Early Endosome Fusion Research Articles

Structure of the endosomal CORVET tethering complex

Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solve the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discover that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane lipid packing defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function.

CORVET-specific subunit levels determine the balance between HOPS/CORVET endosomal tethering complexes

The closely related endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively, and HOPS also mediates the fusion of lysosomes with incoming vesicles including late endosomes and autophagosomes. These heterohexameric complexes share their four core subunits that assemble with additional two, complex-specific subunits. These features and the similar structure of the complexes could allow the formation of hybrid complexes, and the complex specific subunits may compete for binding to the core. Indeed, our biochemical analyses revealed the overlap of binding sites for HOPS-specific VPS41 and CORVET-specific VPS8 on the shared core subunit VPS18. We found that the overexpression of CORVET-specific VPS8 or Tgfbrap1 decreased the amount of core proteins VPS11 and VPS18 that are assembled with HOPS-specific subunits VPS41 or VPS39, indicating reduced amount of assembled HOPS. In line with this, we observed the elevation of both lipidated, autophagosome-associated LC3 protein and the autophagic cargo p62 in these cells, suggesting impaired autophagosome-lysosome fusion. In contrast, overexpression of HOPS-specific VPS39 or VPS41 did not affect the level of assembled CORVET or autophagy. VPS8 or Tgfbrap1 overexpression also induced Cathepsin D accumulation, suggesting that HOPS-dependent biosynthetic delivery of lysosomal hydrolases is perturbed, too. These indicate that CORVET-specific subunit levels fine-tune HOPS assembly and activity in vivo.

P97/VCP Promotes the Recycling of Endocytic Cargo.

The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14, and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.

A dominant negative variant of RAB5B disrupts maturation of surfactant protein B and surfactant protein C

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.

Early Endosomal GTPase Rab5 (Ras-Related Protein in Brain 5) Regulates GPIbβ (Glycoprotein Ib Subunit β) Trafficking and Platelet Production In Vitro.

Maturation of megakaryocytes culminates with extensive membrane rearrangements necessary for proplatelet formation. Mechanisms required for proplatelet extension and origin of membranes are still poorly understood. GTPase Rab5 (Ras-related protein in brain 5) regulates endocytic uptake and homotypic fusion of early endosomes and regulates phosphatidylinositol 3-monophosphate production important for binding of effector proteins during early-to-late endosomal/lysosomal maturation. Approach and Results: To investigate the role of Rab5 in megakaryocytes, we expressed GFP (green fluorescent protein)-coupled Rab5 wild type and its point mutants Q79L (active) and N133L (inactive) in primary murine fetal liver-derived megakaryocytes. Active Rab5 Q79L induced the formation of enlarged early endosomes, while inactive Rab5 N133L caused endosomal fragmentation. Consistently, an increased amount of transferrin internalization in Rab5 Q79L was impaired in Rab5 N133L expressing megakaryocytes, when compared with GFP or Rab5 wild type. Moreover, trafficking of GPIbβ (glycoprotein Ib subunit beta), a subunit of major megakaryocytes receptor and membrane marker, was found to be mediated by Rab5 activity. While GPIbβ was mostly present along the plasma membrane, and within cytoplasmic vesicles in Rab5 wild type megakaryocytes, it accumulated in the majority of Rab5 Q79L enlarged endosomes. Conversely, Rab5 N133L caused mostly GPIbβ plasma membrane retention. Furthermore, Rab5 Q79L expression increased incorporation of the membrane dye (PKH26), indicating higher membrane content. Finally, while Rab5 Q79L increased proplatelet production, inactive Rab5 N133L strongly inhibited it and was coupled with a decrease in late endosomes/lysosomes. Localization of GPIbβ in enlarged endosomes was phosphatidylinositol 3-monophosphate dependent. Taken together, our results demonstrate that Rab5-dependent endocytosis plays an important role in megakaryocytes receptor trafficking, membrane formation, and thrombopoiesis.

