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Based on the CWA Gold Dagger Award-winning novel by H.R.F Keating, The Perfect Murder follows inspector Ghote (Naseeruddin Shah) of the Bombay police as he pursues his most perplexing case: a savage attack on a rich industrialist's private secretary. With the help of a visiting Swedish criminologist (Stellan Skarsgård), Ghote edges closer to the truth, and soon discovers he may not be dealing with murder at all, but something far more complex and profitable.
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In general I really enjoy Merchant Ivory films and like many movies made about or in India. But this one is bizarre. Read morePublished 18 months ago by Johnine
Reminded me of an Indian version of Inspector Clouseau...not quite as bumbling or inept...but definitely entertaining. Read morePublished 21 months ago by S. Hamilton

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text: Brian Patrick O'Toole is an American film producer and screenwriter. O'Toole's work includes co-producing the 2002 horror film Dog Soldiers and his screenwriting debut Cemetery Gates. He writes a monthly column for the prominent American magazine Fangoria and currently works with Black Gate Entertainment, with whom he has written and produced several films, including Basement Jack, Evilution and the upcoming Necropolitan and A Necessary Evil. He wrote the screenplay for the movie version of Atlas Shrugged: Part I.Wikipedia


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Over the years, Kim Harrison's name has come up numerous times in conversations about urban fantasy and paranormal romance. Admittedly, I didn't pay a lot of attention since the synopsis of the first book in The Hollows seemed kind of fluffy, not something I'd care much about. Then, The Drafter was published, and I took notice. This book seemed anything but fluffy. I snatched it up as soon as I could.more
This is the final book in Ms Shupe's Wicked Deceptions trilogy in which the stories are linked by the three male protagonists, who are long-term friends. I enjoyed the first book (The Courtesan Duchess) and felt Ms Shupe was an author to watch.She writes well, her characters are strongly drawn and she can certainly turn up the heat when called for.more
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With the most basic DIY skills, you can recycle throwaway junk into lovely homewares. Design blogger Grace Bonney shows you how
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I like to read econ books, write about the stock market, study football coaching film to pretend I know what's going on and watch British Panel Shows. Which, btw, if you haven't watched British Panel Shows you should start.
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Shows: So so many! Hard to find a favorite. I think, at time of writing, I would have to put the BBC's Sherlock series at the top. I love British Panel Shows (QI, Would I Lie to You, 8 out of 10 Cats etc.
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- Uploaded
- Apr 19, 2001 | 12:53 PM EDT
- Weekly Users' Choice April 25, 2001
- Daily Feature April 20, 2001
If you liked this, check these out!
Plenty more like this here!
Author Comments
Rated 5 / 5 stars
I used to watch this like ten years ago, and now it just fills me with nostalgia. I love the series, and I love Zhu
Rated 3.5 / 5 stars
The fight was cool, woulda been nice if there had been some sound effects though.
Rated 4 / 5 stars
This was sick. A stickman destroying a group of other stickmen with great fast paced combat and creative fighting moves with a well done ending.
Rated 5 / 5 stars
Whoah ! ....Gotta love the slow motion effects and the fast paced combat. I remember watching this way back..It's a great classic.
Rated 5 / 5 stars
and this from where all the stick animators now days became stick animators. zhu the first stick animator! #respect


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I received for pilarfarmer for my non-paper theme. She was such a sweety and sent me two amazing ATC both featuring one of my favorite things in the word - cats!
She quilted this one. SO neat!! Love the little eyes too.
This is the extra one and it reminds me of my kitty, Dahlia. She is a black cat and she sits in the window watching the birds all the time.
Thank you so much pilarfarmer! These are awesome!!!!


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The latest hot and happening news from B-Town for you to catch up on....
New Delhi, Nov 14 (IANS) Actress Preity Zinta promises to return to the small screen with the second season of her celebrity chat show - 'Up, Close and Personal with PZ'.Monday, November 14, 2011 2:55:30 PM
New Delhi, Nov 14 After a not-so-successful stint as an actress, Hrishitaa Bhatt has turned producer and her maiden venture, a comedy called 'Shakal Pe Mat Ja', a tale of mistaken identities, is releasing Friday.Monday, November 14, 2011 2:53:41 PM
New Delhi, Nov 14 (IANS) Bangalore girl Nicole Faria, the only Indian to have ever won the Miss Earth title, says there is no favouritism at international pageants and she believes the problem lies in the selection process at home.Monday, November 14, 2011 1:06:41 PM
New Delhi, Nov 13 Working in Bollywood is a demanding job, says Hema Malini adding that this is the reason why she was not very keen on her daughter Esha Deol joining filmdom as an actress.Sunday, November 13, 2011 7:41:09 PM
New Delhi, Nov 13 (IANS) Manish Paul, who has become one of the most sought after hosts on the small screen, says he doesn't like being restricted by script and tries to improvise on stage.Sunday, November 13, 2011 6:38:08 PM
New Delhi, Nov 13 Filmmaker Satish Kaushik is delighted that Indian cinema has expanded globally and advises young filmmakers to think internationally to tap bigger audiences.Sunday, November 13, 2011 6:29:15 PM
New Delhi, Nov 13 Megastar Amitabh Bachchan, whose daughter-in-law Aishwarya Rai is expected to give birth mid-November, can't wait to see the new arrival and says he is enjoying the hustle bustle in the house.Sunday, November 13, 2011 6:00:42 PM
Kochi, Nov 13 (IANS) Singing legend K.J. Yesudas will celebrate his golden jubilee as a singer Nov 14 as it was on this day half-a-century ago that he recorded his first song which marked his arrival in the film industry.Sunday, November 13, 2011 5:20:36 PM
New Delhi, Nov 13 Actor Shreyas Talpade is excited about shooting for an item song with actress Chitrangada Singh in their new film 'Joker'.Sunday, November 13, 2011 1:09:19 PM
Film: 'Immortals'; Cast: Henry Cavill, Mickey Rourke, Frieda Pinto, John Hurt; Director: Tarsem Singh Dhandwar; Rating: ***Sunday, November 13, 2011 10:46:38 AM
New Delhi, Nov 12 (IANS) Transgender activist Laxmi Narayan Tripathi, who was evicted from 'Bigg Boss 5' Saturday, says she is glad to have left with her 'dignity and self-respect' intact. She predicts troublemaker VJ Pooja Missra will end up being onSaturday, November 12, 2011 11:23:43 PM
New Delhi, Nov 12 (IANS) Transgender activist Laxmi Narayan Tripathi, who was evicted from 'Bigg Boss 5' Friday, says she is glad to have left with her 'dignity and self-respect' intact. She predicts troublemaker VJ Pooja Missra will end up being oneSaturday, November 12, 2011 11:14:15 PM
Kolkata, Nov 12 (IANS) Very few know that Legendary filmmaker Satyajit Ray was equally at ease with still camera as he was with a movie camera. Rare photographs taken by Ray are on display at 17th Kolkata Film Festival.Saturday, November 12, 2011 10:16:38 PM
