Axon guidance (pathfinding) wires the brain during development and is regulated by various attractive and repulsive cues. Semaphorin 3A (Sema3A) is a repulsive cue, inducing the collapse of axon growth cones. In the mammalian forebrain, the corpus callosum is the major commissure that transmits information flow between the two hemispheres, and contralateral axons assemble into well-defined tracts. We found that the patterning of callosal axon projections in rodent layer II and III (L2/3) cortical neurons in response to Sema3A was mediated by the activation of Rab5, a small guanosine triphosphatase (GTPase) that mediates endocytosis, through the membrane fusion protein Rabaptin-5 and the Rab5 guanine nucleotide exchange factor (GEF) Rabex-5. Rabaptin-5 bound directly to Plexin-A1 in the Sema3A receptor complex [an obligate heterodimer formed by Plexin-A1 and neuropilin 1 (NP1)]; Sema3A enhanced this interaction in cultured neurons. Rabaptin-5 bridged the interaction between Rab5 and Plexin-A1. Sema3A stimulated endocytosis from the cell surface of callosal axon growth cones. In utero electroporation to reduce Rab5 or Rabaptin-5 impaired axon fasciculation or caused mistargeting of L2/3 callosal projections in rats. Overexpression of Rabaptin-5 or Rab5 rescued the defective callosal axon fasciculation or mistargeting of callosal axons caused by the loss of Sema3A-Plexin-A1 signaling in rats expressing dominant-negative Plexin-A1 or in NP1-deficient mice. Thus, our findings suggest that Rab5, its effector Rabaptin-5, and its regulator Rabex-5 mediate Sema3A-induced axon guidance during brain development.

Transcellular transport is essential for transmucosal and plasma-to-tissue drug delivery by nanoparticles, whereas its fundamental pathways have not been fully clarified. In this study, an in-depth investigation was conducted into the intracellular itinerary and the transcytosis pathway of wheat germ agglutinin-functionalized nanoparticles (WGA-NP) with various polymer architectures in the Caco-2 cell model. GFP-Rabs, Rab4, Rab5, Rab7, Rab11, GTPases served as key regulators of vesicular transport, and their mutants were transfected to Caco-2 cells respectively to determine the cellular itinerary of WGA-NP and the role of Rabs therein. Transcytosis inhibition experiments indicated that transcellular transport of WGA-NP (PEG(3000)-PLA(40000) formulation) happened in a cytoskeleton-dependent manner and majorly by means of clathrin-mediated mechanism. Intracellular transport, especially the endolysosome pathway was found largely contribute to the transcytosis of WGA-NP. WGA-NP with shorter surface PEG length (2000) resulted in higher cellular association and more colocalization with the clathrin-mediated transport pathway, while that with longer surface PEG length (5000) avoided the clathrin-mediated transport pathway but achieved higher transcytosis after 4 h incubation. WGA-NP with PLGA as the core materials obtained elevated lysosome escape and enhanced transcytosis after 2 h incubation. These findings provided important evidence for the role of polymer architectures in modulating cellular transport of functionalized nanocarriers, and would be helpful in improving carrier design to enhance drug delivery.

We have investigated apolipoprotein E (apoE) recycling in Chinese hamster ovary (CHO) cells, a peripheral cell that does not produce lipoproteins or express apoE. Using a pulse-chase protocol in which cells were pulsed with 125I-apoE-VLDL and chased for different periods, approximately 30% of the apoE internalized during the pulse was resecreted within a 4 h chase in a relatively lipid-free state. The addition of lysosomotropic agents or brefeldin A had no effect on apoE recycling. Unlike previous results with hepatocytes and macrophages, neither apoA-I nor upregulation of ABCA1 stimulated apoE recycling. However, cyclodextrin, which extracts cholesterol from plasma membrane lipid rafts, increased recycling. Confocal studies revealed that apoE, internalized during a 1 h pulse, colocalizes with early endosomal antigen-1, Rab5, Rab11a, and lysobisphosphatidic acid but not with lysosomal-associated membrane protein-1. Colocalization of apoE and Rab11a persisted even after cells had been chased for 1 h, suggesting a pool of apoE within the endosomal recycling compartment (ERC). Our data suggest that apoE recycling in CHO cells is linked to cellular cholesterol removal via the ERC and phospholipid-containing acceptors in a pathway alternative to the ABCA1-apoA-I axis.

Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.

Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5-positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

BACKGROUND

Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined.

METHODS

ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol.

RESULTS

Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry.

CONCLUSIONS

The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

Hepatic endocytosis is characterized by a division of labor between the different cell types with respect to endocytosis, which is mediated by receptors expressed on their cell surface. We have investigated the expression of GTPases of the rab family in rat liver parenchymal and endothelial cells. Small GTPases of the rab protein family control distinct steps of intracellular transport both in the secretory and the endocytic pathway. As controls have been employed the normal rat kidney (NRK) cell line and brain tissue, neuronal cells are known to express high levels of components of the endocytic machinery (clathrin, adaptins, dynamin, uncoating adenosine triphosphatase, etc.). Endothelial cells were found to express four to seven times more rab4, rab5, and rab7 than parenchymal cells. A similar relationship was found between the endocytic rates in the two cell types; the rate of internalization from the plasma membrane of mannose receptors in rat liver endothelial cells was 2.3 pools/min, whereas the corresponding value for internalization of the galactose receptor in parenchymal liver cell was 0.27 pools/min (comparable with the rate of transferrin internalization in NRK cells). Both immunofluorescence and subcellular fractionation experiments showed that rab5 and rab7 were associated with compartments along the endocytic pathway. Brain tissue showed a similar high expression of endocytic components (rab4, rab5, and rab7) as liver endothelial cells, whereas lower values were found in NRK cells. We also analyzed the following proteins involved in endocytosis: clathrin, alpha-adaptin, beta-adaptin, and rabaptin-5. These proteins showed the same pattern of expression as the rab proteins. In conclusion, the results obtained with liver cells corroborate the data obtained in transfected cells and support the notion that rab proteins may be involved in controlling the endocytic rate in liver cells.

C-C chemokine receptor-like 2 (CCRL2) is a non-signaling seven-transmembrane domain (7-TMD) receptor related to the atypical chemokine receptor (ACKR) family. ACKRs bind chemokines but do not activate G protein-dependent signaling or cell functions. ACKRs were shown to regulate immune functions in vivo by their ability to scavenge chemokines from the local environment. This study was performed to investigate whether CCRL2 shares two of the main characteristics of ACKRs, namely the ability to internalize and scavenge the ligands. Cell membrane analysis of CCRL2-transfected cells revealed a weak, constitutive, ligand-independent internalization, and recycling of CCRL2, with a kinetics that was slower than those observed with ACKR3, a prototypic ACKR, or other chemotactic signaling receptors [i.e., chemokine-like receptor 1 and C-X-C motif chemokine receptor 2]. Intracellularly, CCRL2 colocalized with early endosome antigen 1-positive and Rab5-positive vesicles and with recycling compartments mainly characterized by Rab11-positive vesicles. CCRL2-transfected cells and activated mouse blood endothelial cells, that endogenously express CCRL2, were used to investigate the scavenging ability of CCRL2. These experiments confirmed the ability of CCRL2 to bind chemerin, the only recognized ligand, but excluded the ability of CCRL2 to perform scavenging. Collectively, these results identify unique functional properties for this member of the non-signaling 7-TMD receptor family.

