The novel lipid raft adaptor p18 controls endosome dynamics by anchoring the MEK–ERK pathway to late endosomes

The regulation of endosome dynamics is crucial for fundamental cellular functions, such as nutrient intake/digestion, membrane protein cycling, cell migration and intracellular signalling. Here, we show that a novel lipid raft adaptor protein, p18, is involved in controlling endosome dynamics by anchoring the MEK1–ERK pathway to late endosomes. p18 is anchored to lipid rafts of late endosomes through its N‐terminal unique region. p18^?/? mice are embryonic lethal and have severe defects in endosome/lysosome organization and membrane protein transport in the visceral endoderm. p18^?/? cells exhibit apparent defects in endosome dynamics through perinuclear compartment, such as aberrant distribution and/or processing of lysosomes and impaired cycling of Rab11‐positive recycling endosomes. p18 specifically binds to the p14–MP1 complex, a scaffold for MEK1. Loss of p18 function excludes the p14–MP1 complex from late endosomes, resulting in a downregulation of the MEK–ERK activity. These results indicate that the lipid raft adaptor p18 is essential for anchoring the MEK–ERK pathway to late endosomes, and shed new light on a role of endosomal MEK–ERK pathway in controlling endosome dynamics.     

Atg5 regulates late endosome and lysosome biogenesis

Autophagy is an evolutionarily conserved lysosome-based degradation process.Atg5 plays a very important role in autophagosome formation.Here we show that Atg5 is required for biogenesis of late endosomes and lysosomes in an autophagy-independent manner.In Atg5 cells,but not in other essential autophagy genes defecting cells,recycling and retrieval of late endosomal components from hybrid organelles are impaired,causing persistent hybrid organelles and defective formation of late endosomes and lysosomes.Defective retrieval of late endosomal components from hybrid organelles resulting from impaired recruitment of a component of V1-ATPase to acidic organelles blocks the pH-dependent retrieval of late endosomal components from hybrid organelles.Lowering the intracellular pH restores late endosome/lysosome biogenesis in Atg5 cells.Our data demonstrate an unexpected role of Atg5 and shed new light on late endosome and lysosome biogenesis.     

Syntaxin 7 and VAMP-7 are soluble N-ethylmaleimide-sensitive factor attachment protein receptors required for late endosome-lysosome and homotypic lysosome fusion in alveolar macrophages

Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome-lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome-lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome-lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome-lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.     

Rabring7, a novel Rab7 target protein with a RING finger motif

Rab7, a member of the Rab family small G proteins, has been shown to regulate intracellular vesicle traffic to late endosome/lysosome and lysosome biogenesis, but the exact roles of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins that function in the exocytic/endocytic pathway. We have isolated a new Rab7 target protein, Rabring7 (Rab7-interacting RING finger protein), using a CytoTrap system. It contains an H2 type RING finger motif at the C termini. Rabring7 shows no homology with RILP, which has been reported as another Rab7 target protein. GST pull-down and coimmunoprecipitation assays demonstrate that Rabring7 specifically binds the GTP-bound form of Rab7 at the N-terminal portion. Rabring7 is found mainly in the cytosol and is recruited efficiently to late endosomes/lysosomes by the GTP-bound form of Rab7 in BHK cells. Overexpression of Rabring7 not only affects epidermal growth factor degradation but also causes the perinuclear aggregation of lysosomes, in which the accumulation of the acidotropic probe LysoTracker is remarkably enhanced. These results suggest that Rabring7 plays crucial roles as a Rab7 target protein in vesicle traffic to late endosome/lysosome and lysosome biogenesis.     

Kinesin-2 is a motor for late endosomes and lysosomes

The bidirectional nature of late endosome/lysosome movement suggests involvement of at least two distinct motors, one minus-end directed and one plus-end directed. Previous work has identified dynein as the minus-end-directed motor for late endosome/lysosome localization and dynamics. Conventional kinesin (kinesin-1) has been implicated in plus-end-directed late endosome/lysosome movement, but other kinesin family members may also be involved. Kinesin-2 is known to drive the movement of pigment granules, a type of lysosomally derived organelle, and was recently found to be associated with purified late endosomes. To determine whether kinesin-2 might also power endosome movement in non-pigmented cells, we overexpressed dominant negative forms of the KIF3A motor subunit and KAP3 accessory subunit and knocked down KAP3 levels using RNAi. We found kinesin-2 to be required for the normal steady-state localization of late endosomes/lysosomes but not early endosomes or recycling endosomes. Despite the abnormal subcellular distribution of late endosomes/lysosomes, the uptake and trafficking of molecules through the conventional endocytic pathway appeared to be unaffected. The slow time-course of inhibition suggests that both kinesin-2 itself and its attachment to membranes do not turn over quickly.     

