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Spiroplasma eriocheiris is a kind of intracellular pathogen without cell wall and the causative agent of Chinese mitten crab Eriocheir sinensis "tremor disease", which causes significant economic losses in the crustacean aquaculture. However, little is known about the intracellular transport of this pathogen and host innate immune response to this pathogen. Rab GTPases are key regulators for endocytosis and intracellular pathogen trafficking. In this study, we showed that S. eriocheiris infection upregulated the transcription of Rab7 through the downregulation of miR-131-3p. Subsequently, both hemocytes transfected with miR-131-3p mimics and hemocytes derived from Rab7 knockdown crabs exhibited reduced phagocytic activities and increased susceptibility to S. eriocheiris infection. Additionally, Rab7 could interact with the cell shape-determining protein MreB3 of S. eriocheiris, and its overexpression promoted S. eriocheiris internalization and fusion with lysosomes, thereby limiting S. eriocheiris replication in Drosophila S2 cells. Overall, these results demonstrated that Rab7 facilitated host cell phagocytosis and interacted with MreB3 of S. eriocheiris to prevent S. eriocheiris infection. Moreover, miR-131-3p was identified as a negative regulator of this process through its targeting of Rab7. Therefore, targeting miR-131-3p might be an effective strategy for controlling S. eriocheiris in crab aquaculture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Qingguo Meng reports financial support was provided by Jiangsu Provincial Department of Agriculture and Rural Affairs. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chronic hepatitis B virus (HBV) infection can lead to advanced liver pathology. Here, we establish a transgenic murine model expressing a basic core promoter (BCP)-mutated HBV genome. Unlike previous studies on the wild-type virus, the BCP-mutated HBV transgenic mice manifest chronic liver injury that culminates in cirrhosis and tumor development with age. Notably, agonistic anti-Fas treatment induces fulminant hepatitis in these mice even at a negligible dose. As the BCP mutant exhibits a striking increase in HBV core protein (HBc) expression, we posit that HBc is actively involved in hepatocellular injury. Accordingly, HBc interferes with Fis1-stimulated mitochondrial recruitment of Tre-2/Bub2/Cdc16 domain family member 15 (TBC1D15). HBc may also inhibit multiple Rab GTPase-activating proteins, including Rab7-specific TBC1D15 and TBC1D5, by binding to their conserved catalytic domain. In cells under mitochondrial stress, HBc thus perturbs mitochondrial dynamics and prevents the recycling of damaged mitochondria. Moreover, sustained HBc expression causes lysosomal consumption via Rab7 hyperactivation, which further hampers late-stage autophagy and substantially increases apoptotic cell death. Finally, we show that adenovirally expressed HBc in a mouse model is directly cytopathic and causes profound liver injury, independent of antigen-specific immune clearance. These findings reveal an unexpected cytopathic role of HBc, making it a pivotal target for HBV-associated liver disease treatment. The BCP-mutated HBV transgenic mice also provide a valuable model for understanding chronic hepatitis B progression and for the assessment of therapeutic strategies., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

Background: Mitochondrial dysfunction exists in Alzheimer's disease (AD) brain, and damaged mitochondria need to be removed by mitophagy. Small GTPase Rab7 regulates the fusion of mitochondria and lysosome, while TBC1D5 inhibits Rab7 activation. However, it is not clear whether the regulation of Rab7 activity by TBC1D5 can improve mitophagy and inhibit AD progression., Objective: To investigate the role of TBC1D5 in mitophagy and its regulatory mechanism for Rab7, and whether activation of mitophagy can inhibit the progression of AD., Methods: Mitophagy was determined by western blot and immunofluorescence. The morphology and quantity of mitochondria were tracked by TEM. pCMV-Mito-AT1.03 was employed to detect the cellular ATP. Amyloid-β secreted by AD cells was detected by ELISA. Co-immunoprecipitation was used to investigate the binding partner of the target protein. Golgi-cox staining was applied to observe neuronal morphology of mice. The Morris water maze test and Y-maze were performed to assess spatial learning and memory, and the open field test was measured to evaluate motor function and anxiety-like phenotype of experimental animals., Results: Mitochondrial morphology was impaired in AD models, and TBC1D5 was highly expressed. Knocking down TBC1D5 increased the expression of active Rab7, promoted the fusion of lysosome and autophagosome, thus improving mitophagy, and improved the morphology of hippocampal neurons and the impaired behavior in AD mice., Conclusions: Knocking down TBC1D5 increased Rab7 activity and promoted the fusion of autophagosome and lysosome. Our study provided insights into the mechanisms that bring new possibilities for AD therapy targeting mitophagy.