Panaji, Nov 12 (IANS) Films aside, the organisers of the International Film Festival of India (IFFI) aim to leverage the top draw event to beef up international tourism inflows, making it part of the tourism ministry's 'Incredible India' campaign.Saturday, November 12, 2011 10:05:10 PM
Mumbai, Nov 12 (IANS) Renowned dancer and choreographer of Marathi film industry Subal Sarkar died here Saturday following a prolonged illness, a family friend said. He was 76.Saturday, November 12, 2011 7:19:43 PM
New Delhi, Nov 12 (IANS) Playing the role of Milkha Singh in Rakeysh Omprakash Mehra's 'Bhaag Milkha Bhaag', perfectionist actor Farhan Akhtar spent a few days with the legendary athlete to perfect his skills for the movie.Saturday, November 12, 2011 6:51:54 PM
New Delhi, Nov 12 (IANS) Once upon a time, films catering to children remained in the precincts of parallel and serious cinema in Bollywood. Over the years, the genre has undergone a seachange, with many filmmakers entwining emotional and educational theSaturday, November 12, 2011 6:27:46 PM
Srinagar, Nov 12 (IANS) Applauded by a throng of enthusiastic students, a New York rock band, which uses solar energy to power its performances, spread the message of environmental awareness and conservation at a concert here Saturday.Saturday, November 12, 2011 5:54:43 PM
New Delhi, Nov 12 Ranbir Kapoor starrer 'Rockstar' has got off to a flying start with the film managing to earn Rs.11 crore plus on the opening day itself.Saturday, November 12, 2011 5:54:27 PM
Panaji, Nov 12 (IANS) Portuguese director Manuel Gonzales' 'The Consul of Bordeaux' will open the 42nd edition of the International Film Festival of India (IFFI) here Nov 23.Saturday, November 12, 2011 5:40:53 PM
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The recent Volkswagen Group emissions scandal has so far focused on the company's 2.0-liter diesel engines, but may soon broaden to encompass more engines, and more vehicles. The Environmental Protection Agency (EPA) and the California Resource Air Board (CARB) separately announced plans to expand testing to Volkswagen's 3.0-liter engines. Models offering this 6-cylinder engine include […]+ Read More
Tesla has started mounting its home energy lithium-ion battery – dubbed the Powerwall – in select homes. "We are currently in production and have started installing Powerwalls with pilot customers," Tesla spokeswoman Alexis Georgeson told Electrek. "Over the next few weeks we will continue to ramp up volume production." The units being installed are 7 […]+ Read More
Tesla has begun selling an upgraded battery for its two-door Roadster, boosting the battery electric vehicle's range by another 100 miles. "Model S development has helped us apply new battery technology to the Roadster 3.0 packs," a Tesla representative told us. "The cells store 31 percent more energy than the original Roadster cell. Using this […]+ Read More


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Deficiency of Niemann-Pick Type C-1 Protein Impairs Release of Human Immunodeficiency Virus Type 1 and Results in Gag Accumulation in Late Endosomal/Lysosomal Compartments▿
Tang, Yuyang
Leao, Ihid Carneiro
Coleman, Ebony M.
Broughton, Robin Shepard
Hildreth, James E. K.
Journal of Virology
Human immunodeficiency virus type 1 (HIV-1) relies on cholesterol-laden lipid raft membrane microdomains for entry into and egress out of susceptible cells. In the present study, we examine the need for intracellular cholesterol trafficking pathways with respect to HIV-1 biogenesis using Niemann-Pick type C-1 (NPC1)-deficient (NPCD) cells, wherein these pathways are severely compromised, causing massive accumulation of cholesterol in late endosomal/lysosomal (LE/L) compartments. We have found that induction of an NPC disease-like phenotype through treatment of various cell types with the commonly used hydrophobic amine drug U18666A resulted in profound suppression of HIV-1 release. Further, NPCD Epstein-Barr virus-transformed B lymphocytes and fibroblasts from patients with NPC disease infected with a CD4-independent strain of HIV-1 or transfected with an HIV-1 proviral clone, respectively, replicated HIV-1 poorly compared to normal cells. Infection of the NPCD fibroblasts with a vesicular stomatitis virus G-pseudotyped strain of HIV-1 produced similar results, suggesting a postentry block to HIV-1 replication in these cells. Examination of these cells using confocal microscopy showed an accumulation and stabilization of Gag in LE/L compartments. Additionally, normal HIV-1 production could be restored in NPCD cells upon expression of a functional NPC1 protein, and overexpression of NPC1 increased HIV-1 release. Taken together, our findings demonstrate that intact intracellular cholesterol trafficking pathways mediated by NPC1 are needed for efficient HIV-1 production.
Related Articles
Dynamic Movements of Organelles Containing Niemann-Pick C1 Protein: NPC1 Involvement in Late Endocytic EventsV⃞
Ko, Dennis C.
Gordon, Michael D.
Jin, Janet Y.
Scott, Matthew P.
Molecular Biology of the Cell
People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.
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IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion
Desai, Tanay M.
Marin, Mariana
Chin, Christopher R.
Savidis, George
Brass, Abraham L.
Melikyan, Gregory B.
PLoS Pathogens
Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes.
Author Summary
Interferon-induced transmembrane proteins (IFITMs) block infection of many enveloped viruses, including the influenza A virus (IAV) that enters from late endosomes. IFITMs are thought to prevent virus hemifusion (merger of contacting leaflets without formation of a fusion pore) by altering the properties of cell membranes. Here we performed single IAV imaging and found that IFITM3 did not interfere with hemifusion, but prevented complete fusion. Also, contrary to a current view that excess cholesterol in late endosomes of IFITM3-expressing cells inhibits IAV entry, we show that cholesterol-laden endosomes are permissive for virus fusion. The ability of IFITM3 to block the formation of fusion pores implies that this protein stabilizes the cytoplasmic leaflet of endosomal membranes, either directly or indirectly, through altering its physical properties. IFITM3 may also redirect IAV to a non-productive pathway by promoting fusion with intralumenal vesicles of late endosomes instead of their limiting membrane.
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Cholinergic Abnormalities, Endosomal Alterations and Up-Regulation of Nerve Growth Factor Signaling in Niemann-Pick Type C Disease
Cabeza, Carolina
Figueroa, Alicia
Lazo, Oscar M
Galleguillos, Carolina
Pissani, Claudia
Klein, Andrés
Gonzalez-Billault, Christian
Inestrosa, Nibaldo C
Alvarez, Alejandra R
Zanlungo, Silvana
Bronfman, Francisca C
Molecular Neurodegeneration
Neurotrophins and their receptors regulate several aspects of the developing and mature nervous system, including neuronal morphology and survival. Neurotrophin receptors are active in signaling endosomes, which are organelles that propagate neurotrophin signaling along neuronal processes. Defects in the Npc1 gene are associated with the accumulation of cholesterol and lipids in late endosomes and lysosomes, leading to neurodegeneration and Niemann-Pick type C (NPC) disease. The aim of this work was to assess whether the endosomal and lysosomal alterations observed in NPC disease disrupt neurotrophin signaling. As models, we used i) NPC1-deficient mice to evaluate the central cholinergic septo-hippocampal pathway and its response to nerve growth factor (NGF) after axotomy and ii) PC12 cells treated with U18666A, a pharmacological cellular model of NPC, stimulated with NGF.
