Constitutive formation of caveolae in a bacterium.

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.

Macrophages provide a transient muscle stem cell niche via NAMPT secretion.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

Targeted volume correlative light and electron microscopy of an environmental marine microorganism.

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

Smad4 promotes diabetic nephropathy by modulating glycolysis and OXPHOS.

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. TGF-ß1/Smad3 signalling plays a major pathological role in DN; however, the contribution of Smad4 has not been examined. Smad4 depletion in the kidney using anti-Smad4 locked nucleic acid halted progressive podocyte damage and glomerulosclerosis in mouse type 2 DN, suggesting a pathogenic role of Smad4 in podocytes. Smad4 is upregulated in human and mouse podocytes during DN. Conditional Smad4 deletion in podocytes protects mice from type 2 DN, independent of obesity. Mechanistically, hyperglycaemia induces Smad4 localization to mitochondria in podocytes, resulting in reduced glycolysis and oxidative phosphorylation and increased production of reactive oxygen species. This operates, in part, via direct binding of Smad4 to the glycolytic enzyme PKM2 and reducing the active tetrameric form of PKM2. In addition, Smad4 interacts with ATPIF1, causing a reduction in ATPIF1 degradation. In conclusion, we have discovered a mitochondrial mechanism by which Smad4 causes diabetic podocyte injury.

Human Plasminogen Exacerbates Clostridioides difficile Enteric Disease and Alters the Spore Surface.

BACKGROUND & AIMS: The protease plasmin is an important wound healing factor, but it is not clear how it affects gastrointestinal infection-mediated damage, such as that resulting from Clostridioides difficile. We investigated the role of plasmin in C difficile-associated disease. This bacterium produces a spore form that is required for infection, so we also investigated the effects of plasmin on spores. METHODS: C57BL/6J mice expressing the precursor to plasmin, the zymogen human plasminogen (hPLG), or infused with hPLG were infected with C difficile, and disease progression was monitored. Gut tissues were collected, and cytokine production and tissue damage were analyzed by using proteomic and cytokine arrays. Antibodies that inhibit either hPLG activation or plasmin activity were developed and structurally characterized, and their effects were tested in mice. Spores were isolated from infected patients or mice and visualized using super-resolution microscopy; the functional consequences of hPLG binding to spores were determined. RESULTS: hPLG localized to the toxin-damaged gut, resulting in immune dysregulation with an increased abundance of cytokines (such as interleukin [IL] 1A, IL1B, IL3, IL10, IL12B, MCP1, MP1A, MP1B, GCSF, GMCSF, KC, TIMP-1), tissue degradation, and reduced survival. Administration of antibodies that inhibit plasminogen activation reduced disease severity in mice. C difficile spores bound specifically to hPLG and active plasmin degraded their surface, facilitating rapid germination. CONCLUSIONS: We found that hPLG is recruited to the damaged gut, exacerbating C difficile disease in mice. hPLG binds to C difficile spores, and, upon activation to plasmin, remodels the spore surface, facilitating rapid spore germination. Inhibitors of plasminogen activation might be developed for treatment of C difficile or other infection-mediated gastrointestinal diseases.

Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination.