Broad spectrum compounds targeting early stages of rabies virus (RABV) infection

ABMA and its analogue DABMA are two molecules of the adamantane family known to perturbate the endosomal pathway and to inhibit cell infection of several RNA and DNA viruses. Their activity against Rabies Virus (RABV) infection has already been demonstrated in vitro. (Wu et al., 2017, 2019). Here, we describe in more details their mechanism of action by comparison to Arbidol (umifenovir) and Ribavirin, two broad spectrum antivirals against emerging viruses such as Lassa, Ebola, influenza and Hantaan viruses. ABMA and DABMA, delivered 2 h pre-infection, inhibit RABV infection in vitro with an EC50 of 7.8 μM and 14 μM, respectively. They act at post-entry, by causing RABV accumulation within the endosomal compartment and DABMA specifically diminishes the expression of the GTPase Rab7a controlling the fusion of early endosomes to late endosomes or lysosomes. This may suggest that ABMA and DABMA act at different stages of the late endosomal pathway as supported by their different profile of synergy/antagonism with the fusion inhibitor Arbidol. This difference is further confirmed by the RABV mutants induced by successive passages under increasing selective pressure showing a particular involvement of the viral G protein in the DABMA inhibition while ABMA inhibition induces less mutations dispersed in the M, G and L viral proteins. These results suggest new therapeutic perspectives against rabies.

Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

Enlarged early endosomes have been visualized in Alzheimer’s disease (AD) and Down syndrome (DS) using conventional confocal microscopy at a resolution corresponding to endosomal size (hundreds of nm). In order to overtake the diffraction limit, we used super-resolution structured illumination microscopy (SR-SIM) and transmission electron microscopies (TEM) to analyze the early endosomal compartment in DS.By immunofluorescence and confocal microscopy, we confirmed that the volume of Early Endosome Antigen 1 (EEA1)-positive puncta was 13–19% larger in fibroblasts and iPSC-derived neurons from individuals with DS, and in basal forebrain cholinergic neurons (BFCN) of the Ts65Dn mice modelling DS. However, EEA1-positive structures imaged by TEM or SR-SIM after chemical fixation had a normal size but appeared clustered. In order to disentangle these discrepancies, we imaged optimally preserved High Pressure Freezing (HPF)-vitrified DS fibroblasts by TEM and found that early endosomes were 75% denser but remained normal-sized.RNA sequencing of DS and euploid fibroblasts revealed a subgroup of differentially-expressed genes related to cargo sorting at multivesicular bodies (MVBs). We thus studied the dynamics of endocytosis, recycling and MVB-dependent degradation in DS fibroblasts. We found no change in endocytosis, increased recycling and delayed degradation, suggesting a “traffic jam” in the endosomal compartment.Finally, we show that the phosphoinositide PI (3) P, involved in early endosome fusion, is decreased in DS fibroblasts, unveiling a new mechanism for endosomal dysfunctions in DS and a target for pharmacotherapy.

Host membrane lipids are trafficked to membranes of intravacuolar bacterium Ehrlichia chaffeensis

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

PmEEA1, the early endosomal protein is employed by YHV for successful infection in Penaeus monodon

Yellow head disease (YHD) is an infectious disease of Penaeus monodon which is caused by the yellow head virus (YHV). YHV infection invariably leads to 100% shrimp mortality within 3–5 days. Currently, an effective method to prevent or cure shrimp from YHV infection has not been elucidated. Therefore, the molecular mechanism underlying YHV infection should be examined. In this study, early endosome antigen 1 (EEA1) protein that was involved in the tethering step of the vesicle and early endosome fusion was investigated during YHV infection. The open reading frame of P. monodon EEA1 (PmEEA1) was cloned and sequenced (3000 bp). It encoded a putative protein of 999 amino acids and contained the zinc finger C2H2 domain signature at the N-terminus and the FYVE domain at the C-terminus. Suppression of PmEEA1 by specific dsRNA in shrimp showed inhibition of YHV replication after 48 h post YHV injection (hpi). On the other hand, shrimp received only NaCl without any dsRNA showed high YHV levels at approximately one hundred thousand times at 24 hpi and 48 hpi. Moreover, silencing of PmEEA1 by specific dsRNA followed by YHV challenge demonstrated a delay in shrimp mortality from 60 hpi to 168 hpi when compared to the control. These results indicated that YHV required PmEEA1 for trafficking within the infected cells, strongly suggesting that PmEEA1 may be a potential target to control and prevent YHV infection in P. monodon.

Role of RAB5A in FSHR-mediated signal transduction in human granulosa cells.