NPC1-deficient cholinergic cells respond to NGF after axotomy and exhibit increased levels of choline acetyl transferase (ChAT), whose gene is under the control of NGF signaling, compared to wild type cholinergic neurons. This finding was correlated with increased ChAT and phosphorylated Akt in basal forebrain homogenates. In addition, we found that cholinergic neurons from NPC1-deficient mice had disrupted neuronal morphology, suggesting early signs of neurodegeneration. Consistently, PC12 cells treated with U18666A presented a clear NPC cellular phenotype with a prominent endocytic dysfunction that includes an increased size of TrkA-containing endosomes and reduced recycling of the receptor. This result correlates with increased sensitivity to NGF, and, in particular, with up-regulation of the Akt and PLC-γ signaling pathways, increased neurite extension, increased phosphorylation of tau protein and cell death when PC12 cells are differentiated and treated with U18666A.
Our results suggest that the NPC cellular phenotype causes neuronal dysfunction through the abnormal up-regulation of survival pathways, which causes the perturbation of signaling cascades and anomalous phosphorylation of the cytoskeleton.
NGF; Endosomes; Cholesterol; Niemann-Pick type C1; cholinergic system; PC12
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Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O
Kwiatkowska, Katarzyna
Marszałek–Sadowska, Ewelina
Traczyk, Gabriela
Koprowski, Piotr
Musielak, Małgorzata
Ługowska, Agnieszka
Kulma, Magdalena
Grzelczyk, Anna
Sobota, Andrzej
Orphanet Journal of Rare Diseases
Niemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of cholesterol deposits in cells has been widely used for NPC diagnostics. In this report we present an alternative method for cholesterol visualization and estimation using a cholesterol-binding bacterial toxin, perfringolysin O.
To detect cholesterol deposits, a recombinant probe, perfringolysin O fused with glutathione S-transferase (GST-PFO) was prepared. GST-PFO followed by labeled antibodies or streptavidin was applied for immunofluorescence and immunoelectron microscopy to analyze cholesterol distribution in cells derived from NPC patients. The identity of GST-PFO–positive structures was revealed by a quantitative analysis of their colocalization with several organelle markers. Cellular ELISA using GST-PFO was developed to estimate the level of unesterified cholesterol in NPC cells.
GST-PFO recognized cholesterol with high sensitivity and selectivity, as demonstrated by a protein/lipid overlay assay and surface plasmon resonance analysis. When applied to stain NPC cells, GST-PFO decorated abundant deposits of cholesterol in intracellular vesicles that colocalized with filipin-positive structures. These cholesterol deposits were resistant to 0.05%-0.2% Triton X-100 used for cells permeabilization in the staining procedure. GST-PFO-stained organelles were identified as late endosomes/lysosomes based on their colocalization with LAMP-1 and lysobisphosphatidic acid. On the other hand, GST-PFO did not colocalize with markers of the Golgi apparatus, endoplasmic reticulum, peroxisomes or with actin filaments. Only negligible GST-PFO staining was seen in fibroblasts of healthy individuals. When applied to cellular ELISA, GST-PFO followed by anti-GST-peroxidase allowed a semiquantitative analysis of cholesterol level in cells of NPC patients. Binding of GST-PFO to NPC cells was nearly abolished after extraction of cholesterol with methyl-β-cyclodextrin.
Our data indicate that a recombinant protein GST-PFO can be used to detect cholesterol accumulated in NPC cells by immunofluorescence and cellular ELISA. GST-PFO can be a convenient and reliable probe for revealing cholesterol deposits in cells and can be useful in diagnostics of NPC disease.
Niemann-Pick C disease; Lipid storage diseases; Cholesterol-binding proteins; Filipin; Perfringolysin O
Related Articles
Cholesterol Accumulation Sequesters Rab9 and Disrupts Late Endosome Function in NPC1-deficient Cells*
Ganley, Ian G.
Pfeffer, Suzanne R.
The Journal of biological chemistry
Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompaniedby stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent man-nose 6-phosphate receptorswere missorted to the lysosome for degradation, a process that was reversed by overexpression of GFP-tagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking.
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Hrs and SNX3 Functions in Sorting and Membrane Invagination within Multivesicular Bodies
Pons, Véronique
Luyet, Pierre-Philippe
Morel, Etienne
Abrami, Laurence
van der Goot, F. Gisou
Parton, Robert G
Gruenberg, Jean
PLoS Biology
After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.
Author Summary
The cell's genetic program is modulated by extracellular signals that activate cell surface receptors and, in turn, intracellular effectors, to regulate transcription. For cells to function normally, these signals must be turned off to avoid permanent activation—a situation often associated with cancer. For many receptors, signaling is repressed, or down-regulated, in a process that first internalizes and then degrades the receptors. After receptors are removed from the cell surface into structures called early endosomes, they are selectively incorporated within vesicles that form inside the endosome. During this process, endosomal membranes are pulled away from the cytoplasm towards the endosome lumen, against the flow of intracellular membrane traffic, eventually resulting in the formation of a "multivesicular body" (vesicles within vesicles). The common view is that these intralumenal vesicles are then delivered to lysosomes, where they are degraded along with their receptor cargo. We have investigated the mechanisms responsible for the biogenesis of intralumenal vesicles in multivesicular bodies. We find that the small protein SNX3, which binds the signaling lipid phosphatidyl inositol-3-phosphate, is necessary for the formation of intralumenal vesicles, but is not involved in the degradation of the cell surface receptor for EGF. Conversely, we find that Hrs, which also binds phosphatidyl inositol-3-phosphate and mediates receptor sorting into intralumenal vesicles, is essential for lysosomal targeting but dispensable for multivesicular body biogenesis. Phosphatidyl inositol-3-phosphate thus controls the complementary functions of Hrs and SNX3 in the sorting of signaling receptors and multivesicular body biogenesis.
SNX3 plays a direct role in the formation of intralumenal vesicles of multivesicular bodies (MVBs) but is not involved in EGF receptor degradation, whereas Hrs is essential for lysosomal targeting but dispensable for MVB biogenesis. Hence, intralumenal vesicle formation in MVB biogenesis can be uncoupled from lysosomal targeting.
Related Articles
Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function
Reverter, Meritxell
Rentero, Carles
de Muga, Sandra Vilà
Alvarez-Guaita, Anna
Mulay, Vishwaroop
Cairns, Rose
Wood, Peta
Monastyrskaya, Katia
Pol, Albert
Tebar, Francesc
Blasi, Joan
Grewal, Thomas
Enrich, Carlos
Molecular Biology of the Cell
This study shows that impaired cholesterol egress from late endosomes in cells with high annexin A6 levels is associated with altered soluble N-ethylmaleimide–sensitive fusion protein 23 (SNAP23) and syntaxin-4 cellular distribution and assembly and accumulation in Golgi membranes. This correlates with reduced secretion of cargo along the constitutive and SNAP23/syntaxin-4–dependent secretory pathway.
Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4–dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.
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Regulation of the V-ATPase along the Endocytic Pathway Occurs through Reversible Subunit Association and Membrane Localization
Lafourcade, Céline
Sobo, Komla
Kieffer-Jaquinod, Sylvie
Garin, Jérome
van der Goot, F. Gisou
PLoS ONE
The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V0 and the cytoplasmic V1. Here we found that the ratio of membrane associated V1/Vo varies along the endocytic pathway, the relative abundance of V1 being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.
