Abscission, the final step of cytokinesis, cleaves the thin intercellular bridge connecting the two daughter cells 1–6. The scaffold protein ALIX is a core component of the abscission machinery with ...an evolutionarily conserved role in midbody recruitment of ESCRT-III 7–11, which mediates the final cut 1–5, 8–10, 12–14. In mammalian cells, the centralspindlin complex recruits the major midbody organizer CEP55 that directly binds and recruits ALIX and ESCRT-I 7–9, 15–17, which in turn cooperatively recruit ESCRT-III 8, 9, 18. However, CEP55 is missing in Drosophila melanogaster and other invertebrates 6, 9, 19, and it is unknown how the abscission machinery is recruited to the midbody in the absence of CEP55. Here, we address how Drosophila ALIX is recruited to the midbody. Surprisingly, ALIX localizes to the midbody via its V-domain, independently of the GPPX3Y motif in the proline-rich region that recruits human ALIX 8, 9. We elucidate that the centralspindlin component Pavarotti (H.s.MKLP1) interacts with the V-domain of ALIX to recruit it to the midbody. Specifically, our results indicate that an LxxLF motif in Pavarotti directly interacts with a conserved hydrophobic pocket in the ALIX V-domain, which in human ALIX binds (L)YPXnL/LxxLF motifs of virus proteins 20–28. Thus, our study identifies that ALIX is recruited by an analogous mechanism during abscission in Drosophila as during virus budding in mammalian cells and an ancestral role for centralspindlin in recruiting the abscission machinery to the midbody.
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•Drosophila ALIX is recruited to the midbody via its V-domain•Centralspindlin/Pavarotti interacts with and recruits ALIX to the midbody•ALIX interacts with Pavarotti via a conserved hydrophobic pocket in the V-domain•Pavarotti recruits ALIX via a mechanism analogous to virus budding
Lie-Jensen et al. show that, during abscission, Drosophila ALIX is recruited to the midbody via its V-domain. The centralspindlin subunit Pavarotti interacts with a conserved hydrophobic ALIX-V-domain pocket to recruit it to the midbody and promote abscission. This mechanism is analogous to ALIX recruitment during virus budding in human cells.
Overexpression of the transmembrane matrix metalloproteinase MT1-MMP/MMP14 promotes cancer cell invasion. Here we show that MT1-MMP-positive cancer cells turn MT1-MMP-negative cells invasive by ...transferring a soluble catalytic ectodomain of MT1-MMP. Surprisingly, this effect depends on the presence of TKS4 and TKS5 in the donor cell, adaptor proteins previously implicated in invadopodia formation. In endosomes of the donor cell, TKS4/5 promote ADAM-mediated cleavage of MT1-MMP by bridging the two proteases, and cleavage is stimulated by the low intraluminal pH of endosomes. The bridging depends on the PX domains of TKS4/5, which coincidently interact with the cytosolic tail of MT1-MMP and endosomal phosphatidylinositol 3-phosphate. MT1-MMP recruits TKS4/5 into multivesicular endosomes for their subsequent co-secretion in extracellular vesicles, together with the enzymatically active ectodomain. The shed ectodomain converts non-invasive recipient cells into an invasive phenotype. Thus, TKS4/5 promote intercellular transfer of cancer cell invasiveness by facilitating ADAM-mediated shedding of MT1-MMP in acidic endosomes.
Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction ...of the endocytosed fibronectin receptor, α5β1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the α5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with α5β1 integrin. Importantly, we demonstrate that both α5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of α5β1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and α5β1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.
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► α 5β1 integrin is ubiquitinated upon binding to fibronectin ► Fibronectin-bound α5β1 integrin is sorted in multivesicular endosomes ► Lysosomal trafficking of fibronectin and α5β1 integrin requires ESCRTs ► α5-fibronectin degradative sorting is required for fibroblast migration
The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III ...subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations.
The three endosomal sorting complexes required for transport (ESCRTs) are integral to the degradation of endocytosed membrane proteins and multivesicular body (MVB) biogenesis. Here, we review ...evidence that ESCRTs have evolved as a specialized machinery for the degradative sorting of ubiquitinated membrane proteins and we highlight recent studies that have shed light on the mechanisms by which these complexes mediate protein sorting, MVB biogenesis, tumour suppression and viral budding. We also discuss evidence that some ESCRT subunits have evolved additional functions that are unrelated to membrane trafficking.
The ubiquitin-binding protein Hrs and endosomal sorting complex required for transport (ESCRT)-I and ESCRT-III are involved in sorting endocytosed and ubiquitinated receptors to lysosomes for ...degradation and efficient termination of signaling. In this study, we have investigated the role of the ESCRT-II subunit Vps22/EAP30 in degradative protein sorting of ubiquitinated receptors. Vps22 transiently expressed in HeLa cells was detected in endosomes containing endocytosed epidermal growth factor receptors (EGFRs) as well as Hrs and ESCRT-I and ESCRT-III. Depletion of Vps22 by small interfering RNA, which was accompanied by decreased levels of other ESCRT-II subunits, greatly reduced degradation of EGFR and its ligand EGF as well as the chemokine receptor CXCR4. EGFR accumulated on the limiting membranes of early endosomes and aberrantly small multivesicular bodies in Vps22-depleted cells. Phosphorylation and nuclear translocation of extracellular-signal-regulated kinase1/2 downstream of the EGF-activated receptor were sustained by depletion of Hrs or the ESCRT-I subunit Tsg101. In contrast, this was not the case when Vps22 was depleted. These results indicate an important role for Vps22 in ligand-induced EGFR and CXCR4 turnover and suggest that termination of EGF signaling occurs prior to ESCRT-II engagement.
