InteractiveFly: GeneBrief

ALG-2 interacting protein X: Biological Overview | References

Gene name - ALG-2 interacting protein X

Synonyms -

Cytological map position - 98B1-98B1

Function - adaptor protein

Keywords - intracellular protein trafficking - regulates mitotic spindle orientation - forms a complex with Shrub - required for completion of cytokinetic abscission during asymmetric germline stem cell division - associates with the ESCRT machinery assisting with cargo recruitment and intraluminal vesicle formation in multivesicular bodies

Symbol - ALIX

FlyBase ID: FBgn0086346

Genetic map position - chr3R:27,696,466-27,700,110

NCBI classification - Middle V-domain of mammalian Alix and related domains are dimerization and protein interaction modules - Protein-interacting, N-terminal, Bro1-like domain of mammalian Alix and related domains - MAPK-interacting and spindle-stabilizing protein-like

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

ALIX orthologs: Biolitmine

Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. This study shows that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, this study shows that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. It is concluded that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo (Eikenes, 2015).

Cytokinesis is the final step of cell division that leads to the physical separation of the two daughter cells. It is tightly controlled in space and time and proceeds in multiple steps via sequential specification of the cleavage plane, assembly and constriction of the actomyosin-based contractile ring (CR), formation of a thin intercellular bridge and finally abscission that separates the two daughter cells. Studies in a variety of model organisms and systems have elucidated key machineries and signals governing early events of cytokinesis. However, the mechanisms of the final abscission step of cytokinesis are less understood, especially in vivo in the context of different cell types in a multi-cellular organism (Eikenes, 2015).

Recently key insights into the molecular mechanisms and spatiotemporal control of abscission have been gained using a combination of advanced molecular biological and imaging technologies. At late stages of cytokinesis the spindle midzone transforms to densely packed anti-parallel microtubules (MTs) that make up the midbody (MB) and the CR transforms into the midbody ring (MR, diameter of ~1-2 μm). The MR is located at the site of MT overlap and retains several CR components including Anillin, septins (Septins 1, 2 and Peanut in Drosophila melanogaster), myosin-II, Citron kinase (Sticky in Drosophila) and RhoA (Rho1 in Drosophila) and eventually also acquires the centralspindlin component MKLP1 (Pavarotti in Drosophila). In C. elegans embryos the MR plays an important role in scaffolding the abscission machinery even in the absence of MB MTs (Eikenes, 2015).

Studies in human cell lines, predominantly in HeLa and MDCK cells, have shown that components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins play important roles in mediating abscission. Abscission occurs at the thin membrane neck that forms at the constriction zone located adjacent to the MR. An important signal for initiation of abscission is the degradation of the mitotic kinase PLK1 (Polo-like kinase 1) that triggers the targeting of CEP55 (centrosomal protein of 55 kDa) to the MR. CEP55 interacts directly with GPP(3x)Y motifs in the ESCRT-associated protein ALIX (ALG-2-interacting protein X) and in the ESCRT-I component TSG101, thereby recruiting them to the MR. ALIX and TSG101 in turn recruit the ESCRT-III component CHMP4B, which is followed by ESCRT-III polymerization into helical filaments that spiral/slide to the site of abscission. The VPS4 ATPase is thought to promote ESCRT-III redistribution toward the abscission site. Prior to abscission ESCRT-III/CHMP1B recruits Spastin that mediates MT depolymerization at the abscission site. ESCRT-III then facilitates membrane scission of the thin membrane neck, thereby mediating abscission (Eikenes, 2015).

Cytokinesis is tightly controlled by the activation and inactivation of mitotic kinases at several steps to ensure its faithful spatiotemporal progression. Cytokinesis conventionally proceeds to completion via abscission, but is differentially controlled depending on the cell type during the development of metazoan tissues. For example, germ cells in species ranging from insects to humans undergo incomplete cytokinesis leading to the formation of germline cysts in which cells are interconnected via stable intercellular bridges. How cytokinesis is modified to achieve different abscission timing in different cell types is not well understood, but molecular understanding of the regulation of the abscission machinery has started giving some mechanistic insight (Eikenes, 2015).