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A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking
Du, Ximing
Kumar, Jaspal
Ferguson, Charles
Schulz, Timothy A.
Ong, Yan Shan
Hong, Wanjin
Prinz, William A.
Parton, Robert G.
Brown, Andrew J.
Yang, Hongyuan
The Journal of Cell Biology
ORP5 works together with Niemann Pick C-1 to facilitate exit of cholesterol from endosomes and lysosomes.
Oxysterol-binding protein (OSBP) and its related proteins (ORPs) constitute a large and evolutionarily conserved family of lipid-binding proteins that target organelle membranes to mediate sterol signaling and/or transport. Here we characterize ORP5, a tail-anchored ORP protein that localizes to the endoplasmic reticulum. Knocking down ORP5 causes cholesterol accumulation in late endosomes and lysosomes, which is reminiscent of the cholesterol trafficking defect in Niemann Pick C (NPC) fibroblasts. Cholesterol appears to accumulate in the limiting membranes of endosomal compartments in ORP5-depleted cells, whereas depletion of NPC1 or both ORP5 and NPC1 results in luminal accumulation of cholesterol. Moreover, trans-Golgi resident proteins mislocalize to endosomal compartments upon ORP5 depletion, which depends on a functional NPC1. Our results establish the first link between NPC1 and a cytoplasmic sterol carrier, and suggest that ORP5 may cooperate with NPC1 to mediate the exit of cholesterol from endosomes/lysosomes.
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Modulation of Cellular Cholesterol Transport and Homeostasis by Rab11
Hölttä-Vuori, Maarit
Tanhuanpää, Kimmo
Möbius, Wiebke
Somerharju, Pentti
Ikonen, Elina
Molecular Biology of the Cell
To analyze the contribution of vesicular trafficking pathways in cellular cholesterol transport we examined the effects of selected endosomal Rab proteins on cholesterol distribution by filipin staining. Transient overexpression of Rab11 resulted in prominent accumulation of free cholesterol in Rab11-positive organelles that sequestered transferrin receptors and internalized transferrin. Sphingolipids were selectively redistributed as pyrene-sphingomyelin and sulfatide cosequestered with Rab11-positive endosomes, whereas globotriaosyl ceramide and GM2 ganglioside did not. Rab11 overexpression did not perturb the transport of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate–labeled low-density lipoprotein (LDL) to late endosomes or the Niemann-Pick type C1 (NPC1)-induced late endosomal cholesterol clearance in NPC patient cells. However, Rab11 overexpression inhibited cellular cholesterol esterification in an LDL-independent manner. This effect could be overcome by introducing cholesterol to the plasma membrane by using cyclodextrin as a carrier. These results suggest that in Rab11-overexpressing cells, deposition of cholesterol in recycling endosomes results in its impaired esterification, presumably due to defective recycling of cholesterol to the plasma membrane. The findings point to the importance of the recycling endosomes in regulating cholesterol and sphingolipid trafficking and cellular cholesterol homeostasis.
Related Articles
Deficiency of a Niemann-Pick, Type C1-related Protein in Toxoplasma Is Associated with Multiple Lipidoses and Increased Pathogenicity
Lige, Bao
Romano, Julia D.
Bandaru, Veera Venkata Ratnam
Ehrenman, Karen
Levitskaya, Jelena
Sampels, Vera
Haughey, Norman J.
Coppens, Isabelle
PLoS Pathogens
Several proteins that play key roles in cholesterol synthesis, regulation, trafficking and signaling are united by sharing the phylogenetically conserved 'sterol-sensing domain' (SSD). The intracellular parasite Toxoplasma possesses at least one gene coding for a protein containing the canonical SSD. We investigated the role of this protein to provide information on lipid regulatory mechanisms in the parasite. The protein sequence predicts an uncharacterized Niemann-Pick, type C1-related protein (NPC1) with significant identity to human NPC1, and it contains many residues implicated in human NPC disease. We named this NPC1-related protein, TgNCR1. Mammalian NPC1 localizes to endo-lysosomes and promotes the movement of sterols and sphingolipids across the membranes of these organelles. Miscoding patient mutations in NPC1 cause overloading of these lipids in endo-lysosomes. TgNCR1, however, lacks endosomal targeting signals, and localizes to flattened vesicles beneath the plasma membrane of Toxoplasma. When expressed in mammalian NPC1 mutant cells and properly addressed to endo-lysosomes, TgNCR1 restores cholesterol and GM1 clearance from these organelles. To clarify the role of TgNCR1 in the parasite, we genetically disrupted NCR1; mutant parasites were viable. Quantitative lipidomic analyses on the ΔNCR1 strain reveal normal cholesterol levels but an overaccumulation of several species of cholesteryl esters, sphingomyelins and ceramides. ΔNCR1 parasites are also characterized by abundant storage lipid bodies and long membranous tubules derived from their parasitophorous vacuoles. Interestingly, these mutants can generate multiple daughters per single mother cell at high frequencies, allowing fast replication in vitro, and they are slightly more virulent in mice than the parental strain. These data suggest that the ΔNCR1 strain has lost the ability to control the intracellular levels of several lipids, which subsequently results in the stimulation of lipid storage, membrane biosynthesis and parasite division. Based on these observations, we ascribe a role for TgNCR1 in lipid homeostasis in Toxoplasma.
Author Summary
The intracellular parasite Toxoplasma is auxotrophic for several lipids that it scavenges from host organelles. Several studies focused on deciphering the pathways implicated in host lipid delivery to the parasite, but less effort has been devoted to understand how lipids are regulated in Toxoplasma. The 'sterol-sensing domain' (SSD) is conserved across phyla and present in several membrane proteins involved in cholesterol homeostasis, cell signaling and cytokinesis. We studied the role of a SSD-containing protein in Toxoplasma which shows significant similarity with Niemann-Pick type C1 proteins (NPC1). Human NPC disease is typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of the parasite NPC1-related protein (named TgNCR1) in mammalian NPC1 mutant cells suppresses lipid accumulation in lysosomes. However, Toxoplasma never internalizes host cholesterol into lysosomes, which predicts a function for TgNCR1 unrelated to exogenous sterol transport. Indeed, genomic deletion of NCR1 does not result in abnormal levels of cholesterol in the parasites but in the overaccumulation of cholesteryl esters and sphingolipids. TgNCR1-deficient parasites form abundant storage lipid bodies and multiple parasites per cycle of division. This suggests that TgNCR1 functions in monitoring the levels of various lipids within Toxoplasma, which in turn impacts the parasite's lipid homeostasis and growth rate.
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Multiple Cationic Amphiphiles Induce a Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection
Shoemaker, Charles J.
Schornberg, Kathryn L.
Delos, Sue E.
Scully, Corinne
Pajouhesh, Hassan
Olinger, Gene G.
Johansen, Lisa M.
White, Judith M.
PLoS ONE
Ebola virus (EBOV) is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC50 1.6 to 8.0 µM) at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann–Pick C1 protein (NPC1), a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents) inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.
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Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin
Yu, Ting
Lieberman, Andrew P.