Early endosomes and endosomal coatomer are required for autophagy

Autophagy, an intracellular degradative pathway, maintains cell homeostasis under normal and stress conditions. Nascent double-membrane autophagosomes sequester and enclose cytosolic components and organelles, and subsequently fuse with the endosomal pathway allowing content degradation. Autophagy requires fusion of autophagosomes with late endosomes, but it is not known if fusion with early endosomes is essential. We show that fusion of AVs with functional early endosomes is required for autophagy. Inhibition of early endosome function by loss of COPI subunits (β′, β, or α) results in accumulation of autophagosomes, but not an increased autophagic flux. COPI is required for ER-Golgi transport and early endosome maturation. Although loss of COPI results in the fragmentation of the Golgi, this does not induce the formation of autophagosomes. Loss of COPI causes defects in early endosome function, as both transferrin recycling and EGF internalization and degradation are impaired, and this loss of function causes an inhibition of autophagy, an accumulation of p62/SQSTM-1, and ubiquitinated proteins in autophagosomes.     

The role of mVps18p in clustering, fusion, and intracellular localization of late endocytic organelles

Delivery of endocytosed macromolecules to mammalian cell lysosomes occurs by direct fusion of late endosomes with lysosomes, resulting in the formation of hybrid organelles from which lysosomes are reformed. The molecular mechanisms of this fusion are analogous to those of homotypic vacuole fusion in Saccharomyces cerevisiae. We report herein the major roles of the mammalian homolog of yeast Vps18p (mVps18p), a member of the homotypic fusion and vacuole protein sorting complex. When overexpressed, mVps18p caused the clustering of late endosomes/lysosomes and the recruitment of other mammalian homologs of the homotypic fusion and vacuole protein sorting complex, plus Rab7-interacting lysosomal protein. The clusters were surrounded by components of the actin cytoskeleton, including actin, ezrin, and specific unconventional myosins. Overexpression of mVps18p also overcame the effect of wortmannin treatment, which inhibits membrane traffic out of late endocytic organelles and causes their swelling. Reduction of mVps18p by RNA interference caused lysosomes to disperse away from their juxtanuclear location. Thus, mVps18p plays a critical role in endosome/lysosome tethering, fusion, intracellular localization and in the reformation of lysosomes from hybrid organelles.     

Rab9 GTPase regulates late endosome size and requires effector interaction for its stability

Rab9 GTPase resides in a late endosome microdomain together with mannose 6-phosphate receptors (MPRs) and the tail-interacting protein of 47 kDa (TIP47). To explore the importance of Rab9 for microdomain establishment, we depleted the protein from cultured cells. Rab9 depletion decreased late endosome size and reduced the numbers of multilamellar and dense-tubule-containing late endosomes/lysosomes, but not multivesicular endosomes. The remaining late endosomes and lysosomes were more tightly clustered near the nucleus, implicating Rab9 in endosome localization. Cells displayed increased surface MPRs and lysosome-associated membrane protein 1. In addition, cells showed increased MPR synthesis in conjunction with MPR missorting to the lysosome. Surprisingly, Rab9 stability on late endosomes required interaction with TIP47. Rabs are thought of as independent, prenylated entities that reside either on membranes or in cytosol, bound to GDP dissociation inhibitor. These data show that Rab9 stability is strongly influenced by a specific effector interaction. Moreover, Rab9 and the proteins with which it interacts seem critical for the maintenance of specific late endocytic compartments and endosome/lysosome localization.     