Cu ion (Cu) entry into human cells is mediated by CTR1 (also known as SLC31A1), the high-affinity Cu transporter. When extracellular Cu is raised, the cell is protected against excess accumulation by rapid internalization of the transporter. When Cu is lowered, the transporter returns to the membrane. We show in HEK293 cells overexpressing CTR1 that expression of either the C-terminal domain of AP180 (also known as SNAP91), a clathrin-coat assembly protein that sequesters clathrin, or a dominant-negative mutant of dynamin, decreases Cu-induced endocytosis of CTR1, as does a dynamin inhibitor and clathrin knockdown using siRNA. Utilizing imaging, siRNA techniques and a new high-throughput assay for endocytosis employing CLIP-tag methodology, we show that internalized CTR1 accumulates in early sorting endosomes and recycling compartments (containing Rab5 and EEA1), but not in late endosomes or lysosomal pathways. Using live cell fluorescence, we find that upon extracellular Cu removal CTR1 recycles to the cell surface through the slower-recycling Rab11-mediated pathway. These processes enable cells to dynamically alter transporter levels at the plasma membrane and acutely modulate entry as a safeguard against excess cellular Cu.

BACKGROUND

Tumor necrosis factor alpha (TNF-α) plays a central role in the initiation and maintenance of immune responses to periodontopathic bacteria. However, excess TNF-α leads to dysregulated immune responses and progression of periodontitis. Porphyromonas gingivalis (P. gingivalis) invades gingival epithelial cells and then multiplies and survives for a long period. Additionally, increment of TNF-α in periodontal sites is associated with a high prevalence of gram-negative anaerobes such as P. gingivalis. However, it has not been determined whether TNF-α affects invasion of P. gingivalis in periodontal tissues.

RESULTS

We examined the effect of TNF-α on invasion of P. gingivalis in gingival epithelial cells and clarified the mechanism by which TNF-α augments invasion of P. gingivalis. Invasion of P. gingivalis into Ca9-22 cells was augmented by stimulation with TNF-α and it was inhibited by treatment with an antibody to TNF receptor-1. TNF-α increased production of ICAM-1, and P. gingivalis invasion was inhibited by an antibody to ICAM-1 in Ca9-22 cells. Silencing of Rab5 mRNA inhibited P. gingivalis invasion. Furthermore, the JNK inhibitor SP600125 inhibited invasion of P. gingivalis and also decreased the active form of Rab5 in Ca9-22 cells.

CONCLUSIONS

TNF-α augments invasion of P. gingivalis in human gingival epithelial cells through increment of ICAM-1 and activation of Rab5. These phenomena may contribute to persistent infection of P. ginigvalis and prolongation of immune responses in periodontal tissues.

The proteins of the Hedgehog (Hh) family are secreted proteins exerting short- and long-range control over various cell fates in developmental patterning. The Hh gradient in Drosophila wing imaginal discs consists of apical and basolateral secreted pools, but the mechanisms governing the overall establishment of the gradient remain unclear. We investigated the relative contributions of endocytosis and recycling to control the Hh gradient. We show that, upon its initial apical secretion, Hh is re-internalized. We examined the effect of the resistance-nodulation-division transporter Dispatched (Disp) on long-range Hh signaling and unexpectedly found that Disp is specifically required for apical endocytosis of Hh. Re-internalized Hh is then regulated in a Rab5- and Rab4-dependent manner to ensure its long-range activity. We propose that Hh-producing cells integrate endocytosis and recycling as two instrumental mechanisms contributing to regulate the long-range activity of Hh.

The dysregulation of epidermal growth factor receptor (EGFR) signaling has been well documented to contribute to the progression of non-small cell lung cancer (NSCLC), the leading cause of cancer death in the world. EGF-stimulated EGFR activation induces receptor internalization and degradation, which plays an important role in EGFR signaling. This process is frequently deregulated in cancer cells, leading to enhanced EGFR levels and signaling. Our previous study on CMTM7 is only limited to a brief description of the relationship of overexpressed CMTM7 with EGFR-AKT signaling. The biological functions of endogenous CMTM7 and its molecular mechanism remained unclear. In this study, we show that the stable knockdown of CMTM7 augments the malignant potential of NSCLC cells and enhances EGFR-AKT signaling by decreasing EGFR internalization and degradation. Mechanistically, CMTM7 knockdown reduces the activation of Rab5, a protein known to be required for early endosome fusion. In NSCLC, the loss of CMTM7 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, our findings highlight the role of CMTM7 in the regulation of EGFR signaling in tumor cells, revealing CMTM7 as a novel molecule related to Rab5 activation.