Intracellular endosomal trafficking controls the balance between protein degradation and synthesis, i.e., proteostasis, but also many of the cellular signaling pathways that emanate from activated growth factor receptors after endocytosis. Endosomal trafficking, sorting, and motility are coordinated by the activity of small GTPases, including Rab proteins, whose function as molecular switches direct activity at endosomal membranes through effector proteins. Rab7 is particularly important in the coordination of the degradative functions of the pathway. Rab7 effectors control endosomal maturation and the properties of late endosomal and lysosomal compartments, such as coordination of recycling, motility, and fusion with downstream compartments. The spatiotemporal regulation of endosomal receptor trafficking is particularly challenging in neurons because of their enormous size, their distinct intracellular domains with unique requirements (dendrites vs. axons), and their long lifespans as postmitotic, differentiated cells. In Charcot-Marie-Tooth 2B disease (CMT2B), familial missense mutations in Rab7 cause alterations in GTPase cycling and trafficking, leading to an ulcero-mutilating peripheral neuropathy. The prevailing hypothesis to account for CMT2B pathologies is that CMT2B-associated Rab7 alleles alter endocytic trafficking of the neurotrophin NGF and its receptor TrkA and, thereby, disrupt normal trophic signaling in the peripheral nervous system, but other Rab7-dependent pathways are also impacted. Here, using TrkA as a prototypical endocytic cargo, we review physiologic Rab7 effector interactions and control in neurons. Since neurons are among the largest cells in the body, we place particular emphasis on the temporal and spatial regulation of endosomal sorting and trafficking in neuronal processes. We further discuss the current findings in CMT2B mutant Rab7 models, the impact of mutations on effector interactions or balance, and how this dysregulation may confer disease.

Rab7 is a GTPase that controls late endosome and lysosome trafficking. Recent studies have demonstrated that Rab7 is ubiquitinated, a post-translational modification mediated by an enzymatic cascade. To date, only one ubiquitin E3 ligase and one deubiquitinase have been identified in regulating Rab7 ubiquitination. Here, we report that RNF167, a transmembrane endolysosomal ubiquitin ligase, can ubiquitinate Rab7. Using immunoprecipitation and in vitro ubiquitination assays, we demonstrate that Rab7 is a direct substrate of RNF167. Subcellular fractionation indicates that RNF167 activity maintains Rab7's membrane localization. Epifluorescence microscopy in HeLa cells shows that Rab7-positive vesicles are larger under conditions enabling Rab7 ubiquitination by RNF167. Characterization of its ubiquitination reveals that Rab7 must be in its GTP-bound active form for membrane anchoring and, thus, accessible for RNF167-mediated ubiquitin attachment. Cellular distribution analyses of lysosome marker Lamp1 show that vesicle positioning is independent of Rab7 and RNF167 expression and that Rab7 endosomal localization is not affected by RNF167 knockdown. However, both Rab7 and RNF167 depletion affect each other's lysosomal localization. Finally, this study demonstrates that the RNF167-mediated ubiquitination of Rab7 GTPase is impaired by variants of Charcot-Marie-Tooth Type 2B disease. This study identified RNF167 as a new ubiquitin ligase for Rab7 while expanding our knowledge of the mechanisms underlying the ubiquitination of Rab7.

Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Besides possible recycling routes to the plasma membrane and various organelles, previously described endocytic pathways (e.g., clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway) all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs), we identify an endocytic pathway that moves material to nonacidic Lamp1-positive late endosomes. Trafficking via this endocytic route is fully independent of Rab5 and Rab7 but requires the Rab14 protein. The pathway taken by CPPs differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already, as it is not affected by a series of compounds that inhibit macropinocytosis or clathrin-mediated endocytosis. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Liver ischemia-reperfusion (IR) injury is an unavoidable pathological process in transplantation, closely related to poor prognosis. To date, there has been no clear therapeutic measure. We previously reported that mild hypothermia (MH), a widely used therapy, can exert significant protective effect against liver IR injury. Among the multiple mechanisms underlying the therapeutic effect of MH, autophagy flux drew our special attention. In this study, we evaluated the role of autophagy flux in IR injury and thereby explored the relationship between MH and autophagy flux in IR injury. We developed in vivo and in vitro models for hepatic IR injury. By autophagy flux assay with Western blotting and immunofluorescence, we found that MH restricts heavy accumulation of autophagosomes (APs) during IR injury. Activation and blocking of the autophagy flux unraveled that accumulation of APs further aggravated IR injury. Further, MH reduces APs accumulation to restore autophagy flux by regulating the fusion of APs and lysosomes. Besides, MH upregulated the level of Rab7 protein expression that was seriously impaired during IR injury. Inhibition of Rab7 expression increased apoptosis of liver cells and reduced the degree of overlap between APs and lysosomes. The results were reversed upon activation of Rab7. In conclusion, MH can alleviate liver IR injury by regulating the Rab7-mediated APs-lysosomes fusion that reduces APs accumulation. This can provide a theoretical basis for the further application of MH in related clinical diseases., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We examined sequence conservation and signatures of selection in Rab7 proteins across 11 Paramecium aurelia species, and determined the localization patterns of two P. tetraurelia Rab7 paralogs when expressed as GFP fusions in live cells. We found that, while there is a variable number of Rab7 paralogs per genome, Rab7 genes are highly conserved in sequence and appear to be under strong purifying selection across aurelias . Additionally, and surprisingly based on earlier studies, we found that two P. tetraurelia Rab7 proteins have virtually identical localization patterns. Consistent with this, when we examined the gene family of a highly conserved Rab binding partner across aurelias (Rab-Interacting Lysosomal Protein, or RILP), we found that residues in key binding sites in RILPs were absolutely conserved in 13 of 21 proteins, representing genes from 9 of the 11 species examined. Of note, RILP gene number appears to be even more constrained than Rab7 gene number per genome. Abbreviation : WGD: Whole genome duplication.

In a cell line, stably expressing α 1A -adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α 1A -adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α 1A -adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α 1A -adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α 1A -adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α 1A -adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α 1A -adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate., Methods: The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined., Results: PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg 2+ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly., Conclusion: Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum., General Significance: This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules., Competing Interests: Declaration of competing interest The authors declare no conflict of interest associated with this publication., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans -Golgi network (TGN). In the wobbler mouse, a model for human motor neuron (MN) disease, reduction in the levels of Vps54 causes neurodegeneration. However, it is unclear how disruption of the GARP complex leads to MN dysfunction. To better understand the role of Vps54 in MNs, we have disrupted expression of the Vps54 ortholog in Drosophila and examined the impact on the larval neuromuscular junction (NMJ). Surprisingly, we show that both null mutants and MN-specific knockdown of Vps54 leads to NMJ overgrowth. Reduction of Vps54 partially disrupts localization of the t-SNARE, Syntaxin-16, to the TGN but has no visible impact on endosomal pools. MN-specific knockdown of Vps54 in MNs combined with overexpression of the small GTPases Rab5, Rab7, or Rab11 suppresses the Vps54 NMJ phenotype. Conversely, knockdown of Vps54 combined with overexpression of dominant negative Rab7 causes NMJ and behavioral abnormalities including a decrease in postsynaptic Dlg and GluRIIB levels without any effect on GluRIIA. Taken together, these data suggest that Vps54 controls larval MN axon development and postsynaptic density composition through a mechanism that requires Rab7., Competing Interests: Competing interestsThe authors declare no competing financial interests., (© 2020. Published by The Company of Biologists Ltd.)