Snx10 and PIKfyve are required for lysosome formation in osteoclasts

Bone resorption and organelle homeostasis in osteoclasts require specialized intracellular trafficking. Sorting nexin 10 (Snx10) is a member of the sorting nexin family of proteins that plays crucial roles in cargo sorting in the endosomal pathway by its binding to phosphoinositide(3)phosphate (PI3P) localized in early endosomes. We and others have shown previously that the gene encoding sorting Snx10 is required for osteoclast morphogenesis and function, as osteoclasts from humans and mice lacking functional Snx10 are dysfunctional. To better understand the role and mechanisms by which Snx10 regulates vesicular transport, the aim of the present work was to study PIKfyve, another PI3P-binding protein, which phosphorylates PI3P to PI(3,5)P2. PI(3,5)P2 is known to be required for endosome/lysosome maturation, and the inhibition of PIKfyve causes endosome enlargement. Overexpression of Snx10 also induces accumulation of early endosomes suggesting that both Snx10 and PIKfyve are required for normal endosome/lysosome transition. Apilimod is a small molecule with specific, nanomolar inhibitory activity on PIKfyve but only in the presence of key osteoclast factors CLCN7, OSTM1, and Snx10. This observation suggests that apilimod's inhibitory effects are mediated by endosome/lysosome disruption. Here we show that both Snx10 and PIKfyve colocalize to early endosomes in osteoclasts and coimmunoprecipitate in vesicle fractions. Treatment with 10 nM apilimod or genetic deletion of PIKfyve in cells resulted in the accumulation of early endosomes, and in the inhibition of osteoclast differentiation, lysosome formation, and secretion of TRAP from differentiated osteoclasts. Snx10 and PIKfyve also colocalized in gastric zymogenic cells, another cell type impacted by Snx10 mutations. Apilimod-specific inhibition of PIKfyve required Snx10 expression, as it did not inhibit lysosome biogenesis in Snx10-deficient osteoclasts. These findings suggest that Snx10 and PIKfyve are involved in the regulation of endosome/lysosome homeostasis via the synthesis of PI(3,5)P2 and may point to a new strategy to prevent bone loss.     

Vaccinia Virus Infection Requires Maturation of Macropinosomes

The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live‐cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome‐associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow‐up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late‐penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration.      

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system

The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. In conclusion: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.     

The Rab7 effector WDR91 promotes autophagy-lysosome degradation in neurons by regulating lysosome fusion

The effectors of the Rab7 small GTPase play multiple roles in Rab7-dependent endosome-lysosome and autophagy-lysosome pathways. However, it is largely unknown how distinct Rab7 effectors coordinate to maintain the homeostasis of late endosomes and lysosomes to ensure appropriate endolysosomal and autolysosomal degradation. Here we report that WDR91, a Rab7 effector required for early-to-late endosome conversion, is essential for lysosome function and homeostasis. Mice lacking Wdr91 specifically in the central nervous system exhibited behavioral defects and marked neuronal loss in the cerebral and cerebellar cortices. At the cellular level, WDR91 deficiency causes PtdIns3P-independent enlargement and dysfunction of lysosomes, leading to accumulation of autophagic cargoes in mouse neurons. WDR91 competes with the VPS41 subunit of the HOPS complex, another Rab7 effector, for binding to Rab7, thereby facilitating Rab7-dependent lysosome fusion in a controlled manner. WDR91 thus maintains an appropriate level of lysosome fusion to guard the normal function and survival of neurons.     

Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.     

Quantitative analysis of male germline stem cell differentiation reveals a role for the p53-mTORC1 pathway in spermatogonial maintenance

p53 protects cells from DNA damage by inducing cell-cycle arrest upon encountering genomic stress. Among other pathways, p53 elicits such an effect by inhibiting mammalian target of rapamycin complex 1 (mTORC1), the master regulator of cell proliferation and growth. Although recent studies have indicated roles for both p53 and mTORC1 in stem cell maintenance, it remains unclear whether the p53-mTORC1 pathway is conserved to mediate this process under normal physiological conditions. Spermatogenesis is a classic stem cell-dependent process in which undifferentiated spermatogonia undergo self-renewal and differentiation to maintain the lifelong production of spermatozoa. To better understand this process, we have developed a novel flow cytometry (FACS)-based approach that isolates spermatogonia at consecutive differentiation stages. By using this as a tool, we show that genetic loss of p53 augments mTORC1 activity during early spermatogonial differentiation. Functionally, loss of p53 drives spermatogonia out of the undifferentiated state and causes a consistent expansion of early differentiating spermatogonia until the stage of preleptotene (premeiotic) spermatocyte. The frequency of early meiotic spermatocytes is, however, dramatically decreased. Thus, these data suggest that p53-mTORC1 pathway plays a critical role in maintaining the homeostasis of early spermatogonial differentiation. Moreover, our FACS approach could be a valuable tool in understanding spermatogonial differentiation.     