We previously identified that ngr1 allele deletion limits the severity of experimental autoimmune encephalomyelitis (EAE) by preserving axonal integrity. However, whether this favorable outcome observed in EAE is a consequence of an abrogated neuronal-specific pathophysiological mechanism, is yet to be defined. Here we show that, Cre-loxP-mediated neuron-specific deletion of ngr1 preserved axonal integrity, whereas its re-expression in ngr1-/- female mice potentiated EAE-axonopathy. As a corollary, myelin integrity was preserved under Cre deletion in ngr1flx/flx , retinal ganglion cell axons whereas, significant demyelination occurred in the ngr1-/- optic nerves following the re-introduction of NgR1. Moreover, Cre-loxP-mediated axon-specific deletion of ngr1 in ngr1flx/flx mice also demonstrated efficient anterograde transport of fluorescently-labeled ChTxß in the optic nerves of EAE-induced mice. However, the anterograde transport of ChTxß displayed accumulation in optic nerve degenerative axons of EAE-induced ngr1-/- mice, when NgR1 was reintroduced but was shown to be transported efficiently in the contralateral non- recombinant adeno-associated virus serotype 2-transduced optic nerves of these mutant mice. We further identified that the interaction between the axonal motor protein, Kinesin-1 and collapsin response mediator protein 2 (CRMP2) was unchanged upon Cre deletion of ngr1 Whereas, this Kinesin-1/CRMP2 association was reduced when NgR1 was re-expressed in the ngr1-/- optic nerves. Our data suggest that NgR1 governs axonal degeneration in the context of inflammatory-mediated demyelination through the phosphorylation of CRMP2 by stalling axonal vesicular transport. Moreover, axon-specific deletion of ngr1 preserves axonal transport mechanisms, blunting the induction of inflammatory demyelination and limiting the severity of EAE.SIGNIFICANCE STATEMENT Multiple sclerosis (MS) is commonly induced by aberrant immune-mediated destruction of the protective sheath of nerve fibers (known as myelin). However, it has been shown that MS lesions do not only consist of this disease pattern, exhibiting heterogeneity with continual destruction of axons. Here we investigate how neuronal NgR1 can drive inflammatory-mediated axonal degeneration and demyelination within the optic nerve by analyzing its downstream signaling events that govern axonal vesicular transport. We identify that abrogating the NgR1/pCRMP2 signaling cascade can maintain Kinesin-1-dependent anterograde axonal transport to limit inflammatory-mediated axonopathy and demyelination. The ability to differentiate between primary and secondary mechanisms of axonal degeneration may uncover therapeutic strategies to limit axonal damage and progressive MS.

Efficient Transmission Electron Microscopy Characterization of Cell-Nanostructure Interfacial Interactions.

Engineered nano-bio interfaces driven by tunable vertically configured nanostructures have recently emerged as a powerful tool for cellular manipulations and interrogations. Yet the interplay between substrate topography and cellular behavior is highly complex and not fully understood. A new experimental design is proposed that enables generation of ultrathin sections (lamellae) of cell-nanostructure imprints with minimal artifacts. We demonstrate the potential of such lamellae for efficient transmission electron microscopy (TEM) characterization of interfacial interactions between adherent cells and vertically aligned Si nanostructures. This approach will advance understanding of cellular responses to extracellular biophysical and biochemical cues-which is likely to facilitate the design of improved cellular manipulation technologies.

FLNC myofibrillar myopathy results from impaired autophagy and protein insufficiency.

Myofibrillar myopathy is a progressive muscle disease characterized by the disintegration of muscle fibers and formation of protein aggregates. Causative mutations have been identified in nine genes encoding Z-disk proteins, including the actin binding protein filamin C (FLNC). To investigate the mechanism of disease in FLNCW2710X myopathy we overexpressed fluorescently tagged FLNC or FLNCW2710X in zebrafish. Expression of FLNCW2710X causes formation of protein aggregates but surprisingly, our studies reveal that the mutant protein localizes correctly to the Z-disk and is capable of rescuing the fiber disintegration phenotype that results from FLNC knockdown. This demonstrates that the functions necessary for muscle integrity are not impaired, and suggests that it is the formation of protein aggregates and subsequent sequestration of FLNC away from the Z-disk that results in myofibrillar disintegration. Similar to those found in patients, the aggregates in FLNCW2710X expressing fish contain the co-chaperone BAG3. FLNC is a target of the BAG3-mediated chaperone assisted selective autophagy (CASA) pathway and therefore we investigated its role, and the role of autophagy in general, in clearing protein aggregates. We reveal that despite BAG3 recruitment to the aggregates they are not degraded via CASA. Additionally, recruitment of BAG3 is sufficient to block alternative autophagy pathways which would otherwise clear the aggregates. This blockage can be relieved by reducing BAG3 levels or by stimulating autophagy. This study therefore identifies both BAG3 reduction and autophagy promotion as potential therapies for FLNCW2710X myofibrillar myopathy, and identifies protein insufficiency due to sequestration, compounded by impaired autophagy, as the cause.

Cathepsin D and its newly identified transport receptor SEZ6L2 can modulate neurite outgrowth.