RAB7 is a small GTPase that belongs to the Rab family, and as a vesicle trafficking factor it is shown to regulate the transport to late endocytic compartments, autophagosome maturation and organelle function. In present study, we showed the critical roles of RAB7 GTPase on actin dynamics and mitochondria function in oocyte meiosis. RAB7 mainly accumulated at cortex and spindle periphery during oocyte maturation. RAB7 depletion caused the failure of polar body extrusion and asymmetric division, and Rab7 exogenous mRNA supplement could rescue the defects caused by RAB7 RNAi. Based on mass spectrometry analysis, we found that RAB7 associated with several actin nucleation factors and mitochondria-related proteins in oocytes. The depletion of RAB7 caused the decrease of actin dynamics, which further affected meiotic spindle migration to the oocyte cortex. In addition, we found that RAB7 could maintain mitochondrial membrane potential and the mitochondrial distribution in mouse oocytes, and this might be due to its effects on the phosphorylation of DRP1 at Ser616 domain. Taken together, our data indicated that RAB7 transported actin nucleation factor for actin polarization, which further affected the phosphorylation of DRP1 for mitochondria dynamics and the meiotic spindle migration in mouse oocytes., (© 2020 Federation of American Societies for Experimental Biology.)

Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

Endocytosis has been proposed to modulate cell signaling activities. However, the role of endocytosis in embryogenesis, which requires coordination of multiple signaling inputs, has remained less understood. We previously showed that mouse embryos lacking a small guanosine triphosphate (GTP)-binding protein Rab7 implicated in endocytic flow are defective in gastrulation. Here, we investigate how subcellular defects associated with Rab7 deficiency are related to the observed developmental defects. Rab7-deficient embryos fail to organize mesodermal tissues due to defects in Wnt-β-catenin signaling. Visceral endoderm (VE)-specific ablation of Rab7 results in patterning defects similar to systemic Rab7 deletion. Rab7 mutants accumulate the Wnt antagonist Dkk1 in the extracellular space and in intracellular compartments throughout the VE epithelium. These data indicate that Rab7-dependent endocytosis regulates the concentration and availability of extracellular Dkk1, thereby relieving the epiblast of antagonism. This intercellular mechanism therefore organizes distinct spatiotemporal patterns of canonical Wnt activity during the peri-gastrulation stages of embryonic development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Endosomes and lysosomes harbor Rab5 and Rab7 on their surface as key proteins involved in their identity, biogenesis, and fusion. Rab activation requires a guanine nucleotide exchange factor (GEF), which is Mon1-Ccz1 for Rab7. During endosome maturation, Rab5 is replaced by Rab7, though the underlying mechanism remains poorly understood. Here, we identify the molecular determinants for Rab conversion in vivo and in vitro, and reconstitute Rab7 activation with yeast and metazoan proteins. We show (i) that Mon1-Ccz1 is an effector of Rab5, (ii) that membrane-bound Rab5 is the key factor to directly promote Mon1-Ccz1 dependent Rab7 activation and Rab7-dependent membrane fusion, and (iii) that this process is regulated in yeast by the casein kinase Yck3, which phosphorylates Mon1 and blocks Rab5 binding. Our study thus uncovers the minimal feed-forward machinery of the endosomal Rab cascade and a novel regulatory mechanism controlling this pathway., Competing Interests: LL, AB, EH, NF, YH, AP, KA, DK, CU No competing interests declared, (© 2020, Langemeyer et al.)

The small GTPase Rab7 is the main regulator of membrane trafficking at late endosomes. This small GTPase regulates endosome-to-trans Golgi Network trafficking of sorting receptors, membrane fusion of late endosomes to lysosomes, and autophagosomes to lysosomes during autophagy. Rab7, like all Rab GTPases, binds downstream effectors coordinating several divergent pathways. How cells regulate these interactions and downstream functions is not well understood. Recent evidence suggests that Rab7 function can be modulated by the combination of several post-translational modifications that facilitate interactions with one effector while preventing binding to another one. In this review, we discuss recent data on how phosphorylation, palmitoylation and ubiquitination modulate the ability of this small GTPase to orchestrate membrane trafficking at the late endosomes.