PLoS Genetics
Cholesterol availability is rate-limiting for myelination, and prior studies have established the importance of cholesterol synthesis by oligodendrocytes for normal CNS myelination. However, the contribution of cholesterol uptake through the endocytic pathway has not been fully explored. To address this question, we used mice with a conditional null allele of the Npc1 gene, which encodes a transmembrane protein critical for mobilizing cholesterol from the endolysosomal system. Loss of function mutations in the human NPC1 gene cause Niemann-Pick type C disease, a childhood-onset neurodegenerative disorder in which intracellular lipid accumulation, abnormally swollen axons, and neuron loss underlie the occurrence of early death. Both NPC patients and Npc1 null mice exhibit myelin defects indicative of dysmyelination, although the mechanisms underlying this defect are incompletely understood. Here we use temporal and cell-type-specific gene deletion in order to define effects on CNS myelination. Our results unexpectedly show that deletion of Npc1 in neurons alone leads to an arrest of oligodendrocyte maturation and to subsequent failure of myelin formation. This defect is associated with decreased activation of Fyn kinase, an integrator of axon-glial signals that normally promotes myelination. Furthermore, we show that deletion of Npc1 in oligodendrocytes results in delayed myelination at early postnatal days. Aged, oligodendocyte-specific null mutants also exhibit late stage loss of myelin proteins, followed by secondary Purkinje neuron degeneration. These data demonstrate that lipid uptake and intracellular transport by neurons and oligodendrocytes through an Npc1-dependent pathway is required for both the formation and maintenance of CNS myelin.
Author Summary
The myelin sheath in the central nervous system is a specialized extension of the oligodendrocyte plasma membrane that serves as an electrical insulator to ensure proper nerve conduction. To accomplish this, myelin is enriched in lipids, particularly unesterified cholesterol, which is an essential and limiting component for myelin formation. Here we determine the contribution of exogenously derived cholesterol to myelination by using a conditional null mutant of the mouse Npc1 gene. Npc1 encodes a transmembrane protein critical for mobilizing exogenously derived cholesterol from late endosomes and lysosomes, and is mutated in patients with Niemann-Pick type C disease, a degenerative disorder caused by impaired intracellular lipid trafficking. We show that mice lacking Npc1 in either neurons or oligodendrocytes exhibit a defect in myelin formation in selected brain regions, with an arrest in oligodendrocyte maturation. In addition, mice with Npc1 deficiency in oligodendrocytes, when aged, show progressive motor dysfunction with myelin breakdown and secondary Purkinje neuron loss. Taken together, our findings demonstrate the role of Npc1 in mediating reciprocal signaling between neurons and glia, and highlight the importance of exogenous cholesterol for CNS myelin formation and maintenance.
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Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments
Padilla-Parra, Sergi
Marin, Mariana
Kondo, Naoyuki
Melikyan, Gregory B.
PLoS Pathogens
Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH4Cl. Removal of NH4Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH4Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH4Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps – fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes.
Author Summary
Endosomal trafficking and regulation of retrovirus fusion is poorly understood, due in part to heterogeneity of viral particles and their asynchronous entry into an endocytic pathway. Here, we used an avian retrovirus that enters host cells in a receptor- and low pH-dependent manner. This feature allowed capturing the virus in intracellular compartments through raising the endosomal pH. Virus fusion was synchronously initiated upon permitting endosome acidification and visualized in real-time by single particle imaging. We found that different receptor isoforms directed virus into distinct sub-cellular compartments supporting quick release of the viral core. Through tracking individual sub-viral particles released from endosomes, we found that the full length receptor mediated core delivery into the cytoplasm. By contrast, fusion mediated by the GPI-anchored receptor released the core into another endosomal compartment, from which the core entered the cytosol through an additional temperature-dependent step. These findings demonstrate different permissiveness of endosomal compartments to viral fusion and the existence of a post-fusion step leading to the cytosolic release of cores and initiation of infection.
Related Articles
ACAT1-associated Late Endosomes/Lysosomes Significantly Improve Impaired Intracellular Cholesterol Metabolism and the Survival of Niemann-Pick Type C Mice
Kamikawa, Masashi
Lei, XiaoFeng
Fujiwara, Yukio
Nishitsuji, Kazuchika
Mizuta, Hiroshi
Takeya, Motohiro
Sakashita, Naomi
Acta Histochemica et Cytochemica
We previously demonstrated that macrophages exhibit endoplasmic reticulum fragmentation under cholesterol-rich conditions, which results in the generation of acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1)-associated late endosomes/lysosomes (ACAT1-LE). ACAT1-LE efficiently esterify free cholesterol in loco, even with abnormal egress of free cholesterol from late endosomes. Because impaired free cholesterol transport from late endosomes results in Niemann-Pick type C disease (NPC), the induction of ACAT1-LE is a potential therapeutic intervention for NPC. To examine the effects of ACAT1-LE induction on intracellular cholesterol metabolism, we incubated bone marrow-derived macrophages possessing NPC phenotype (npc1–/–) with methyl-β-cyclodextrin-cholesterol complex (mβCD-cho), a cholesterol donor. Immunofluorescence confocal microscopy revealed that mβCD-cho treatment of npc1–/– macrophages resulted in significant colocalization of signals from ACAT1 and lysosome-associated membrane protein 2, a late endosome/lysosome marker. npc1–/– macrophages contained significant amounts of free cholesterol with negligible amounts of cholesteryl ester, while wild-type macrophages possessed the same amounts of both cholesterols. mβCD-cho treatment also induced marked restoration of cholesterol esterification activity. mβCD-cho administration in neonate npc1–/– mice improved survival. These results indicate that ACAT1-LE induction in npc1–/– mice corrects impaired intracellular cholesterol metabolism and that restoring cholesterol esterification improves prognosis of npc1–/–. These data suggest that ACAT1-LE induction is a potential alternative therapeutic strategy for NPC.
Niemann-Pick type C disease; acyl-coenzyme A: cholesterol acyltransferase 1; late endosomes; cholesterol; methyl-β-cyclodextrin
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Inhibition of cholesterol recycling impairs cellular PrPSc propagation
Gilch, Sabine
Bach, Christian
Lutzny, Gloria
Vorberg, Ina
Schätzl, Hermann M.
Cellular and Molecular Life Sciences
The infectious agent in prion diseases consists of an aberrantly folded isoform of the cellular prion protein (PrPc), termed PrPSc, which accumulates in brains of affected individuals. Studies on prion-infected cultured cells indicate that cellular cholesterol homeostasis influences PrPSc propagation. Here, we demonstrate that the cellular PrPSc content decreases upon accumulation of cholesterol in late endosomes, as induced by NPC-1 knock-down or treatment with U18666A. PrPc trafficking, lipid raft association, and membrane turnover are not significantly altered by such treatments. Cellular PrPSc formation is not impaired, suggesting that PrPSc degradation is increased by intracellular cholesterol accumulation. Interestingly, PrPSc propagation in U18666A-treated cells was partially restored by overexpression of rab 9, which causes redistribution of cholesterol and possibly of PrPSc to the trans-Golgi network. Surprisingly, rab 9 overexpression itself reduced cellular PrPSc content, indicating that PrPSc production is highly sensitive to alterations in dynamics of vesicle trafficking.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-009-0158-4) contains supplementary material, which is available to authorized users.