The Drosophila female germline represents a powerful system to address mechanisms controlling cytokinesis and abscission in vivo. Each Drosophila female germline stem cell (fGSC) divides asymmetrically with complete cytokinesis to give rise to another fGSC and a daughter cell cystoblast (CB). Cytokinesis during fGSC division is delayed so that abscission takes place during the G2 phase of the following cell cycle (about 24 hours later). The CB in turn undergoes four mitotic divisions with incomplete cytokinesis giving rise to a 16-cell cyst in which the cells remain interconnected by stable intercellular bridges called ring canals (RCs). One of the 16 cells with four RCs will become specified as the oocyte and the cyst becomes encapsulated by a single layer follicle cell epithelium to form an egg chamber. Drosophila male GSCs (mGSCs) also divide asymmetrically with complete cytokinesis to give rise to another mGSC and a daughter cell gonialblast (GB). Anillin, Pavarotti, Cindr, Cyclin B and Orbit are known factors localizing at RCs/MRs and/or MBs during complete cytokinesis in fGSCs and/or mGSCs. It has been recently reported that Aurora B delays abscission and that Cyclin B promotes abscission in Drosophila germ cells and that mutual inhibitions between Aurora B and Cyclin B/Cdk-1 control the timing of abscission in Drosophila fGSCs and germline cysts. However, little is known about further molecular mechanisms controlling cytokinesis and abscission in Drosophila fGSCs (Eikenes, 2015).

This study has characterize the roles of ALIX and the ESCRT-III component Shrub during cytokinesis in Drosophila fGSCs. ALIX and Shrub are required for completion of abscission in fGSCs. They co-localize during this process, and their direct interaction is required for abscission with normal kinetics. This study thus shows that a complex between ALIX and Shrub is required for abscission in fGSCs and provide evidence of an evolutionarily conserved functional role of the ALIX/ESCRT-III pathway in mediating cytokinetic abscission in the context of a multi-cellular organism (Eikenes, 2015).

Loss of ALIX or/and Shrub function or inhibition of their interaction delays abscission in fGSCs leading to the formation of stem cysts in which the fGSC remains interconnected to chains of daughter cells via MRs. As abscission eventually takes place a cyst of e.g. 2 germ cells may pinch off and subsequently undergo four mitotic divisions to give rise to a germline cyst with 32 germ cells. Consistently, loss of ALIX or/and Shrub or interference with their interaction caused a high frequency of egg chambers with 32 germ cells during Drosophila oogenesis. It was also found that ALIX controls cytokinetic abscission in both fGSCs and mGSCs and thus that ALIX plays a universal role in cytokinesis during asymmetric GSC division in Drosophila. Taken together this study provides evidence that the ALIX/ESCRT-III pathway is required for normal abscission timing in a living metazoan tissue (Eikenes, 2015).

The results together with findings in other models underline the evolutionary conservation of the ESCRT system and associated proteins in cytokinetic abscission. Specifically, ESCRT-I or ESCRT-III have been implicated in abscission in a subset of Archaea (ESCRT-III), in A. thaliana (elch/tsg101/ESCRT-I) and in C. elegans (tsg101/ESCRT-I). In S. cerevisiae, Bro1 (ALIX) and Snf7 (CHMP4/ESCRT-III) have also been suggested to facilitate cytokinesis. In cultured Drosophila cells, Shrub/ESCRT-III mediates abscission and in human cells in culture ALIX, TSG101/ESCRT-I and CHMP4B/ESCRT-III promote abscission. ALIX and the ESCRT system thus act in an ancient pathway to mediate cytokinetic abscission (Eikenes, 2015).