The rare autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) is associated with mutations in the RAB7A gene, involved in the late endocytic pathway. CMT2B is characterized by predominant sensory loss, ulceromutilating features, with lesser-to-absent motor deficits. We characterized clinically and genetically a family harboring a novel pathogenic RAB7A variant and performed structural and functional analysis of the mutant protein. A 39-year-old woman presented with early-onset walking difficulties, progressive distal muscle wasting and weakness in lower limbs and only mild sensory signs. Electrophysiology demonstrated an axonal sensorimotor neuropathy. Nerve biopsy showed a chronic axonal neuropathy with moderate loss of all caliber myelinated fibers. Next-generation sequencing (NGS) technology revealed in the proband and in her similarly affected father the novel c.377A>G (p.K126R) heterozygous variant predicted to be deleterious. The mutation affects the biochemical properties of RAB7 GTPase, causes altered interaction with peripherin, and inhibition of neurite outgrowth, as for previously reported CMT2B mutants. However, it also shows differences, particularly in the epidermal growth factor receptor degradation process. Altogether, our findings indicate that this RAB7A variant is pathogenic and widens the phenotypic spectrum of CMT2B to include predominantly motor CMT2. Alteration of the receptor degradation process might explain the different clinical presentations in this family.

Degradation of endocytosed proteins involves the formation of transient connections between late endosomes and lysosomes in a process called "kiss and run." Genes and proteins controlling this mechanism are unknown. Here, we identify the small guanosine triphosphatase (GTPase) RabX1 as an organizer of a late endosomal compartment that forms dynamic tubular connections to lysosomes. By analyzing trafficking of the adhesion protein Fasciclin2 in the Drosophila follicular epithelium, we show that a reduction of RabX1 function leads to defects in Fasciclin2 degradation. RabX1 mutants fail to form normal lysosomes and accumulate Fasciclin2 in a swelling late-endosomal compartment. RabX1 protein localizes to late endosomes, where it induces the formation of tubular connections to lysosomes. We propose that these tubules facilitate influx of lysosomal content into late endosomes and that this influx leads to the formation of endolysosomes, in which Fasciclin2 is degraded. We show that the formation of RabX1 tubules is dependent on the V-ATPase proton pump. Moreover, we provide evidence that V-ATPase activity is upregulated during epithelial differentiation. This upregulation intensifies RabX1 tubulation and thereby boosts the capacity of the endolysosomal pathway. Enhanced endolysosomal capacity is required for the removal of Fasciclin2 from the epithelium, which is part of a developmental program promoting epithelial morphogenesis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for osmotic water reabsorption by kidney collecting ducts. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane of collecting duct principal cells, where it increases water permeability and, hence, water reabsorption. Despite continuing efforts, gaps remain in our knowledge of vasopressin-regulated AQP2 trafficking. Here, we studied the functions of two retromer complex proteins, small GTPase Rab7 and vacuolar protein sorting 35 (Vps35), in vasopressin-induced AQP2 trafficking in a collecting duct cell model (mpkCCD cells). We showed that upon vasopressin removal, apical AQP2 returned to Rab5-positive early endosomes before joining Rab11-positive recycling endosomes. In response to vasopressin, Rab11-associated AQP2 trafficked to the apical plasma membrane before Rab5-associated AQP2 did so. Rab7 knockdown resulted in AQP2 accumulation in early endosomes and impaired vasopressin-induced apical AQP2 trafficking. In response to vasopressin, Rab7 transiently colocalized with Rab5, indicative of a role of Rab7 in AQP2 sorting in early endosomes before trafficking to the apical membrane. Rab7-mediated apical AQP2 trafficking in response to vasopressin required GTPase activity. When Vps35 was knocked down, AQP2 accumulated in recycling endosomes under vehicle conditions and did not traffic to the apical plasma membrane in response to vasopressin. We conclude that Rab7 and Vps35 participate in AQP2 sorting in early endosomes under vehicle conditions and apical membrane trafficking in response to vasopressin.