Cell division is strictly regulated by a diversity of proteins and lipids to ensure proper duplication and segregation of genetic material and organelles. Here we report a novel role of the putative ...lipid transporter ACAT-related protein required for viability 1 (Arv1) during telophase. We observed that the subcellular localization of Arv1 changes according to cell cycle progression and that Arv1 is recruited to the cleavage furrow in early telophase by epithelial protein lost in neoplasm (EPLIN). At the cleavage furrow Arv1 recruits myosin heavy chain 9 (MYH9) and myosin light chain 9 (MYL9) by interacting with IQ-motif-containing GTPase-activating protein (IQGAP1). Consequently the lack of Arv1 delayed telophase-progression, and a strongly increased incidence of furrow regression and formation of multinuclear cells was observed both in human cells in culture and in follicle epithelial cells of egg chambers of Drosophila melanogaster in vivo. Interestingly, the cholesterol-status at the cleavage furrow did not affect the recruitment of either IQGAP1, MYH9 or MYL. These results identify a novel function for Arv1 in regulation of cell division through promotion of the contractile actomyosin ring, which is independent of its lipid transporter activity.
A ubiquitin-binding endosomal protein machinery is responsible for sorting endocytosed membrane proteins into intraluminal vesicles of multivesicular endosomes (MVEs) for subsequent degradation in ...lysosomes. The Hrs–STAM complex and endosomal sorting complex required for transport (ESCRT)-I, -II and -III are central components of this machinery. Here, we have performed a systematic analysis of their importance in four trafficking pathways through endosomes. Neither Hrs, Tsg101 (ESCRT-I), Vps22/EAP30 (ESCRT-II), nor Vps24/CHMP3 (ESCRT-III) was required for ligand-mediated internalization of epidermal growth factor (EGF) receptors (EGFRs) or for recycling of cation-independent mannose 6-phosphate receptors (CI-M6PRs) from endosomes to the
trans-Golgi network (TGN). In contrast, both Hrs and ESCRT subunits were equally required for degradation of both endocytosed EGF and EGFR. Whereas depletion of Hrs or Tsg101 caused enhanced recycling of endocytosed EGFRs, this was not the case with depletion of Vps22 or Vps24. Depletion of Vps24 instead caused a strong increase in the levels of CI-M6PRs and a dramatic redistribution of the Golgi and the TGN. These results indicate that, although Hrs–STAM and ESCRT-I, -II and -III have a common function in degradative protein sorting, they play differential roles in other trafficking pathways, probably reflecting their functions at distinct stages of the endocytic pathway.
Lysosomal degradation of signalling receptors such as the epidermal growth factor (EGF) receptor (EGFR) is an important mechanism for termination of cell signalling. Such degradation involves the ...endosomal sorting of ubiquitylated receptors into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs) that move along microtubules to fuse with perinuclear lysosomes. The Rab7-interacting lysosomal protein RILP is interesting in this context as it interacts with Vps22 (also known as EAP30) and Vps36 (also known as EAP45), subunits of the endosomal sorting complex required for transport II (ESCRT-II), as well as with the dynein-dynactin motor complex. Because previous functional studies of RILP have been based on its overexpression, we have asked here whether RILP is required for endocytic trafficking of receptors. Depletion of RILP caused elevated levels of four late-endosomal molecules, lyso-bisphosphatidic acid, Lamp1, CD63 and cation-independent mannose-6-phosphate receptors. Electron microscopy showed that endosomes of RILP-depleted cells were morphologically distinct from normal late endosomes and had a strongly reduced content of ILVs. As in Vps22-depleted cells, ligand-mediated degradation of EGFRs was strongly inhibited in RILP-depleted cells, in which endocytosed EGFRs were found to accumulate in early endosomes. By contrast, endocytosis and recycling of transferrin receptors occurred normally in RILP-depleted cells. These results establish that RILP, like the ESCRT proteins, is required for biogenesis of MVEs and degradative trafficking of EGFRs but not for trafficking of transferrin receptors through early endosomes. We propose that RILP might coordinate the biogenesis of MVEs with dynein-mediated motility.
The small GTPase Rab5 controls the fusogenic properties of early endosomes through GTP-dependent recruitment and activation of effector proteins. Expression of a GTPase-defective mutant, Rab5(Q79L), ...is known to cause formation of enlarged early endosomes. The ability of Rab5-GTP to recruit multiple effectors raises the question whether the Rab5(Q79L)-induced giant endosomes simply represent enlarged early endosomes or whether they have a more complex phenotype. In this report, we have addressed this issue by generating a HEp2 cell line with inducible expression of Rab5(Q79L) and performing ultrastructural analysis of Rab5(Q79L)-induced endosomes. We find that Rab5(Q79L) not only induces formation of enlarged early endosomes but also causes enlargement of later endocytic profiles. Most strikingly, Rab5(Q79L) causes formation of enlarged multivesicular endosomes with a large number of intraluminal vesicles, and endosomes that contain both early and late endocytic markers are frequently observed. In addition, we observe defects in the sorting of the EGF receptor and the transferrin receptor through this compartment.
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