Cholesterol; Recycling; Prion; PrP; NPC-1; Rab 9
Related Articles
Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content
Mundy, Dorothy I.
Li, Wei Ping
Luby-Phelps, Katherine
Anderson, Richard G. W.
Molecular Biology of the Cell
Caveolin-1 traffics to late endosomal/lysosomal membranes in response to manipulations of the cholesterol content of cells, suggesting that caveolin functions in the egress of cholesterol from this organelle. Cavicles associate with the periphery of the lysosome as they do with caveosomes, but these are separate organelles.
Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking.
Related Articles
Adenovirus RID-α activates an autonomous cholesterol regulatory mechanism that rescues defects linked to Niemann-Pick disease type C
Cianciola, Nicholas L.
Carlin, Cathleen R.
The Journal of Cell Biology
Viral subversion of cholesterol homeostasis provides insights into sterol trafficking, autophagy, and lysosomal storage diseases.
Host–pathogen interactions are important model systems for understanding fundamental cell biological processes. In this study, we describe a cholesterol-trafficking pathway induced by the adenovirus membrane protein RID-α that also subverts the cellular autophagy pathway during early stages of an acute infection. A palmitoylation-defective RID-α mutant deregulates cholesterol homeostasis and elicits lysosomal storage abnormalities similar to mutations associated with Niemann-Pick type C (NPC) disease. Wild-type RID-α rescues lipid-sorting defects in cells from patients with this disease by a mechanism involving a class III phosphatidylinositol-3-kinase. In contrast to NPC disease gene products that are localized to late endosomes/lysosomes, RID-α induces the accumulation of autophagy-like vesicles with a unique molecular composition. Ectopic RID-α regulates intracellular cholesterol trafficking at two distinct levels: the egress from endosomes and transport to the endoplasmic reticulum necessary for homeostatic gene regulation. However, RID-α also induces a novel cellular phenotype, suggesting that it activates an autonomous cholesterol regulatory mechanism distinct from NPC disease gene products.
Related Articles
In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101
Falguières, Thomas
Luyet, Pierre-Philippe
Bissig, Christin
Scott, Cameron C.
Velluz, Marie-Claire
Gruenberg, Jean
Molecular Biology of the Cell
Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively.
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Treatment of Niemann–Pick Type C Disease by Histone Deacetylase Inhibitors
Helquist, Paul
Maxfield, Frederick R.
Wiech, Norbert L.
Wiest, Olaf
Niemann–Pick type C disease (NPC) is a devastating, recessive, inherited disorder that causes accumulation of cholesterol and other lipids in late endosomes and lysosomes. Mutations in 2 genes, NPC1 and NPC2, are responsible for the disease, which affects about 1 in 120,000 live births. About 95 % of patients have mutations in NPC1, a large polytopic membrane protein that is normally found in late endosomes. More than 200 missense mutations in NPC1 have been found in NPC patients. The disease is progressive, typically leading to death before the age of 20 years, although some affected individuals live well into adulthood. The disease affects peripheral organs, including the liver, spleen, and lungs, but the most severe symptoms are associated with neurological disease. There are some palliative treatments that slow progression of NPC disease. Recently, it was found that histone deacetylase (HDAC) inhibitors that are effective against HDACs 1, 2, and 3 can reduce the cholesterol accumulation in fibroblasts derived from NPC patients with mutations in NPC1. One example is vorinostat. As vorinostat is a Food and Drug Administration–approved drug for treatment of cutaneous T-cell lymphoma, this opens up the possibility that HDAC inhibitors could be repurposed for treatment of this rare disease. The mechanism of action of the HDAC inhibitors requires further study, but these drugs increase the level of the NPC1 protein. This may be due to post-translational stabilization of the NPC1 protein, allowing it to be transported out of the endoplasmic reticulum.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-013-0217-2) contains supplementary material, which is available to authorized users.
Histone deacetylase inhibitors; Cholesterol; Lysosomes; Lipids; Proteostasis
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Annexin A6-Balanced Late Endosomal Cholesterol Controls Influenza A Replication and Propagation
Musiol, Agnes
Gran, Sandra
Ehrhardt, Christina
Ludwig, Stephan
Grewal, Thomas
Gerke, Volker
Rescher, Ursula
Influenza is caused by influenza A virus (IAV), an enveloped, negative-stranded RNA virus that derives its envelope lipids from the host cell plasma membrane. Here, we examined the functional role of cellular cholesterol in the IAV infection cycle. We show that shifting of cellular cholesterol pools via the Ca2+-regulated membrane-binding protein annexin A6 (AnxA6) affects the infectivity of progeny virus particles. Elevated levels of cellular AnxA6, which decrease plasma membrane and increase late endosomal cholesterol levels, impaired IAV replication and propagation, whereas RNA interference-mediated AnxA6 ablation increased viral progeny titers. Pharmacological accumulation of late endosomal cholesterol also diminished IAV virus propagation. Decreased IAV replication caused by upregulated AnxA6 expression could be restored either by exogenous replenishment of host cell cholesterol or by ectopic expression of the late endosomal cholesterol transporter Niemann-Pick C1 (NPC1). Virus released from AnxA6-overexpressing cells displayed significantly reduced cholesterol levels. Our results show that IAV replication depends on maintenance of the cellular cholesterol balance and identify AnxA6 as a critical factor in linking IAV to cellular cholesterol homeostasis.
Influenza A virus (IAV) is a major public health concern, and yet, major host-pathogen interactions regulating IAV replication still remain poorly understood. It is known that host cell cholesterol is a critical factor in the influenza virus life cycle. The viral envelope is derived from the host cell membrane during the process of budding and, hence, equips the virus with a special lipid-protein mixture which is high in cholesterol. However, the influence of host cell cholesterol homeostasis on IAV infection is largely unknown. We show that IAV infection success critically depends on host cell cholesterol distribution. Cholesterol sequestration in the endosomal compartment impairs progeny titer and infectivity and is associated with reduced cholesterol content in the viral envelope.
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Rab8-dependent Recycling Promotes Endosomal Cholesterol Removal in Normal and Sphingolipidosis Cells
Linder, Matts D.
Uronen, Riikka-Liisa
Hölttä-Vuori, Maarit
van der Sluijs, Peter
Peränen, Johan
Ikonen, Elina
Molecular Biology of the Cell
The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann–Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits.
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ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease
Schweitzer, Jill Kuglin
Pietrini, Sean D.
D'Souza-Schorey, Crislyn
PLoS ONE
In human Niemann-Pick Type C (NPC) disease, endosomal trafficking defects lead to an accumulation of free cholesterol and other lipids in late endosome/lysosome (LE/LY) compartments, a subsequent block in cholesterol esterification and significantly reduced cholesterol efflux out of the cell. Here we report that nucleotide cycling or cellular knockdown of the small GTP-binding protein, ARF6, markedly impacts cholesterol homeostasis. Unregulated ARF6 activation attenuates the NPC phenotype at least in part by decreasing cholesterol accumulation and restoring normal sphingolipid trafficking. These effects depend on ARF6-stimulated cholesterol efflux out of the endosomal recycling compartment, a major cell repository for free cholesterol. We also show that fibroblasts derived from different NPC patients displayed varying levels of ARF6 that is GTP-bound, which correlate with their response to sustained ARF6 activation. These studies support emerging evidence that early endocytic defects impact NPC disease and suggest that such heterogeneity in NPC disease could result in diverse responses to therapeutic interventions aimed at modulating the trafficking of lipids.