The endolysosomal system fulfils a myriad of cellular functions predicated on regulated membrane identity progressions, collectively termed maturation. Mature or "late" endosomes are designated by small membrane-bound GTPases Rab7 and Arl8b, which can either operate independently or collaborate to form a joint compartment. Whether, and how, Rab7 and Arl8b resolve this hybrid identity compartment to regain functional autonomy is unknown. Here, we report that Arl8b employs its effector SKIP to instigate inactivation and removal of Rab7 from select membranes. We find that SKIP interacts with Rab7 and functions as its negative effector, delivering the cognate GAP, TBC1D15. Recruitment of TBC1D15 to SKIP occurs via the HOPS complex, whose assembly is facilitated by contacts between Rab7 and the KMI motif of SKIP. Consequently, SKIP mediates reinstatement of single identity Arl8b sub-compartment through an ordered Rab7-to-Arl8b handover, and, together with Rab7's positive effector RILP, enforces spatial, temporal and morphological compartmentalization of endolysosomal organelles., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The Rab family is the most significant subfamily of small GTP-binding proteins. These proteins have widespread intracellular localization and play an important role in many biological processes. Rab7 plays a crucial role in the innate immune system of crustaceans. In the present study, we cloned and characterized Rab7 from Chinese mitten crab (Eriocheir sinensis), designated EsRab7. The full-length of the EsRab7 cDNA sequence is 1,257 bp and contains a 618-bp open reading frame encoding a 205-amino acid polypeptide. Bioinformatics analysis showed that the Rab7 protein was highly conserved during evolution. Quantitative real-time PCR showed the highest tissue expression in muscle, followed by hepatopancreas. EsRab7 was significantly upregulated in hemocytes after stimulation by Gram-positive Staphylococcus aureus or Gram-negative Vibrio parahaemolyticus. Further studies showed that EsRab7 knockdown during bacterial stimulation resulted in decreased bacterial phagocytosis. In addition, EsRab7 regulated the expression of antimicrobial peptides via the Toll signaling pathway. Collectively, these results demonstrate that EsRab7 plays critical roles in antimicrobial function in the Chinese mitten crab., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Synaptic connections in the brain are continuously weakened or strengthened in response to changes in neuronal activity. This process, known as synaptic plasticity, is the cellular basis for learning and memory, and is thought to be altered in several neuronal disorders. An important aspect of synaptic plasticity is the tightly controlled trafficking and synaptic targeting of the AMPA-type glutamate receptors, which are the major mediators of fast excitatory transmission in the brain. This review addresses the role of Rab GTPases in AMPA receptor trafficking in neurons under basal conditions and during activity-induced synaptic plasticity, especially during long-term potentiation (LTP) and long-term depression (LTD). We highlight the importance of the tight spatio-temporal control of Rab activity and suggest that this is critical for proper neuronal functions. We also discuss how abnormal AMPA receptor trafficking and malfunctioning of Rabs can lead to neurologic disorders or memory problems.