A vesicle surface tyrosine kinase regulates phagosome maturation

Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle-associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation.     

PI3P signaling regulates receptor sorting but not transport in the endosomal pathway

While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.     

A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments

Rab5 is a small GTPase that plays roles in the homotypic fusion of early endosomes and regulation of intracellular vesicle transport. We show here that expression of GFP-tagged GTPase-deficient form of Rab5b (Rab5bQ79L) in NRK cells results in the sequential formation of three morphologically and functionally distinct types of endosomes. Expression of GFP-Rab5bQ79L initially caused a homotypic fusion of early endosomes accompanying a redistribution of the TGN-resident cargo molecules, and subsequent fusion with late endosomes/lysosomes, leading to the formation of giant hybrid organelles with features of early endosomes and late endosomes/lysosomes. Surprisingly, the giant endosomes gradually fragmented and shrunk, leading to the accumulation of early endosome clusters and concurrent reformation of late endosomes/lysosomes, a process accelerated by treatment with a phosphatidylinositol-3-kinase (PI(3)K) inhibitor, wortmannin. We postulate that such sequential processes reflect the biogenesis and maintenance of late endosomes/lysosomes, presumably via direct fusion with early endosomes and subsequent fission from hybrid organelles. Thus, our findings suggest a regulatory role for Rab5 in not only the early endocytic pathway, but also the late endocytic pathway, of membrane trafficking in coordination with PI(3)K activity.     

Vps33B is required for delivery of endocytosed cargo to lysosomes

Lysosomes are the main degradative compartments of eukaryotic cells. The CORVET and HOPS tethering complexes are well known for their role in membrane fusion in the yeast endocytic pathway. Yeast Vps33p is part of both complexes, and has two mammalian homologues: Vps33A and Vps33B. Vps33B is required for recycling of apical proteins in polarized cells and a causative gene for ARC syndrome. Here, we investigate whether Vps33B is also required in the degradative pathway. By fluorescence and electron microscopy we show that Vps33B depletion in HeLa cells leads to significantly increased numbers of late endosomes that together with lysosomes accumulate in the perinuclear region. Degradation of endocytosed cargo is impaired in these cells. By electron microscopy we show that endocytosed BSA‐gold reaches late endosomes, but is decreased in lysosomes. The increase in late endosome numbers and the lack of internalized cargo in lysosomes are indicative for a defect in late endosomal–lysosomal fusion events, which explains the observed decrease in cargo degradation. A corresponding phenotype was found after Vps33A knock down, which in addition also resulted in decreased lysosome numbers. We conclude that Vps33B, in addition to its role in endosomal recycling, is required for late endosomal–lysosomal fusion events.      

GSK-3-TSC axis governs lysosomal acidification through autophagy and endocytic pathways

Impaired lysosomal activity, which results in defective protein processing, waste accumulation, and protein aggregation, is implicated in a number of disease pathologies. Acidification of lysosomes is a crucial process required for lysosome function. Previously we showed that inhibition of glycogen synthase kinase-3 (GSK-3) enhanced lysosomal acidification in both normal and pathological conditions. However, how GSK-3 integrates into the lysosome networking is unknown. Here we show that inhibition of mTORC1 and increased autophagic activity are downstream to GSK-3 inhibition and contribute to lysosomal acidification. Strikingly, lysosomal acidification is also restored by GSK-3 inhibition in the absence of functional autophagy, and, independently of mTORC1. This is facilitated by increased endocytic traffic: We show that GSK-3 inhibition enhanced material internalization, increased recruitment of active Rab5 into endosomes, and increased Rab7/RILP clustering into lysosomes, all processes required for late endosome maturation. Consistently, in cells defective in endocytic traffic caused by either constitutively active Rab5, or, deletion of the Niemann-Pick C1 protein, GSK-3 inhibition could not restore lysosomal acidification. Finally we found that the tuberous sclerosis complex, TSC, is required for lysosomal acidification and is activated by GSK-3 inhibition. Thus, the GSK-3/TSC axis regulates lysosomal acidification via both the autophagic and endocytic pathways. Our study provides new insights into the therapeutic potential of GSK-3 inhibitors in treating pathological conditions associated with impaired cellular clearance.