Despite the fact that an essential role of ALIX in promoting cytokinetic abscission during asymmetric GSC division was found in the Drosophila female and male germlines, strong bi-nucleation directly attributed to cytokinesis failure was found in Drosophila alix mutants in the somatic cell types that were examined. This might have multiple explanations. One possibility is that maternally contributed alix mRNA may support normal cytokinesis and development. Whereas ALIX and CHMP4B depletion in cultured mammalian cells causes a high frequency of bi- and multi-nucleation it is also possible that cells do not readily become bi-nucleate upon failure of the final step of cytokinetic abscission in the context of a multi-cellular organism. Consistent with the observations of a high frequency of stem cysts upon loss of ALIX and Shrub in the germline, Shrub depletion in cultured Drosophila cells resulted in chains of cells interconnected via intercellular bridges/MRs due to multiple rounds of cell division with failed abscission. Moreover, loss of ESCRT-I/tsg101 function in the C. elegans embryo did not cause furrow regression. These and the current observations suggest that ALIX- and Shrub/ESCRT-depleted cells can halt and are stable at the MR stage for long periods of time and from which cleavage furrows may not easily regress, at least not in these cell types and in the context of a multi-cellular organism. It is also possible that redundant mechanisms contribute to abscission during symmetric cytokinesis in somatic Drosophila cells. Further studies should address the general involvement of ALIX and ESCRT-III in cytokinetic abscission in somatic cells in vivo (Eikenes, 2015).

Different cell types display different abscission timing, intercellular bridge morphologies and spatiotemporal control of cytokinesis. In fGSCs it was found that ALIX and Shrub co-localize throughout late stages of cytokinesis and abscission. In human cells ALIX localizes in the central region of the MB, whereas CHMP4B at first localizes at two cortical ring-like structures adjacent to the central MB region and then progressively distributes also at the constriction zone where it promotes abscission. ALIX and CHMP4B are thus found at discrete locations within the intercellular bridge as cells approach abscission in human cultured cells. In contrast, ESCRT-III localizes to a ring-like structure during cytokinesis in Archaea, resembling the Shrub localization at MRs was observed in Drosophila fGSCs. Moreover, ALIX and Shrub are present at MRs for a much longer time (from G1/S) prior to abscission (in G2) in fGSCs than in human cultured cells. Here, ALIX and CHMP4B are increasingly recruited about an hour before abscission and then CHMP4B acutely increases at the constriction zones shortly (~30 min) before the abscission event (Eikenes, 2015).

How may ALIX and Shrub be recruited to the MR/MB in Drosophila cells in the absence of CEP55 that is a major recruiter of ALIX and ultimately CHMP4/ESCRT-III in human cells? Curiously, a GPP(3x)Y consensus motif was detected within the Drosophila ALIX sequence (GPPPGHY, aa 808-814) resembling the CEP55-interacting motif in human ALIX (GPPYPTY, aa 800-806). Whether Drosophila ALIX is recruited to the MR/MB via a protein(s) interacting with this motif or other domains is presently uncharacterized. Accordingly, alternative pathways of ALIX and ESCRT recruitment have been reported, as well as suggested in C. elegans, where CEP55 is also missing. Further studies are needed to elucidate mechanisms of recruitment and spatiotemporal control of ALIX and ESCRT-III during cytokinesis in fGSCs and different cell types in vivo (Eikenes, 2015).

This study found that the direct interaction between ALIX and Shrub is required for completion of abscission with normal kinetics in fGSCs. This is consistent with findings in human cells in which loss of the interaction between ALIX and CHMP4B causes abnormal midbody morphology and multi-nucleation. Following ALIX-mediated recruitment of CHMP4B/ESCRT-III to cortical rings adjacent to the MR in human cells, ESCRT-III extends in spiral-like filaments to promote membrane scission. Due to the discrete localizations of ALIX and CHMP4B during abscission in human cells ALIX has been proposed to contribute to ESCRT-III filament nucleation. In vitro studies have shown that the interaction between ALIX and CHMP4B may release autoinhibitory intermolecular interactions within both proteins and promote CHMP4B polymerization. Specifically, ALIX dimers can bundle pairs of CHMP4B filaments in vitro. Moreover, in yeast, the interaction of the ALIX homologue Bro1 with Snf7 (CHMP4 homologue) enhances the stability of ESCRT-III polymers. There is a high degree of evolutionary conservation of ALIX and ESCRT-III proteins and because ALIX and Shrub co-localize and interact to promote abscission in fGSCs it is possible that ALIX can facilitate Shrub filament nucleation and/or polymerization during this process (Eikenes, 2015).