How, in the absence of a functional mannose 6-phosphate (Man-6-P)-signal-dependent transport pathway, some acid hydrolases remain sorted to endolysosomes in the brain is poorly understood. We demonstrate that cathepsin D binds to mouse SEZ6L2, a type 1 transmembrane protein predominantly expressed in the brain. Studies of the subcellular trafficking of SEZ6L2, and its silencing in a mouse neuroblastoma cell line reveal that SEZ6L2 is involved in the trafficking of cathepsin D to endosomes. Moreover, SEZ6L2 can partially correct the cathepsin D hypersecretion resulting from the knockdown of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase in HeLa cells (i.e. in cells that are unable to synthesize Man-6-P signals). Interestingly, cleavage of SEZ6L2 by cathepsin D generates an N-terminal soluble fragment that induces neurite outgrowth, whereas its membrane counterpart prevents this. Taken together, our findings highlight that SEZ6L2 can serve as receptor to mediate the sorting of cathepsin D to endosomes, and suggest that proteolytic cleavage of SEZ6L2 by cathepsin D modulates neuronal differentiation.

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.

Termination of autophagy and reformation of lysosomes regulated by mTOR.

Autophagy is an evolutionarily conserved process by which cytoplasmic proteins and organelles are catabolized. During starvation, the protein TOR (target of rapamycin), a nutrient-responsive kinase, is inhibited, and this induces autophagy. In autophagy, double-membrane autophagosomes envelop and sequester intracellular components and then fuse with lysosomes to form autolysosomes, which degrade their contents to regenerate nutrients. Current models of autophagy terminate with the degradation of the autophagosome cargo in autolysosomes, but the regulation of autophagy in response to nutrients and the subsequent fate of the autolysosome are poorly understood. Here we show that mTOR signalling in rat kidney cells is inhibited during initiation of autophagy, but reactivated by prolonged starvation. Reactivation of mTOR is autophagy-dependent and requires the degradation of autolysosomal products. Increased mTOR activity attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell-a process we identify in multiple animal species. Thus, an evolutionarily conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation.

The HOPS proteins hVps41 and hVps39 are required for homotypic and heterotypic late endosome fusion.

The homotypic fusion and protein sorting (HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo-lysosomal pathway of human cells. By expressing hemagglutinin (HA)-tagged constructs, we show by immunoelectron microscopy (immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)-mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal-lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells.

Apoptosis-linked gene-2 (ALG-2)/Sec31 interactions regulate endoplasmic reticulum (ER)-to-Golgi transport: a potential effector pathway for luminal calcium.

Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery.

Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function.

Nemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.

The Sec7 guanine nucleotide exchange factor GBF1 regulates membrane recruitment of BIG1 and BIG2 guanine nucleotide exchange factors to the trans-Golgi network (TGN).

Three Sec7 guanine nucleotide exchange factors (GEFs) activate ADP-ribosylation factors (ARFs) to facilitate coating of transport vesicles within the secretory and endosomal pathways. GBF1 recruits COPI to pre-Golgi and Golgi compartments, whereas BIG1 and BIG2 recruit AP1 and GGA clathrin adaptors to the trans-Golgi network (TGN) and endosomes. Here, we report a functional cascade between these GEFs by showing that GBF1-activated ARFs (ARF4 and ARF5, but not ARF3) facilitate BIG1 and BIG2 recruitment to the TGN. We localize GBF1 ultrastructurally to the pre-Golgi, the Golgi, and also the TGN. Our findings suggest a model in which GBF1 localized within pre-Golgi and Golgi compartments mediates ARF activation to facilitate recruitment of COPI to membranes, whereas GBF1 localized at the TGN mediates ARF activation that leads to the recruitment of BIG1 and BIG2 to the TGN. Membrane-associated BIG1/2 then activates ARFs that recruit clathrin adaptors. In this cascade, an early acting GEF (GBF1) activates ARFs that mediate recruitment of late acting GEFs (BIG1/2) to coordinate coating events within the pre-Golgi/Golgi/TGN continuum. Such coordination may optimize the efficiency and/or selectivity of cargo trafficking through the compartments of the secretory pathway.

Zebrafish models of BAG3 myofibrillar myopathy suggest a toxic gain of function leading to BAG3 insufficiency.