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Caveolin-1 is ubiquitinated and targeted to intralumenal vesicles in endolysosomes for degradation
Hayer, Arnold
Stoeber, Miriam
Ritz, Danilo
Engel, Sabrina
Meyer, Hemmo H.
Helenius, Ari
The Journal of Cell Biology
Identification of the pathway by which caveolin-1 is degraded when caveolae assembly is compromised suggests that "caveosomes" may be endosomal accumulations of the protein awaiting degradation.
Caveolae are long-lived plasma membrane microdomains composed of caveolins, cavins, and a cholesterol-rich membrane. Little is known about how caveolae disassemble and how their coat components are degraded. We studied the degradation of caveolin-1 (CAV1), a major caveolar protein, in CV1 cells. CAV1 was degraded very slowly, but turnover could be accelerated by compromising caveolae assembly. Now, CAV1 became detectable in late endosomes (LE) and lysosomes where it was degraded. Targeting to the degradative pathway required ubiquitination and the endosomal sorting complex required for transport (ESCRT) machinery for inclusion into intralumenal vesicles in endosomes. A dual-tag strategy allowed us to monitor exposure of CAV1 to the acidic lumen of individual, maturing LE in living cells. Importantly, we found that "caveosomes," previously described by our group as independent organelles distinct from endosomes, actually correspond to late endosomal compartments modified by the accumulation of overexpressed CAV1 awaiting degradation. The findings led us to a revised model for endocytic trafficking of CAV1.
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Your soup was stellar, the conversation was charming, and the dessert was a true delight, but the kudos of the celebration go to these cute toast-shaped coasters! Crafted from cork that mimics multi-grain, this collection of eight is a staple detail of your cutting-edge apartment decor. Whether you pop these sweet mini-slices on table in your breakfast nook or stack them by the tea set in your den, they're 'browned' to prompt a clink of your coffee mug or sparkling mimosa!
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With the oral arguments over President Obama's health care law out of the way, Democrats and Republicans are bracing for the political fallout expected this June when the Supreme Court hands down its ruling.
Whatever the court decides, it's sure to roil the political waters just months before the November elections, leaving the White House and Democrats scrambling to frame any outcome as a win for them and prompting wide speculation over how much an overturn of parts or all of the law would damage Mr. Obama's re-election prospects.
"I can tell you that there is no contingency plan that's in place," White House spokesman Josh Earnest said Wednesday, responding to a question of what the administration will do if the court partially or fully overturns the law. "We're focused on implementing the law and we are confident that the law is constitutional."
Meanwhile, Republicans face the distinct possibility that the court could uphold the law, pulling their claims that it's unconstitutional right out from under them. But they tried to paint the justices' apparent skepticism of the individual mandate as a victory in and of itself, being gleeful that the hearing seemed to go well for their side.
"Democrats are playing defense right now because of the questions the Supreme Court was asking and it feels like it's fallen right into the hands of Republicans," said GOP strategist Ron Bonjean.
Mr. Bonjean said that if the court upholds the law, Republicans will try to leverage it to their advantage, telling voters they need to win the presidency and a majority in the Senate to overturn it.
The key would be how closely the court splits the vote, said Democratic strategist Paul Goldman. If the court upholds the law 5-4, Republicans can still point to the fact that four justices opposed it.
"It takes away their argument that you violated the Constitution, but it doesn't take away their argument that it was a bad law and that it was an over-the-top use of government authority," he said.
The court spent three days hearing challenges to the law, with the swing-vote justices appearing skeptical of the individual mandate that requires Americans to have health insurance.
The administration rushed to defend Solicitor General Donald Verrilli after some commentators criticized his defense of the Affordable Care Act, with CNN contributor Jeffrey Toobin calling it a "train wreck" for Mr. Obama.
Senate Majority Leader Harry Reid jumped to Mr. Verrilli's defense, telling reporters Tuesday that just because the justices asked tough questions, it doesn't mean they've already decided to knock the mandate down.
"It's good to speculate as to what might happen, but believe me, those nine men and women are extremely smart, and a lot of times they probe with those questions, not in any way to tip their mitt as to how they're going to vote on it," Mr. Reid said.
Some Democrats went so far as to embrace the possibility of an overturn, with former President Clinton strategist James Carville telling CNN's Wolf Blitzer that overturning the health care law would be a political touchdown for Democrats. Health care costs would escalate and Republicans would carry the blame, he said.
"Just as a professional Democrat, there's nothing better to me than overturning this thing 5-4 and then the Republican Party will own the health care system for the foreseeable future," Mr. Carville said. "And I really believe that. That is not spin."
© Copyright 2015 The Washington Times, LLC. Click here for reprint permission.


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Michelle Collins Print Blouse
Michelle Collins opted for a cool bold print when she paired this black-and-white houndstooth top with an orange skirt.
Michelle Collins Pencil Skirt
A bright orange pencil skirt topped off Michelle Collins' bold look at the Lorraine's High Street Fashion Awards.
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The White House has begun making contingency plans for the withdrawal of Harriet Miers as President Bush's choice to fill a seat on the Supreme Court, conservative sources said yesterday.
"White House senior staff are starting to ask outside people, saying, 'We're not discussing pulling out her nomination, but if we were to, do you have any advice as to how we should do it?' " a conservative Republican with ties to the White House told The Washington Times.
The White House denied making such calls.
"Absolutely not true," White House spokesman Trent Duffy said.
But the conservative political consultant said that he had received such a query from Sara Taylor, director of the Office of White House Political Affairs.
Miss Taylor denied making any such calls.
A second Republican, who is the leader of a conservative interest group and has ties to the White House, confirmed that calls are being made to a select group of conservative activists who are not employed by the government.
"The political people in the White House are very worried about how she will do in the hearings," the second conservative leader said. "I think they have finally awakened."
"Absolutely false," Miss Taylor said. "Some of these conspiracy theories have risen to a new level."
The White House also said yesterday that Miss Miers will carry on with all previously planned meetings with senators on Capitol Hill and is still working to schedule new ones.
"They're continuing to work to schedule meetings," White House spokesman Jim Dyke said.
The Times reported yesterday that Senate Republican lawyers said no new meetings with Miss Miers would be scheduled — at least until after the hearings.
A conservative political consultant with ties to the White House said the president and his political team once thought Democrats would go easy on Miss Miers, a friend of Mr. Bush's and his personal counsel. The theory was that Democrats see her as the best they could expect in the way of Bush appointments to the high court.
"But now Democrats smell blood in water," said the Republican, adding that he received a call from Miss Taylor seeking contingency advice on how to handle a possible decision by Miss Miers to withdraw her name or a decision by the president to withdraw the nomination.
"So there are some in the White House and some Republicans in the Senate who are worried the Democrats can now build a case that she is not competent enough or knowledgeable enough to be a justice on the Supreme Court," he said. "Really, that is the most damaging case you can build against a nominee."