The current findings indicate that accurate control of the levels and interaction of ALIX and Shrub ensure proper abscission timing in fGSCs. Their reduced levels or interfering with their complex formation caused delayed abscission kinetics. How cytokinesis is modified to achieve a delay in abscission in Drosophila fGSCs and incomplete cytokinesis in germline cysts is not well understood. Aurora B plays an important role in controlling abscission timing both in human cells and the Drosophila female germline. During Drosophila germ cell development Aurora B contributes to mediating a delay of abscission in fGSCs and a block in cytokinesis in germline cysts. Bam expression has also been proposed to block abscission in germline cysts. It will be interesting to investigate mechanisms regulating the levels, activity and complex assembly of ALIX and Shrub and other abscission regulators at MRs/MBs to gain insight into how the abscission machinery is modified to control abscission timing in fGSCs (Eikenes, 2015).

Intercellular bridge MTs in fGSC-CB pairs were degraded in G1/S when the fusome adopted bar morphology. Abscission in G2 thus appears to occur independently of intercellular bridge MTs in Drosophila fGSCs. This has also been described in C. elegans embryonic cells where the MR scaffolds the abscission machinery as well as in Archaea that lack the MT cytoskeleton]. In mammalian and Drosophila S2 cells in culture, on the other hand, intercellular bridge MTs are present until just prior to abscission (Eikenes, 2015).

It is interesting to note a resemblance of the stem cysts that appeared upon loss of ALIX and Shrub function to germline cysts in that the MRs remained open for long periods of time similar to RCs. Some modification of ALIX and Shrub levels/recruitment may thus contribute to incomplete cytokinesis in Drosophila germline cysts under normal conditions. Because stem cysts were detected in the case when ALIX weakly interacted with Shrub it is also possible that inhibition of their complex assembly/activity may contribute to incomplete cytokinesis in germline cysts. Abscission factors, such as ALIX and Shrub, may thus be modified and/or inhibited during incomplete cytokinesis in germline cysts. Such a scenario has been shown in the mouse male germline where abscission is blocked by inhibition of CEP55-mediated recruitment of the abscission machinery, including ALIX, to stable intercellular bridges. Altogether these data thus suggest that ALIX and Shrub are essential components of the abscission machinery in Drosophila GSCs, and it is speculated that their absence or inactivation may contribute to incomplete cytokinesis. More insight into molecular mechanisms controlling abscission timing and how the abscission machinery is modified in different cellular contexts will give valuable information about mechanisms controlling complete versus incomplete cytokinesis in vivo (Eikenes, 2015).

In summary, this study reports that a complex between ALIX and Shrub is required for completion of cytokinetic abscission with normal kinetics during asymmetric Drosophila GSC division, giving molecular insight into the mechanics of abscission in a developing tissue in vivo (Eikenes, 2015).

Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics

The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. This study reports a novel role for the multifunctional adaptor protein centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of gamma-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule-associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, these results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division (Malerod, 2018).

During cell division, the mitotic spindle (MS) that forms between the two centrosomes ensures faithful segregation of the chromosomes between the two daughter cells, positions the cleavage furrow, and is anchored to the cell cortex to ensure proper spindle orientation. Different subpopulations of microtubules (MTs); the kinetochore, interpolar/astral, and astral MTs, are involved in controlling each process, respectively. Correct orientation of the MS ensures proper segregation of molecules defining cell fate and is important during asymmetric stem cell division to generate one daughter cell which self-renews and one which undergoes differentiation. The orientation of the MS further defines the cleavage plane of the cell and thereby its position within the tissue, exemplified by the planar division of epithelial cells to generate a monolayered epithelium. The precise orientation of the MS can be influenced by internal cues (cell polarity determinants) or external cues (neighboring cells or extracellular matrix) and is cell type-dependent (Malerod, 2018).