Rab GTPases act as molecular switches regulating various aspects of membrane trafficking. Among them, Rab5 and Rab7 play central roles in the endolysosomal network. Although many effectors downstream of Rab7 have been elucidated, our present understanding of the mechanism regulating Rab7 activity is extremely limited. It has only recently been accepted that the Mon1-Ccz1 complex is a Rab7 guanine nucleotide exchange factor, but it still remains unclear what the location where Mon1-Ccz1 works with Rab7 is. To address what kind of change or switch exists in the regulatory mechanism upstream of Rab7 during its transition from the late endosome to lysosome, we examined Rab7 activity in steady-state cells and during EGF-induced macropinocytosis using a newly developed FRET sensor. A combination of a Rab7 sensor and confocal FRET imaging techniques revealed that the activation of Rab7 on late endosomes depends on Mon1-Ccz1 and is implicated in late-endosome-lysosome fusion. In contrast, Rab7 activity on lysosomes was independent of Mon1-Ccz1 and active Rab7 played a role in perinuclear clustering of lysosomes.

The yeast Rab5 homologue, Vps21p, is known to be involved both in the vacuolar protein sorting (VPS) pathway from the trans-Golgi network to the vacuole, and in the endocytic pathway from the plasma membrane to the vacuole. However, the intracellular location at which these two pathways converge remains unclear. In addition, the endocytic pathway is not completely blocked in yeast cells lacking all Rab5 genes, suggesting the existence of an unidentified route that bypasses the Rab5-dependent endocytic pathway. Here we show that convergence of the endocytic and VPS pathways occurs upstream of the requirement for Vps21p in these pathways. We also identify a previously unidentified endocytic pathway mediated by the AP-3 complex. Importantly, the AP-3-mediated pathway appears mostly intact in Rab5-disrupted cells, and thus works as an alternative route to the vacuole/lysosome. We propose that the endocytic traffic branches into two routes to reach the vacuole: a Rab5-dependent VPS pathway and a Rab5-independent AP-3-mediated pathway.

Hyphal growth in filamentous fungi is supported by the uptake (endocytosis) and recycling of membranes and associated proteins at the growing tip. An increasing body of published evidence in various fungi demonstrates that this process is of essential importance for fungal growth and pathogenicity. Here, we introduce fluorescent markers to visualize the endocytic pathway in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein (eGFP) to the actin-binding protein fimbrin (ZtFim1), which is located in actin patches that are formed at the plasma membrane and are participating in endocytic uptake at the cell surface. In addition, we tagged early endosomes by eGFP-labelling a Rab5-homologue (ZtRab5) and late endosomes and vacuoles by expressing eGFP-Rab7 homologue (ZtRab7). Using fluorescent dyes and live cell imaging we confirmed the dynamic behavior and localization of these markers. This set of molecular tools enables an in-depth phenotypic analysis of Z. tritici mutant strains thereby supporting new strategies towards the goal of controlling wheat against Z. tritici.

Rab proteins are small molecular mass GTP-ases involved in the regulation of vescicular transport. The ability of rab proteins to carry out their role in intracellular membrane traffic requires the post-translational attachment to their C-terminus of a geranylgeranyl group, an isoprenoid lipid moiety derived from mevalonate. Here we report that depletion of intracellular mevalonate by lovastatin in FRTL-5 thyroid cells specifically resulted in a four-fold increase of Rab5 and Rab7 protein levels. This increase was reversed within 4 h upon addition of mevalonate. Similarly lovastatin also induced, at same extent, mRNA levels. Lovastatin effect was not common to other prenylated proteins. Moreover incubation with cycloheximide abolished the observed increase in lovastatin treated cells, suggesting that the effect is mediated by newly synthesized protein. These findings demonstrate that Rab5 and Rab7 expression are regulated by the isoprenoid pathway.