Polycystic ovary syndrome, a common condition characterized by endocrine dysfunction, menstrual irregularity, anovulation and polycystic ovaries, affects 5-7% of reproductive-age women. RAB5B, which is identified by a genome-wide association study as a risk locus for this syndrome, encodes a small GTPase involved in control of receptor internalization and early endosome fusion. We found that RAB5A mRNA levels in luteinized granulosa cells of obese patients with polycystic ovary syndrome were lower than in those of obese women without the syndrome. RAB5A regulated follicle-stimulating hormone (FSH)-mediated translocation of the FSH receptor (FSHR) from the membrane to the cytoplasm and the subsequent FSH-FSHR signaling pathway. We showed that RAB5A negatively regulated aromatase expression and estradiol synthesis in human granulosa cells in association with changes in FSHR levels by way of the cAMP/PKA/CREB pathway. The regulation of FSHR by RAB5A may have been associated with two transcription factors, USF1 and USF2. In conclusion, RAB5A gene was abnormally expressed in luteinized granulosa cells of obese patients with polycystic ovary syndrome, which may help explain high FSHR levels found in this syndrome.

Recognition and stabilization of geranylgeranylated human Rab5 by the GDP Dissociation Inhibitor (GDI)

ABSTRACTThe small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

VPS18 recruits VPS41 to the human HOPS complex via a RING–RING interaction

Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting), to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING (really interesting new gene) domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

Sorting nexin (MoVps17) is required for fungal development and plant infection by regulating endosome dynamics in the rice blast fungus.

Vps17 is a sorting nexin (SNX) and a component of the retromer, a protein complex mediating retrograde vesicle transport between endosomes and the trans-Golgi network. However, its role in the development and pathogenicity of filamentous fungi such as the rice blast fungus (Magnaporthe oryzae) remains unclear. We investigate the functional relationship between the SNX and the cargo-selective complex (CSC) of the fungal retromer by genetic analysis, live cell imaging and immunological assay. Our data show that the MoVps17 null mutation causes defects in growth, development and pathogenicity in M. oryzae. MoVps17 is localized to endosomes depending on the activity of phosphatidylinositol 3-kinase (PI3K), a key enzyme for fungal development and infection. Both PX and BAR domains of MoVps17 are essential for its endosomal localization and function. Furthermore, our yeast two-hybrid assays show that MoVps17 and MoVps5 can interact. Lastly, live cell imaging suggests that MoVps17 can regulate early endosome fusion and budding as well as endocytosis. Taken together, our results suggest that MoVps17 specifically functions as a retromer component with CSC and also plays a distinct role in the regulation of endosome dynamics during fungal development and plant infection.

Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B.

Herpes simplex virus (HSV)-encoded glycoprotein B (gB) is the most abundant protein in the viral envelope and promotes fusion of the virus with the cellular membrane. In the present study, we found that gB impacts on the major histocompatibility complex (MHC)-II pathway of antigen presentation by fostering homotypic fusion of early endosomes and trapping MHC-II molecules in these altered endosomes. By using an overexpression approach, we demonstrated that transient expression of gB induces giant vesicles of early endosomal origin, which contained Rab5, early endosomal antigen 1 (EEA1), and large amounts of MHC-II molecules [human leukocyte antigen (HLA)-DR, and HLA-DM], but no CD63. In HSV-1-infected and stably transfected cell lines that expressed lower amounts of gB, giant endosomes were not observed, but strongly increased amounts of HLA-DR and HLA-DM were found in EEA1+ early endosomes. We used these giant vesicles as a model system and revealed that gB interacts with Rab5 and EEA1, and that gB-induced homotypic fusion of early endosomes to giant endosomes requires phosphatidylinositol 3-phosphate, the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptors, and the cytosolic gB sequence 889YTQVPN894 We conclude that gB expression alters trafficking of molecules of the HLA-II processing pathway, which leads to increased retention of MHC-II molecules in early endosomal compartments, thereby intercepting antigen presentation.-Niazy, N., Temme, S., Bocuk, D., Giesen, C., König, A., Temme, N., Ziegfeld, A., Gregers, T. F., Bakke, O., Lang, T., Eis-Hübinger, A. M., Koch, N. Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B.

ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN, an Interacting Partner of RAB5 GTPases, Regulates Membrane Trafficking to Protein Storage Vacuoles in Arabidopsis.