Regardless of the molecular mechanisms setting the orientation, the MS is anchored to the cell cortex by the astral MTs radiating from the centrosomes. The centrosome is the major MT-organizing center in most cell types and nucleates astral MTs and the other MT subpopulations of the MS. The centrosome is composed of a centriole pair and the surrounding pericentriolar material (PCM), generated by dynamic assembly of proteins found to stabilize each other via positive feedback loops. During mitosis, the centrosome matures when the PCM expands extensively due to recruitment of scaffold and MT nucleating proteins, which promote MS formation. The γ-tubulin ring complexes (γTuRCs) of the PCM, composed of γ-tubulin and associated proteins (γ-tubulin complex proteins, GCPs), nucleate MT filaments at the centrosome. The ring of γ-tubulin within γTuRC resembles the MT geometry and serves as a template for assembly of α/β-tubulin-dimers, which polymerize into long filaments, MTs. Although the centrosomes represent the major centers for MT nucleation, MTs may alternatively be formed at the Golgi, chromosomes, nuclear envelope, plasma membrane, and pre-existing MTs. Importantly, γ-tubulin seems to be implicated in the nucleation process regardless of the intracellular localization (Malerod, 2018).

Microtubules of the MS, including the astral MTs, are dynamic and their timely assembly and disassembly is tightly controlled by proteins regulating nucleation, severing, and stability of the filaments. MT stability is regulated by MT-associated proteins. These proteins stabilize MTs by binding to the growing plus-end of the filaments to prevent catastrophe, or alternatively, by decorating the MTs to prevent interaction with severing proteins. Furthermore, the γTuRC itself has also been reported to modulate the stability of MTs by interacting with motor proteins such as dynein, kinesin-5, and kinesin-14 as well as the plus-end tracking protein EB1 (Malerod, 2018).

Astral MT regulation occurs at several levels to achieve proper MS orientation: (1) astral MT nucleation at the centrosomes, (2) astral MT dynamics and stability, and (3) astral MT anchoring and behavior at the cell cortex. Aberrant regulation of astral MTs has been shown to correlate with spindle misorientation. For example, centrosomal proteins regulating γTuRC-mediated nucleation of MTs and MAPs controlling MT stability have been shown to regulate spindle orientation in their capacity of modulating MT dynamics. Despite the emerging insight into how astral MT formation is controlled to ensure proper MS orientation, the molecular mechanisms are incompletely understood (Malerod, 2018).

The multifunctional adaptor protein ALG-2-interacting protein X (ALIX) has been shown to localize to centrosomes in interphase and during cell division. However, the biological roles of centrosomal ALIX are not known. Extensive research has implicated ALIX in a diversity of cellular processes, such as apoptosis, endocytosis and endosome biogenesis, cell adhesion, virus release, plasma membrane repair, and cytokinesis. Specifically, ALIX controls cytokinesis by participating in recruiting abscission-promoting proteins of the endosomal sorting complex required for transport (ESCRT) to the midbody. The current study has investigated the role of centrosomal ALIX during cell division. ALIX is shown to localize to the PCM, where it interacts with and stabilizes γTuRC, thus promoting efficient nucleation of astral MTs. In addition, centrosomal ALIX recruits MAP1S, which stabilizes the newly formed MTs radiating from the centrosomes. It is concluded that ALIX facilitates efficient formation of astral MTs by stimulating their nucleation and stabilization, which promotes correct MS orientation during both asymmetric and symmetric cell division (Malerod, 2018).

This study has unraveled a novel role of ALIX located at the centrosomes during cell division in regulation of MS orientation by modulating the formation of astral MTs. ALIX is recruited to the PCM via DSpd-2/Cep192, which recruit PCM components (including Cnn/Cep215, γ-tubulin, and Dgrip91/GCP3), to promote nucleation of astral MTs. Notably, even though DSpd-2/Cep192 appears to be a major recruiter of ALIX to centrosomes, the fact that ALIX was still partially detected at centrosomes in the absence of DSpd-2/Cep192 indicates that additional recruitment mechanisms exist. Centrosomal protein of 55 kDa (CEP55), which localizes to centrosomes during early phases of cell division and moves to and recruits ALIX to the midbody during cytokinesis, represents a possible additional recruiter of ALIX to centrosomes in human cells. However, because CEP55 orthologues lack in lower eukaryotes, such as D. melanogaster (and C. elegans), other proteins could also participate in recruiting ALIX to centrosomes. Interestingly, a direct interaction between DSpd-2/Cep192 and γ-tubulin has not been elucidated. Based on the current results, it is therefore tempting to speculate that ALIX serves a scaffolding role at the interface between DSpd-2/Cep192 and γTuRC, since it was found that ALIX binds DSpd-2, γ-tubulin, and Dgrip91 in vitro. Thus, the results provide mechanistic insight into DSpd-2/Cep192-mediated regulation of astral MT formation and proper orientation of the MS during metaphase in Drosophila and human cells (Malerod, 2018).

The current model shows the MS orientation in cells with or without ALIX. The PCM protein DSpd-2/Cep192 recruits ALIX to the PCM, where ALIX recruits γ-tubulin of the γTuRC at the centrosomes, thus facilitating nucleation of astral MTs. Furthermore, ALIX recruits MAP1S to the centrosomes, in close proximity to the newly formed MTs which are then stabilized by MAP1S. Thus, ALIX facilitates both nucleation of and stabilization of astral MTs emanating from the centrosomes, thus promoting efficient formation of stable astral MTs which mediate anchoring to the cell cortex and thus correct positioning of the MS. By this mechanism, ALIX is one of several molecules controlling MS orientation, providing robustness to correctly orient the MS during cell division (Malerod, 2018).

Astral MTs seem to be equally essential for correct spindle orientation in diverse cell types, in difference to internal polarity cues or external signals provided by neighboring cells. Interestingly, that loss of ALIX induced spindle misorientation in a variety of cell types, including stem cells (NBs and mGSCs) and epithelial cells (SOPs, FECs, and Caco-2 cells), corresponds well with the current data showing that ALIX controls spindle orientation by facilitating the formation of astral MTs and indicates a general role of ALIX in this process. Furthermore, the defective localization of Miranda and aPKC in alix mutant NBs reflects the compromised formation of astral MTs, rather than aberrant cell polarity, since astral MTs have previously been shown to stabilize these determinants at the basal and apical membranes, respectively (Malerod, 2018).

ALIX was shown to maintain the epithelial blood-cerebrospinal fluid barrier by facilitating assembly of tight junctions, which were recently reported to control spindle orientation in Caco-2 cyst cells. In general, cell-cell contacts such as tight junctions seem to control MS orientation in epithelial cells by F-actin, an essential component of the cell cortex facilitating capture of astral MTs. In human epithelial cells, ALIX might thus affect both the formation of astral MTs, as has been shown in this study, and their anchoring to the cell cortex. Also in Drosophila SOPs, septate junctions, resembling tight junctions, regulate the MS orientation. Whether ALIX regulates septate junctions in Drosophila epithelial cells remains to be elucidated, but the current data showing that cold-induced depolymerization of MTs potentiated the spindle misorientation only in wild-type FECs, and not in alix1 FECs, suggest that ALIX regulates the MS orientation by MT-dependent mechanisms (Malerod, 2018).