Membrane fusion in the endocytic pathway is mediated by a protein machinery consistent of Rab GTPases, tethering factors and SNAREs. In yeast, the endosomal CORVET and lysosomal HOPS tethering complexes share 4 of their 6 subunits. The 2 additional subunits in each complex - Vps3 and Vps8 for CORVET, and the homologous Vps39 and Vps41 for HOPS - bind directly to Rab5 and Rab7, respectively. In humans, all subunits for HOPS have been described. However, human CORVET remains poorly characterized and a homolog of Vps3 is still missing. Here we characterize 2 previously identified Vps39 isoforms, hVps39-1/hVam6/TLP and hVps39-2/TRAP1, in yeast and HEK293 cells. None of them can compensate the loss of the endogenous yeast Vps39, though the specific interaction of hVps39-1 with the virus-specific LT protein was reproduced. Both human Vps39 proteins show a cytosolic localization in yeast and mammalian cells. However, hVps39-2/TRAP1 strongly co-localizes with co-expressed Rab5 and interacts directly with Rab5-GTP in vitro. We conclude that hVps39-2/TRAP1 is an endosomal protein and an effector of Rab5, suggesting a role of the protein as a subunit of the putative human CORVET complex.

The small GTPase Rab5 is one of the key regulators of early endocytic traffic and, like other GTPases, cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle is controlled by a guanine nucleotide dissociation inhibitor (GDI), which functions as a Rab vehicle in the cytosol. GDI extracts from membranes the inactive GDP-bound form of the Rab. Then, the cytosolic GDI:Rab complex is delivered to the appropriate target membrane, where the Rab protein is reloaded, presumably via a GDI displacement factor (Pfeffer and Aivazian, 2004). We previously reported that the formation of the GDI:Rab5 complex is stimulated by the mitogen-activated protein kinase p38 (Cavalli et al., 2001). Mol. Cell7, 421-432.]. Selective activation of p38 MAPK increases endocytic rates in vivo, presumably allowing more efficient internalization of cell surface components for repair, storage, or degradation. These observations emphasize the possibility that external stimuli contribute to the regulation of membrane traffic. Here, we describe how to monitor the ability of GDI to extract Rab5 from early endosomal membranes in vitro and the role of p38 MAPK in this process. In addition, we detail how to investigate the possible role of p38 MAPK in the regulation of endocytosis in vivo.

A defining characteristic of HIV-1 infection is the ability of the virus to persist within the host. Specifically, MHC-I downregulation by the HIV-1 accessory protein Nef is of critical importance in preventing infected cells from cytotoxic T-cell mediated killing. Nef downregulates MHC-I by modulating the host membrane trafficking machinery, resulting in the endocytosis and eventual sequestration of MHC-I within the cell. In the current report, we utilized the intracellular protein-protein interaction reporter system, bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy, to track the Nef/MHC-I interaction and determine its subcellular localization in cells. We demonstrate that this interaction occurs upon Nef binding the MHC-I cytoplasmic tail early during endocytosis in a Rab5-positive endosome. Disruption of early endosome regulation inhibited Nef-dependent MHC-I downregulation, demonstrating that Nef hijacks the early endosome to sequester MHC-I within the cell. Furthermore, super-resolution imaging identified that the Nef:MHC-I BiFC complex transits through both early and late endosomes before ultimately residing at the trans-Golgi network. Together we demonstrate the importance of the early stages of the endocytic network in the removal of MHC-I from the cell surface and its re-localization within the cell, which allows HIV-1 to optimally evade host immune responses.