Mutations in the co-chaperone Bcl2-associated athanogene 3 (BAG3) can cause myofibrillar myopathy (MFM), a childhood-onset progressive muscle disease, characterized by the formation of protein aggregates and myofibrillar disintegration. In contrast to other MFM-causing proteins, BAG3 has no direct structural role, but regulates autophagy and the degradation of misfolded proteins. To investigate the mechanism of disease in BAG3-related MFM, we expressed wild-type BAG3 or the dominant MFM-causing BAG3 (BAG3(P209L)) in zebrafish. Expression of the mutant protein results in the formation of aggregates that contain wild-type BAG3. Through the stimulation and inhibition of autophagy, we tested the prevailing hypothesis that impaired autophagic function is responsible for the formation of protein aggregates. Contrary to the existing theory, our studies reveal that inhibition of autophagy is not sufficient to induce protein aggregation. Expression of the mutant protein, however, did not induce myofibrillar disintegration and we therefore examined the effect of knocking down Bag3 function. Loss of Bag3 resulted in myofibrillar disintegration, but not in the formation of protein aggregates. Remarkably, BAG3(P209L) is able to rescue the myofibrillar disintegration phenotype, further demonstrating that its function is not impaired. Together, our knockdown and overexpression experiments identify a mechanism whereby BAG3(P209L) aggregates form, gradually reducing the pool of available BAG3, which eventually results in BAG3 insufficiency and myofibrillar disintegration. This mechanism is consistent with the childhood onset and progressive nature of MFM and suggests that reducing aggregation through enhanced degradation or inhibition of nucleation would be an effective therapy for this disease.

Basal spot junctions of Drosophila epithelial tissues respond to morphogenetic forces and regulate Hippo signaling.

Organ size is controlled by numerous factors including mechanical forces, which are mediated in part by the Hippo pathway. In growing Drosophila epithelial tissues, cytoskeletal tension influences Hippo signaling by modulating the localization of key pathway proteins to different apical domains. Here, we discovered a Hippo signaling hub at basal spot junctions, which form at the basal-most point of the lateral membranes and resemble adherens junctions in protein composition. Basal spot junctions recruit the central kinase Warts via Ajuba and E-cadherin, which prevent Warts activation by segregating it from upstream Hippo pathway proteins. Basal spot junctions are prominent when tissues undergo morphogenesis and are highly sensitive to fluctuations in cytoskeletal tension. They are distinct from focal adhesions, but the latter profoundly influences basal spot junction abundance by modulating the basal-medial actomyosin network and tension experienced by spot junctions. Thus, basal spot junctions couple morphogenetic forces to Hippo pathway activity and organ growth.

Disruption of the Man-6-P targeting pathway in mice impairs osteoclast secretory lysosome biogenesis.

Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo-lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain two types of lysosomes. The major species is a secretory lysosome containing cathepsin K and tartrate-resistant acid phosphatase (TRAP), two hydrolases critical for bone resorption. These secretory lysosomes are shown to fuse with the plasma membrane, allowing the regulated release of acid hydrolases at the site of bone resorption. The other type of lysosome contains cathepsin D, but little cathepsin K or TRAP. Osteoclasts from Gnptab(-/-) (gene encoding GlcNAc-1-phosphotransferase α, ß-subunits) mice, which lack a functional mannose 6-phosphate (Man-6-P) targeting pathway, show increased secretion of cathepsin K and TRAP and impaired secretory lysosome formation. However, cathepsin D targeting was intact, showing that osteoclasts have a Man-6-P-independent pathway for selected acid hydrolases.

Lysosomal membrane protein composition, acidic pH and sterol content are regulated via a light-dependent pathway in metazoan cells.

In metazoans, lysosomes are characterized by a unique tubular morphology, acidic pH, and specific membrane protein (LAMP) and lipid (cholesterol) composition as well as a soluble protein (hydrolases) composition. Here we show that perturbation to the eye-color gene, light, results in impaired lysosomal acidification, sterol accumulation, altered endosomal morphology as well as compromised lysosomal degradation. We find that Drosophila homologue of Vps41, Light, regulates the fusion of a specific subset of biosynthetic carriers containing characteristic endolysosomal membrane proteins, LAMP1, V0-ATPase and the cholesterol transport protein, NPC1, with the endolysosomal system, and is then required for the morphological progression of the multivesicular endosome. Inhibition of Light results in accumulation of biosynthetic transport intermediates that contain these membrane cargoes, whereas under similar conditions, endosomal delivery of soluble hydrolases, previously shown to be mediated by Dor, the Drosophila homologue of Vps18, is not affected. Unlike Dor, Light is recruited to endosomes in a PI3P-sensitive fashion wherein it facilitates fusion of these biosynthetic cargoes with the endosomes. Depletion of the mammalian counterpart of Light, hVps41, in a human cell line also inhibits delivery of hLAMP to endosomes, suggesting an evolutionarily conserved pathway in metazoa.

Neuron specific Rab4 effector GRASP-1 coordinates membrane specialization and maturation of recycling endosomes.

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.