The current study suggests a dual role for ALIX during astral MT formation: (1) by promoting nucleation via γ-tubulin recruitment and (2) by stabilization of MTs via stabilizing MAP1S at the centrosomes. Although MAP1S is predominantly associated along MTs, it has also been shown to concentrate at the centrosomes. Here, MAP1S has been suggested to stabilize newly formed MT filaments, which likely explains the reduced regrowth of MTs observed at early time points after cold-induced depolymerization in MAP1S-depleted cells. Accordingly, this study found that ectopically expressed MAP1S was unable to rescue the reduced number of astral MTs observed in ALIX-deficient cells, thus arguing against that MAP1S influences the nucleation of MTs as such. Rather, MAP1S significantly increased the length of astral MTs in ALIX knockdown cells, supporting the hypothesis that ALIX facilitates MT stability via MAP1S. It is envisioned that ALIX stabilizes MAP1S adjacent to the PCM, close to the ends of the newly formed MTs emanating from the centrosomes. A simultaneous interaction of MAP1S with both MTs and ALIX seems plausible since the MT-interacting domain is located in the light chain of MAP1S, whereas ALIX seems to bind the heavy chain (Malerod, 2018).

In summary, the current study identifies a novel evolutionarily conserved role of centrosomal ALIX in promoting astral MT formation to orient the MS. The reduced, rather than absent, recruitment of γ-tubulin, MAP1S and consequently appearance of astral MTs in ALIX-deficient cells, clearly suggests that ALIX represents one of several mechanisms to ensure formation of astral MTs. Thus, ALIX provides robustness to correctly orient the MS during asymmetric and planar cell division (Malerod, 2018).

Tumor susceptibility gene 101 regulates predisposition to apoptosis via ESCRT machinery accessory proteins

PESCRT proteins are implicated in myriad cellular processes, including endosome formation, fusion of autophagosomes/amphisomes with lysosomes, and apoptosis. The role played by these proteins in either facilitating or protecting against apoptosis is unclear. In this study, while trying to understand how deficiency of Mahogunin RING finger 1 (MGRN1) affects cell viability, a novel role was uncovered for its interactor, the ESCRT-I protein TSG101: it directly participates in mitigating ER stress-mediated apoptosis. The association of TSG101 with ALIX prevents predisposition to apoptosis, whereas ALIX-ALG-2 interaction favors a death phenotype. Altered Ca(2+) homeostasis in cells and a simultaneous increase in the protein levels of ALIX and ALG-2 are required to elicit apoptosis by activating ER stress-associated caspase 4/12. It was further demonstrated that in the presence of membrane-associated, disease-causing prion protein (Ctm)PrP, increased ALIX and ALG-2 levels are detected along with ER stress markers and associated caspases in transgenic brain lysates and cells. These effects were rescued by overexpression of TSG101. This is significant because MGRN1 deficiency is closely associated with neurodegeneration and prenatal and neonatal mortality, which could be due to excess cell death in selected brain regions or myocardial apoptosis during embryonic development (Kaul, 2017).


Plenty of SH3s (POSH) functions as a scaffold protein for the Jun N-terminal kinase (JNK) signal transduction pathway, which leads to cell death in mammalian cultured cells and Drosophila. This study shows that POSH forms a complex with Apoptosis-linked gene-2 (ALG-2) and ALG-2-interacting protein (ALIX/AIP1) in a calcium-dependent manner. Overexpression of ALG-2 or ALIX in developing imaginal eye discs results in roughened or melanized eyes, respectively. These phenotypes are enhanced by co-overexpression of POSH. It was found that overexpression of either gene could induce ectopic JNK activation, suggesting that POSH/ALG-2/ALIX may function together in the regulation of the JNK pathway (Tsuda, 2006).

The present study demonstrates that POSH can form a complex with ALG-2 and ALIX both in vitro and in Drosophila cultured cells. Transgenic studies revealed that overexpression of ALG-2 in imaginal eye discs using GMR-GAL4 resulted in a roughened eye, similar to that induced by POSH. However, when both ALG-2 and POSH are overexpressed, the eye size is dramatically reduced, suggesting that ALG-2 in combination with POSH can kill the cells during development. Overexpression of ALIX produced eyes with melanotic pigments, which is indicative of neuronal degeneration. Flies overexpressing both ALIX and POSH show increased amounts of melanotic pigments, with no effect on the eye size. The phenotypic differences between ALG-2 and ALIX suggest that the cell death occurs more rapidly in eye discs overexpressing ALG-2/POSH than in those overexpressing ALIX/POSH (Tsuda, 2006).