Cadmium (Cd) is nephrotoxic. Circulating Cd-metallothionein complexes (CdMT) are filtered by the kidney, reabsorbed by proximal tubule cells (PTC) via receptor-mediated endocytosis, and trafficked to lysosomes which results in apoptosis. ADP-ribosylation factors (Arfs) regulate vesicular trafficking. Arf1 is traditionally associated with the secretory pathway, but has been recently found involved in endocytotic trafficking in PTC. Hence, the role of Arf1 was investigated in MT-1 and transferrin (Tf) endocytosis, and in CdMT-1-induced cell death in a PTC line by overexpressing Arf1-wildtype (WT) or dominant-negative mutant Arf1-T31N. Endogenous Arf1 distribution in PTC was punctate throughout the cytosol, but was not detected in the plasma membrane. Arf1 colocalized with markers for sorting to late endosomes (Rab7, CLC6). Arf1 weakly overlapped with the late endosomal/lysosomal marker CLC7, but not with markers for early (Rab5, CLC5) and recycling endosomes (Rab11). Arf1-T31N significantly attenuated CdMT-1 toxicity by ∼60% when compared to Arf1-WT. However, overexpression of Arf1-T31N did not prevent internalization of Alexa Fluor 546-coupled Tf or MT-1 which accumulated in an EEA1-positive early endocytotic compartment, but not in Arf1-WT overexpressing cells. We conclude that Arf1 is involved in trafficking of protein-metal complexes, including CdMT, to late endosomes/lysosomes in renal PTC.

Macropinocytosis, the internalization of extracellular fluid and material by plasma membrane ruffles, is critical for antigen presentation, cell metabolism, and signaling. Macropinosomes mature through homotypic and heterotypic fusion with endosomes and ultimately merge with lysosomes. The molecular underpinnings of this clathrin-independent endocytic pathway are largely unknown. Here, we show that the filamentous septin GTPases associate preferentially with maturing macropinosomes in a phosphatidylinositol 3,5-bisphosphate-dependent manner and localize to their contact/fusion sites with macropinosomes/endosomes. Septin knockdown results in large clusters of docked macropinosomes, which persist longer and exhibit fewer fusion events. Septin depletion and overexpression down-regulates and enhances, respectively, the delivery of fluid-phase cargo to lysosomes, without affecting Rab5 and Rab7 recruitment to macropinosomes/endosomes. In vitro reconstitution assays show that fusion of macropinosomes/endosomes is abrogated by septin immunodepletion and function-blocking antibodies and is induced by recombinant septins in the absence of cytosol and polymerized actin. Thus, septins regulate fluid-phase cargo traffic to lysosomes by promoting macropinosome maturation and fusion with endosomes/lysosomes.

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

The small GTPase Rab5 has been well defined to control the vesicle-mediated plasma membrane protein transport to the endosomal compartment. However, its function in the internalization of vascular endothelial (VE)-cadherin, an important component of adherens junctions, and as a result regulating the endothelial cell polarity and barrier function remain unknown. Here, we demonstrated that lipopolysaccharide (LPS) simulation markedly enhanced the activation and expression of Rab5 in human pulmonary microvascular endothelial cells (HPMECs), which is accompanied by VE-cadherin internalization. In parallel, LPS challenge also induced abnormal cell polarity and dysfunction of the endothelial barrier in HPMECs. LPS stimulation promoted the translocation of VE-cadherin from the plasma membrane to intracellular compartments, and intracellularly expressed VE-cadherin was extensively colocalized with Rab5. Small interfering RNA (siRNA)-mediated depletion of Rab5a expression attenuated the disruption of LPS-induced internalization of VE-cadherin and the disorder of cell polarity. Furthermore, knockdown of Rab5 inhibited the vascular endothelial hyperpermeability and protected endothelial barrier function from LPS injury, both in vitro and in vivo. These results suggest that Rab5 is a critical mediator of LPS-induced endothelial barrier dysfunction, which is likely mediated through regulating VE-cadherin internalization. These findings provide evidence, implicating that Rab5a is a potential therapeutic target for preventing endothelial barrier disruption and vascular inflammation.