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|Reactions Classified By Conversion Type → Simple Reactions → Chemical Reactions
|Reactions Classified By Substrate → Small-Molecule Reactions
EC Number: 2.3.1.-
Enzymes and Genes:
|Mycobacterium tuberculosis H37Rv :
|antigen 85A
: fbpA
antigen 85B : fbpB
antigen 85C : fbpC
In Pathway: mycolate biosynthesis
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.
Mass balance status: Marked as unbalanced.
Enzyme Commission Primary Name: 2.3.1 -- Transferring groups other than amino-acyl groups
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MandM Direct is one of the UK's leading fashion and sports retailers selling your favourite top brands - Bench, Firetrap, Timberland, Fred Perry, adidas, Nike, Puma, Henleys, Berghaus and loads more, all with savings of up to 75% off UK recommended retail prices.
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Panned for breaking nearly every theatrical commandment by, uh, everyone, the 2004 musical production "The Ten Commandments," starring Val Kilmer and performed in Los Angeles, was tucked away where all bad performances go quietly to die: YouTube.0
Nearly two millennia after becoming the site of a mass Jewish suicide, the ancient fortress Masada might finally yield a "Survivor."0
The Beatles' "Abbey Road" is temporarily being transplanted to Israel.0
Chalk it up to the recession; it's making all of us behave in unexpected ways. The Workmen's Circle/Arbeter Ring, a not-for-profit organization founded a century ago by socialist, Yiddish-speaking immigrants, recently launched its own credit card. With the organization's logo in the upper-left corner, the Visa Platinum card offers new users the standard 0% annual percentage rate for the first six months. The Workmen's Circle gets $50 whenever someone signs up and makes his first purchase, and then 0.3% of whatever the cardholder spends after that. "It's such an easy way to share with the Workmen's Circle/Arbiter Ring and continue our mission of progressive and cultural Jewish identity building," the Workmen's Circle's executive director, Ann Toback, told The Shmooze. "I thought it was a win-win all around."0
Two influential Orthodox organizations, Agudath Israel and Torah Umesorah – The National Society for Hebrew Day Schools, have come out against a New York State bill that would create a one-year window in which child sex abuse victims could sue their molesters, no matter how long ago the abuse happened.0
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DNA Computing Models
Sir Francis Crick would undoubtedly be at the front of the line ordering this fascinating book. Being one of the discoverers of DNA, he would be amazed at how his work has been applied to mankind's most important invention, the computer. DNA contains the genetic instructions for the biological development of cellular life forms or viruses. DNA computing uses DNA as a substrate for storing information, while molecular biological operations are used to manipulate this information.
DNA Computing Models begins with a comprehensive introduction to the field of DNA computing. This book emphasizes computational methods to tackle central problems of DNA computing, such as controlling living cells, building patterns, and generating nanomachines. DNA Computing Models presents laboratory-scale human-operated models of computation, including a description of the first experiment of DNA computation conducted by Adleman in 1994. It provides molecular-scale autonomous models of computation and addresses the design of computational devices working in living cells. It also addresses the important problem of proper word design for DNA computing.
DNA Computing Models is designed for researchers and advanced-level students in computers science, bioengineering and molecular biology as a reference or secondary text book. This book is also suitable for practitioners in industry.less
294 pages; ISBN 9780387736372
Read online, or download in secure PDF format
Title: DNA Computing Models
Author: Zoya Ignatova; Israel Martínez-Pérez; Karl-Heinz Zimmermann
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Welcome to WebmasterWorld Guest from 184.108.40.206
Moderators: buckworks & webwork
Did I miss something? When did all this happen?
Is there a way to find decent cheap domain names somewhere or is it all a gamble now?
C'est la vie. Hindsight is easier than foresight for us mere mortals.
You can still get $7 names, as long as they are not registered already. ;-)
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LOS ANGELES (MarketWatch) -- Japan stocks moved higher early Tuesday, with some major exporters among the leading gainers amid a slightly weaker yen, and with foreign buying reportedly helping support the market. The blue chip Nikkei Stock Average NIK, +1.64% added 0.5% to 8,722.68, outperforming a 0.3% gain for the broader Topix, with Dow Jones Newswires reporting six foreign brokerages bought a net 3.5 million shares ahead of the open. With the U.S. dollar rising about the 79.60-yen level, some export majors rose, including 1.2% gains for Hitachi Ltd. 6501, +3.27% HTHIF, +3.28% and Bridgestone Corp. 5108, +3.06% BRDCF, -3.55% a 2.2% advance for Nintendo Co. 7974, +2.64% NTDOF, -3.12% and a 1.8% gain for Fanuc Corp. 6954, +4.21% FANUF, +1.69% Shares of Olympus Corp. 7733, -0.39% OCPNF, +6.40% rose 5.8% after reporting a swing to a larger-than-expected profit for the fiscal second quarter.
Read the full story: Asia trades mixed, with Japan bouncing back


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Just when it looked on Sunday, July 1st as though Vancouver would be celebrating Canada Day and NHL Free Agency in a very low key way, a big bottle of Crown Royal was broken out late on with the signing of defenceman Jason Garrison from the Florida Panthers.
(They are of course officially the exclusive supplier of Vancouver Canucks players for the past ten years or so).
The big blue liner is a native of beach community White Rock, BC, west of Vancouver and reports suggest that he may have left some money on the table for the opportunity to sign with his home province team.
So, is it a good move or not?
Let's take a look at possible pros and cons for you the reader to consider.
Love 'emDetermination
Garrison has shown impressive determination in his hockey journey to date. He did not make major junior, and settled instead for hockey in the lower level BCHL with the Nanaimo Clippers, then went undrafted in his year of eligibility, and opted to take the US college route, joining the University of Minnesota-Duluth.
He eventually earned an entry level contract with the Florida Panthers.2-Way Play
He prides himself on his two way playand his numbers are notable for 2011/12 with 33 points in 77 games (including 16 goals that is a record for a Florida defender). The goals total additionally encompasses 9 power play markers, which is significant for a team that needs power play support.
Jason Garrison prides himself on his two way play. (Photo Ronald C. Modra/Sports Imagery/Getty Images)
Garrison also managed a +6 rating while limiting himself to a respectable 32 penalty minutes.
The newest Canuck also brings size with a 6' 2" frame that packs a solid 220 lbs that can hit and stand up opposition forwards.
Hate 'emLack of Experience
Garrison has less than three full years of playing time leading to a fair inexperience factor in the NHL. While he did have 16 goals last regular season, his previous best year was a mere 5 goals.
Hopefully, last season was not just a flash in the pan.Hometown Pressure
The large new contract ($27.6 million over 6 years) brings a certain amount of pressure on the newbie, which could be compounded by the burden of attempting to perform well in his hometown.
The media glare is several million watts dimmer in Florida too, and it will be revealing to see how Garrison handles it in the seething cauldron of Vancouver Canucks fandom with its suffocating media attention (NWSB excepted of course).
With Garrison apparently planned to pair with Alex Edler, will he be able to find chemistry with the error prone Edler (who had one of the most frustrating seasons to date by a Canucks premier talent)?
Over to you readers: Where do you stand on the Garrison signing?
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