It was further demonstrated that ALG-2 and ALIX can activate the JNK signaling pathway, similar to the case for POSH. Since neither ALIX nor ALG-2 has a putative kinase domain, overexpression of either protein together with POSH may facilitate ectopic JNK activation by affecting the subcellular localizations of JNK components. In fact, ALG-2 has been shown to interact with ASK1, thereby regulating its subcellular localization and JNK activation in mammalian cells (Tsuda, 2006).

ALG-2 and ALIX are both thought to be involved in membrane trafficking. ALG-2 interacts with Annexin XI and VII, both of which play roles in vesicular trafficking and exocytosis. In contrast, ALIX binds to CHMP4b, a human homolog of yeast Snf7, which is involved in multivesicular body (MVB) sorting. Furthermore, ALG-2 and ALIX both bind to Tsg101, a component of ESCRT-I. ESCRT-I cooperates with two other complexes, ESCRT-II and ESCRT-III, to drive MVB formation. MVB sorting is thought to be topologically identical to the budding of HIV and other retroviruses from the plasma membrane. Indeed, ALIX has been shown to associate with HIV-1 Gag protein, which is required for promoting membrane fission events, and recruit the ESCRT machinery to permit budding. Recently, POSH was also shown to be required for sorting of HIV-1 Gag protein to the plasma membrane. Furthermore, POSH interacts with hepatocyte growth factor-regulated tyrosine kinase substrate, which is known to play a central role in MVB formation, and modulates its stability. These findings suggest that the POSH/ALIX/ALG-2 complex may have a role in membrane trafficking and virus budding (Tsuda, 2006).

Functions of Alix orthologs in other species

Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor

Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, it was found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. This study reports that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. This study shows that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. It was also shown that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, these results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef (Amorim, 2014).

Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release

The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in Drosophila. This study identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues in vivo and in vitro. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1DeltaPTAP, this study demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSHV14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1DeltaPTAP L-domain mutant (YPXnL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPXnL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release. Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPXnL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable (Votteler, 2009).


Search PubMed for articles about Drosophila Alix1

Amorim, N. A., da Silva, E. M., de Castro, R. O., da Silva-Januario, M. E., Mendonca, L. M., Bonifacino, J. S., da Costa, L. J. and daSilva, L. L. (2014). Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor. J Biol Chem 289(40): 27744-27756. PubMed ID: 25118280

Eikenes, A. H., Malerod, L., Christensen, A. L., Steen, C. B., Mathieu, J., Nezis, I. P., Liestol, K., Huynh, J. R., Stenmark, H. and Haglund, K. (2015). ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo. PLoS Genet 11: e1004904. PubMed ID: 25635693

Kaul, Z. and Chakrabarti, O. (2017). Tumor susceptibility gene 101 regulates predisposition to apoptosis via ESCRT machinery accessory proteins. Mol Biol Cell 28(15): 2106-2122. PubMed ID: 28539405

Malerod, L., Le Borgne, R., Lie-Jensen, A., Eikenes, A. H., Brech, A., Liestol, K., Stenmark, H. and Haglund, K. (2018). Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics. EMBO J. 37(13): PubMed ID: 29858227

Tsuda, M., Seong, K. H. and Aigaki, T. (2006). POSH, a scaffold protein for JNK signaling, binds to ALG-2 and ALIX in Drosophila. FEBS Lett. 580(13): 3296-300. 16698022

Votteler, J., Iavnilovitch, E., Fingrut, O., Shemesh, V., Taglicht, D., Erez, O., Sorgel, S., Walther, T., Bannert, N., Schubert, U. and Reiss, Y. (2009). Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release. BMC Biochem 10: 12. PubMed ID: 19393081

Biological Overview

date revised: 21 January 2019