RAB5 GTPases act as molecular switches that regulate various endosomal functions in animal cells, including homotypic fusion of early endosomes, endosomal motility, endosomal signaling, and subcompartmentalization of the endosomal membrane. RAB5 proteins fulfill these diverse functions through interactions with downstream effector molecules. Two canonical RAB5 members, ARA7 and RAB HOMOLOG1 (RHA1), are encoded in the Arabidopsis thaliana genome. ARA7 and RHA1 play crucial roles in endocytic and vacuolar trafficking pathways. Plant RAB5 GTPases function via interactions with effector molecules, whose identities and functions are currently unclear. In this study, we searched for canonical RAB5 effector molecules of Arabidopsis and identified a candidate, which we called ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN (EREX). The intimate genetic interaction between EREX and RAB5 members, the results from subcellular colocalization experiments, and the direct interaction observed in an in vitro pull-down assay strongly suggest that EREX is a genuine effector of canonical RAB5s in Arabidopsis. We further found that close homologs of EREX play partially redundant functions with EREX in the transport of seed storage proteins. Our results indicate that canonical plant RAB5s acquired distinct effector molecules from those of non-plant systems to fulfill their functions.

Vicenistatin induces early endosome-derived vacuole formation in mammalian cells.

Homotypic fusion of early endosomes is important for efficient protein trafficking and sorting. The key controller of this process is Rab5 which regulates several effectors and PtdInsPs levels, but whose mechanisms are largely unknown. Here, we report that vicenistatin, a natural product, enhanced homotypic fusion of early endosomes and induced the formation of large vacuole-like structures in mammalian cells. Unlike YM201636, another early endosome vacuolating compound, vicenistatin did not inhibit PIKfyve activity in vitro but activated Rab5-PAS pathway in cells. Furthermore, vicenistatin increased the membrane surface fluidity of cholesterol-containing liposomes in vitro, and cholesterol deprivation from the plasma membrane stimulated vicenistatin-induced vacuolation in cells. These results suggest that vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity.

Molecular mechanism of Ras-related protein Rab-5A and effect of mutations in the catalytically active phosphate-binding loop

Ras-related protein (Rab-5a) is primarily involved in the regulation of early endosome fusion during endocytosis and takes part in the budding process. During GTP hydrolysis, Rab5a was spotted in the cytoplasmic side of early endosomes in association with the GTP. Previous study suggested that the substitution of alanine with proline at position 30 of Rab5a reduces the GTPase activity around 12-fold, while, with arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold. Most of the other substitutions at this position show a little or no effect on the GTPase activity. In this paper, structure analysis and molecular dynamics (MD) simulation studies of human Rab5a and its mutants have been extensively carried out. The effect of binding of a non-hydrolyzable GTP analog guanosine-5′-(β, γ)-imidotriphosphate (GppNHp) with Rab5a and its mutants are described. The objective of the current study is to perform a detailed examination of structural flexibility of Rab5a and its mutants p.Ala30Pro and p.Ala30Arg using MD simulations. Our observations suggest that mutant p.Ala30Arg stabilize the protein molecule when bound to GppNHp which offers additional contacts. Despite an in silico approach, this study provides a deep insight into the impact of mutation on the structure, function, stability, and mechanism of binding of GppNHp to the Rab5a at molecular level.

Numb regulates vesicular docking for homotypic fusion of early endosomes via membrane recruitment of Mon1b.

Numb is an endocytic protein that plays crucial roles in diverse cellular processes such as asymmetric cell division, cell migration and differentiation. However, the molecular mechanism by which Numb regulates endocytic trafficking is poorly understood. Here, we demonstrate that Numb is a docking regulator for homotypic fusion of early endosomes (EEs). Numb depletion causes clustered but unfused EEs, which can be rescued by overexpressing cytosolic Numb 65 and Numb 71 but not plasma membrane-attached Numb 66 or Numb 72. Time-lapse analysis reveals that paired vesicles tend to tether but not fuse with each other in the absence of Numb. We further show that Numb binds to another docking regulator, Mon1b, and is required for the recruitment of cytosolic Mon1b to the EE membrane. Consistent with this, deletion of Mon1b causes similar defects in EE fusion. Our study thus identifies a novel mechanism by which Numb regulates endocytic sorting by mediating EE fusion.

Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering.

Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion.

Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion.