the sorting of membrane proteins into Golgi derived COPI vesicles, and provide the first in vivo evidence for a role of GTP hydrolysis by arf1 in the sorting of cargo into COPI coated vesicles and buds.
Europe PMC
Immunofluorescence microscopy.
Pepperkok, R., Girod, A., Simpson, J. & Rietdorf, J.
In "Monoclonal Antibodies", Shepherd, P. & Dean, C. (eds.), Oxford Univ.Press, pp. 355-370
Dyskerin localizes to the nucleolus and its mislocalization is unlikely to play a role in the pathogenesis of dyskeratosis congenita.
Heiss, N.S., Girod, A., Salowsky, R., Wiemann, S., Pepperkok, R. & Poustka, A.
Hum Mol Genet 1999 Dec;8(13):2515-24
Mutations in the DKC1 gene are responsible for causing the bone marrow failure syndrome, dyskeratosis congenita (DKC; OMIM 305000). The majority of mutations identified to date are missense mutations and are clustered in exons 3, 4 and 11. It is predicted that the corresponding protein dyskerin is a nucleolar phosphoprotein which functions in both pseudo-uridylation and cleavage of precursor rRNA. Dyskerin contains multiple putative nuclear localization signals (NLSs) at the N-terminus (KKHKKKKERKS) and C-terminus [KRKR(X)(17)KKEKKKSKKDKKAK(X)(17)- KKKKKKKKAKEVELVSE]. By fusing dyskerin with the enhanced green fluorescent protein (EGFP) and by following a time course of expression in mammalian cell lines, we showed that full-length dyskerin initially localizes to the nucleoplasm and subsequently accumulates in the nucleoli. A co-localization to the coiled bodies was observed in some cells where dyskerin-EGFP had translocated to the nucleoli. Analysis of a series of mutant constructs indicated that whereas the most C- terminal lysine-rich clusters [KKEKKKS-KKDKKAK(X)(17)KKKKKKKKAKEVELVSE] influence the rate of nucleoplasmic and nucleolar accumulation, the KRKR sequence is primarily responsible for the nuclear import. Nucleolar localization was maintained when either the N- or C-terminal motifs were mutated, but not when all NLSs were removed. We conclude that the intranuclear localization of dyskerin is accomplished by the synergistic effect of a number of NLSs and that the nucleolar localization signals are contained within the NLSs. Further, examination of dyskerin-EGFP fusions mimicking mutations detected in patients indicated that the intracellular mislocalization of dyskerin is unlikely to cause DKC.
Europe PMC
Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum.
Girod, A., Storrie, B., Simpson, J.C., Johannes, L., Goud, B., Roberts, L.M., Lord, J.M., Nilsson, T. & Pepperkok, R.
Nat Cell Biolog 1999 Nov;1(7):423-430
The cytosolic coat-protein complex COP-I interacts with cytoplasmic 'retrieval' signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL- containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.
Europe PMC
Intracellular movement of green fluorescent protein-tagged phosphatidylinositol 3-kinase in response to growth factor receptor signaling.
Gillham, H., Golding, M.C., Pepperkok, R. & Gullick, W.J.
J Cell Biol 1999 Aug 23;146(4):869-80
Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein-tagged p85 subunit that phosphatidylinositol 3- kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein-tagged fragments of p85 we show that binding to the receptor requires the NH(2)-terminal part of the protein as well as its SH2 domains.
Europe PMC
Segregation of COPI-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to-Golgi transport complexes.
Shima, D.T., Scales, S.J., Kreis, T.E. & Pepperkok, R.
Curr Biol 1999 Jul 29-Aug 12;9(15):821-4
Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in the selective packaging of anterograde cargo into coated transport vesicles budding from the ER [1]. In mammalian cells, these vesicles coalesce to form tubulo-vesicular transport complexes (TCs), which shuttle anterograde cargo from the ER to the Golgi complex [2] [3] [4]. In contrast, COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER [1] [5] [6] [7]. The binding of COPI to COPII-coated TCs [3] [8] [9], however, has led to the proposal that COPI binds to TCs and specifically packages recycling proteins into retrograde vesicles for return to the ER [3] [9]. To test this hypothesis, we tracked fluorescently tagged COPI and anterograde-transport markers simultaneously in living cells. COPI predominated on TCs shuttling anterograde cargo to the Golgi complex and was rarely observed on structures moving in directions consistent with retrograde transport. Furthermore, a progressive segregation of COPI-rich domains and anterograde-cargo-rich domains was observed in the TCs. This segregation and the directed motility of COPI-containing TCs were inhibited by antibodies that blocked COPI function. These observations, which are consistent with previous biochemical data [2] [9], suggest a role for COPI within TCs en route to the Golgi complex. By sequestering retrograde cargo in the anterograde-directed TCs, COPI couples the sorting of ER recycling proteins [10] to the transport of anterograde cargo.
Europe PMC
Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy.
Pepperkok, R., Squire, A., Geley, S. & Bastiaens, P.I.
Curr Biol 1999 Mar 11;9(5):269-72
The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.
Europe PMC
The KDEL retrieval system is exploited by Pseudomonas exotoxin A, but not by Shiga-like toxin-1, during retrograde transport from the Golgi complex to the endoplasmic reticulum.
Jackson, M.E., Simpson, J.C., Girod, A., Pepperkok, R., Roberts, L.M. & Lord, J.M.
J Cell Sci 1999 Feb;112 ( Pt 4):467-75
To investigate the role of the KDEL receptor in the retrieval of protein toxins to the mammalian cell endoplasmic reticulum (ER), lysozyme variants containing AARL or KDEL C-terminal tags, or the human KDEL receptor, have been expressed in toxin-treated COS 7 and HeLa cells. Expression of the lysozyme variants and the KDEL receptor was confirmed by immunofluorescence. When such cells were challenged with diphtheria toxin (DT) or Escherichia coli Shiga-like toxin 1 (SLT-1), there was no observable difference in their sensitivities as compared to cells which did not express these exogenous proteins. By contrast, the cytotoxicity of Pseudomonas exotoxin A (PE) is reduced by expressing lysozyme-KDEL, which causes a redistribution of the KDEL receptor from the Golgi complex to the ER, and cells are sensitised to this toxin when they express additional KDEL receptors. These data suggest that, in contrast to SLT-1, PE can exploit the KDEL receptor in order to reach the ER lumen where it is believed that membrane transfer to the cytosol occurs. This contention was confirmed by microinjecting into Vero cells antibodies raised against the cytoplasmically exposed tail of the KDEL receptor. Immunofluorescence confirmed that these antibodies prevented the retrograde transport of the KDEL receptor from the Golgi complex to the ER, and this in turn reduced the cytotoxicity of PE, but not that of SLT-1, to these cells.
Europe PMC
Fluorescence microscopy of living vertebrate cells.
Pepperkok, R., & Shima, G.
In "Protein Localisation by Fluorescence Microscopy", V.J. Allan (ed.), Oxford Univ. Press, pp. 109-132
Three distinct steps in transport of vesicular stomatitis virus glycoprotein from the ER to the cell surface in vivo with differential sensitivities to GTP gamma S.
Pepperkok, R., Lowe, M., Burke, B. & Kreis, T.E.
J Cell Sci 1998 Jul;111 ( Pt 13):1877-88
Microinjected GTP gamma S revealed three distinct steps in the exocytic transport of the temperature sensitive glycoprotein of vesicular stomatitis virus (ts-O45-G) from the ER to the cell surface in intact Vero cells. While COPII dependent export of ts-O45-G from the ER is blocked in cells injected with recombinant protein of a dominant mutant of SAR1a (SAR1a[H79G]) inhibited in GTP hydrolysis, neither injected GTP gamma S nor antibodies against beta-COP (anti-EAGE) interfere with this transport step significantly. In contrast, transport to the Golgi complex is blocked by 50 microM GTP gamma S, a dominant mutant of ARF1 (ARF1[Q71L]) inhibited in GTP hydrolysis, or microinjected anti-EAGE, but injected Sar1a[H79G]p has no effect. Microinjection of GTP gamma S or expression of ARF[Q71L] rapidly induces accumulation of COPI coated vesicular structures lacking ts-O45-G. Finally, transport of ts-O45-G from the trans-Golgi network (TGN) to the cell surface is inhibited only by high concentrations of GTP gamma S (500 microM). Interestingly, this step is only partially brefeldin A sensitive, and injected antibodies against beta-COP and p200/myosin II, a TGN membrane associated protein, have no effect. These data provide first strong in vivo evidence for at least three distinct steps in the exocytic pathway of mammalian cells regulated by different sets of GTPases and coat proteins. COPII, but not COPI, is required for ER export of ts-O45-G. COPI plays a role in subsequent transport to the Golgi complex, and a so far unidentified GTP gamma S sensitive coat appears to be involved in transport from the TGN to the cell surface.
Europe PMC
Computer-automated and semiautomated capillary microinjection of macromolecules into living cells
Pepperkok, R., Saffrich, R. & Ansorge, W.
In "Cell Biology: A Laboratory Handbook, 2nd Edition", Vol 4, Celis, J.E. (ed.), 1998, Academic Press
Cellular micro-biochemistry.
Pepperkok, R., Rosorius, O., & Scheel, J.
In "Springer Lab Manual: Microinjection and transgenesis of cultured cells and embryos", Cid-Arregui, C. & Garcia-Carranca, A. (eds). Springer, Heidelberg, pp. 145-155
Electroporation of cells
Herr, S., Pepperkok, R., Saffrich, R., Wieman, S. & Ansorge, W.
In "Cell Biology: A Laboratory Handbook, 2nd Edition", Vol 4, Celis, J.E. (ed.), 1998, Academic Press
Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI.
Scales, S.J., Pepperkok, R. & Kreis, T.E.
Cell 1997 Sep 19;90(6):1137-48
Exocytic transport from the endoplasmic reticulum (ER) to the Golgi complex has been visualized in living cells using a chimera of the temperature-sensitive glycoprotein of vesicular stomatitis virus and green fluorescent protein (ts-G-GFP[ct]). Upon shifting to permissive temperature, ts-G-GFP(ct) concentrates into COPII-positive structures close to the ER, which then build up to form an intermediate compartment or transport complex, containing ERGIC-53 and the KDEL receptor, where COPII is replaced by COPI. These structures appear heterogenous and move in a microtubule-dependent manner toward the Golgi complex. Our results suggest a sequential mode of COPII and COPI action and indicate that the transport complexes are ER-to-Golgi transport intermediates from which COPI may be involved in recycling material to the ER.
Europe PMC
Dissociation of coatomer from membranes is required for brefeldin A- induced transfer of Golgi enzymes to the endoplasmic reticulum.
Scheel, J., Pepperkok, R., Lowe, M., Griffiths, G. & Kreis, T.E.
J Cell Biol 1997 Apr 21;137(2):319-33
Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the "EAGE"-peptide of beta-COP, inhibit BFA-induced redistribution of beta-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing beta-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45- G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only approximately 50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTP(gamma)S. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.
Europe PMC
Microinjection and electroporation of macromolecules into live cells.
Pepperkok, R.
In "Springer Lab Manual Cell Cycle-Materials and Methods", M. Pagano (ed.) Springer, Heidelberg, pp. 75-87
Capillary microinjection is one of the most efficient methods for introducing macromolecules into living mammalian cells (Proctor 1992). Samples can be delivered directly into either the nucleus or the cytoplasm. In synchronized cell cultures, the molecules can be transferred at well defined stages of the cell cycle and modifications of culture conditions are possible, before, during, or after injection. Therefore, microinjection of target specific inhibitors (e.g., antibodies, synthetic peptides, antisense mRNAs) or activators (e.g., proteins, peptides, cDNAs under the control of a constitutive promoter) has been a useful tool to study the in vivo function of proteins in cell proliferation and cell cycle control.
Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo.
Pepperkok, R., Scheel, J., Horstmann, H., Hauri, H.P., Griffiths, G. & Kreis, T.E.
Cell 1993 Jul 16;74(1):71-82
Microinjection of antibodies against a synthetic peptide of a non- clathrin-coated vesicle-associated coat protein, beta-COP, blocks transport of a temperature-sensitive vesicular stomatitis virus glycoprotein (ts-O45-G) to the cell surface. Transport is inhibited upon release of the viral glycoprotein from temperature blocks at 39.5 degrees C (endoplasmic reticulum [ER]) and 15 degrees C (intermediate compartment), but not at 20 degrees C (trans-Golgi network). Ts-O45-G is arrested in tubular membrane structures containing p53 at the interface of the ER and the Golgi stack. This is consistent with inhibition of acquisition of endoglycosidase H resistance of ts-O45-G in injected cells. Secretion of endogenous proteins and maturation of cathepsin D are also inhibited. These data provide in vivo evidence that beta-COP has an important function in biosynthetic membrane traffic in mammalian cells.
Europe PMC
CLIP-170, a Cytoplasmic Linker Protein Mediating Interaction of Endosomes with Microtubules.
Scheel, J., Rickard, J., Pierre, P., Henning, D., Karecla, P.I., Pepperkok, R., Joggerst-Thomalla, B., Sawyer, A. Parton, R.G. & Kreis, T.E.
In Nato ASI Series 74, pp. 145-157, "Molecular Mechanisms of Membrane Traffic", Morre, D.J., Howell, K.E. & Bergeron, J.J.M. (eds.) (1993), Springer Verlag, Berlin/Heidelberg.
Microtubules play a key role in the dynamic spatial organization of the cytoplasmic matrix. They are involved in regulating the structure and positioning of intracellular organelles like the Golgi apparatus and endosomes, and they provide the tracks for directed movement of such organelles [Kreis, 1990]. Membrane-bounded cytoplasmic organelles attach to microtubules, move along or remain stably associated with them, and eventually detach. These interactions of organelles with microtubules appear to be specific. For example, endosomes, but not Golgi elements, reverse their direction of translocation along microtubules upon acidification of the cytoplasm [Heuser, 1989; Parton et al., 1991]. These various interactions require different levels of regulation; specificity and timing of binding and release, positioning, and direction of movement. We postulate, therefore, that different proteins must be involved in the regulation of these interactions of cytoplasmic organelles with microtubules.
A fully automated image acquisition and analysis system for low light level fluorescence microscopy.
Herr, S., Bastian, T., Pepperkok, R., Boulin, C. & Ansorge, W.
Meth Mol Cell Biol 1993 40 164-170
?-COP, a Coat Protein of Nonclathrin-Coated Vesicles of the Golgi Complex, is Involved in Transport of Vesicular Stomatitis Virus Glycoprotein.
Duden, R., Storrie, B., Pepperkok, R., Scheel, J., Joggerst-Thomalla, B., Sawyer, A., Horstmann, H., Griffiths, G. & Kreis, T.E.
In Nato ASI Series 74, pp. 117-126, "Molecular Mechanisms of Membrane Traffic", Morre, D.J., Howell, K.E. & Bergeron, J.J.M. (eds.) (1993), Springer Verlag, Berlin/Heidelberg.
The Golgi complex is a polarized cytoplasmic organelle that is generally considered to be built up of at least three functionally distinct, membrane bounded subcompartments, the cis-Golgi network (CGN), the stacked Golgi cisternae and the trans-Golgi network (TGN) [Mellman and Simons, 1992]. It receives material from the endoplasmic reticulum (ER) and endosomes, transports cargo through its subcompartments and delivers it to the plasma membrane and endosomes. Vesicular carriers are believed to mediate the vectorial transport of this material from one membrane bounded subcompartment to the next. Recently, the idea that transport may also occur by transient tubular connections has also been discussed [Klausner et al., 1992; Kreis, 1992]; until now, however, the evidence for this mechanism of membrane transport is less convincing than for vesicular transport.