Objective: To investigate whether CD137-CD137L signaling could affect the secretion of mouse vascular smooth muscle cells (VSMCs) -derived exosomes through autophagy mediated Rab7 pathway. Methods: Primary thoracic aorta VSMCs from C57BL/6J mouse were obtained by tissue block adherence method. VSMCs between the third to fifth passages were used and VSMCs were divided into 4 groups: control group, CD137 agonist group, lentivirus control group, Rab7 lentiviral interference group. VSMCs in CD137 agonist group were treated with recombinant protein of CD137L (10 μg/ml), VSMCs in lentivirus control group were treated with lentiviral followed by recombinant protein of CD137L (10 μg/ml), VSMCs in Rab7 lentiviral interference group were treated with Rab7 lentiviral intervention followed by recombinant protein of CD137L (10 μg/ml). Western blot was used to detect the protein expression of LC3Ⅱ, p62, Rab7, CD9, CD81 and Hsc70. Fluorescence microscopy was used to track the changes of autophagy in cells infected with mRFP-GFP-LC3. Transmission electron microscope was used to observe the morphology and size of VSMCs-derived exosomes. The nanoparticle tracking analysis(NTA) was used to detect the concentration and size of exosomes in each group. Results: (1) The expressions of Rab7, LC3Ⅱ and p62 protein in VSMCs of CD137 activation group were significantly higher than those in control group (all P< 0.05). The expressions of Rab7, LC3Ⅱ and p62 protein in Rab7 lentivirus interference group was lower than in CD137 activation group (all P< 0.05), while the expressions were similar between the lentivirus control group and the CD137 activation group (all P> 0.05). (2) The total number of fluorescent spots and yellow fluorescent spots in the VSMCs of the CD137 activation group were higher than those in the control group (all P< 0.05), and the number of yellow fluorescent spots was higher than that of the red fluorescent spots in the VSMCs of the CD137 activation group ((50.3±0.9) vs. (10.3±1.5)/cell). The total numbers of fluorescent spots and yellow fluorescent spots in VSMCs of Rab7 lentivirus interference group were lower than those of CD137 activation group (both P< 0.05), and the number of red fluorescent spots in VSMCs was higher than that of yellow fluorescent spots ((40.7±4.0) and (10.7±1.2)/cell) in the Rab7 lentiviral interference group. The total numbers of fluorescent spots and yellow fluorescent spots in the VSMCs were similar between the lentivirus control group and the CD137 activation group (all P> 0.05). (3) Under transmission electron microscopy, the size of the VSMCs-derived exosomes was about 30-150 nm. The exosome markers (CD9, CD81) could be detected in vesicles by Western blot. NTA results showed that the concentration of VSMCs-derived exosomes was significantly higher in the CD137-activated group than in the control group ( P< 0.05), which was significantly lower in the Rab7 lentiviral interference group than in the CD137-activation group ( P< 0.05) and was similar between the lentivirus control group and the CD137 activation group ( P> 0.05). The expression of Hsc70 protein in exosomes secreted by CD137 activation group was higher than that in the control group ( P< 0.05). The expression of Hsc70 protein in exosomes was lower in Rab7 lentivirus interference group than in the CD137 activation group ( P< 0.05), which was similar between the lentivirus control group and the CD137 activation group ( P> 0.05). The expression of LC3Ⅱ protein in exosome was higher in CD137 activation group than in control group ( P< 0.05), which was lower in Rab7 lentivirus interference group than in CD137 activation group ( P< 0.05), which was similar between the lentivirus control group and the CD137 activation group ( P> 0.05). Conclusion: The CD137-CD137L signaling may affect the secretion of mouse VSMCs-derived exosomes through modulating the Rab7 pathway mediated autophagy.

Vacuoles are essential organelles in plants, playing crucial roles, such as cellular material degradation, ion and metabolite storage, and turgor maintenance. Vacuoles receive material via the endocytic, secretory, and autophagic pathways. Membrane fusion is the last step during which prevacuolar compartments (PVCs) and autophagosomes fuse with the vacuole membrane (tonoplast) to deliver cargoes. Protein components of the canonical intracellular fusion machinery that are conserved across organisms, including Arabidopsis thaliana , include complexes, such as soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), that catalyze membrane fusion, and homotypic fusion and vacuole protein sorting (HOPS), that serve as adaptors which tether cargo vesicles to target membranes for fusion under the regulation of RAB-GTPases. The mechanisms regulating the recruitment and assembly of tethering complexes are not well-understood, especially the role of RABs in this dynamic regulation. Here, we report the identification of the small synthetic molecule Endosidin17 (ES17), which interferes with synthetic, endocytic, and autophagic traffic by impairing the fusion of late endosome compartments with the tonoplast. Multiple independent target identification techniques revealed that ES17 targets the VPS35 subunit of the retromer tethering complex, preventing its normal interaction with the Arabidopsis RAB7 homolog RABG3f. ES17 interference with VPS35-RABG3f interaction prevents the retromer complex to endosome anchoring, resulting in retention of RABG3f. Using multiple approaches, we show that VPS35-RABG3f-GTP interaction is necessary to trigger downstream events like HOPS complex assembly and fusion of late compartments with the tonoplast. Overall, our results support a role for the interaction of RABG3f-VPS35 as a checkpoint in the control of traffic toward the vacuole., Competing Interests: The authors declare no competing interest.

Mutations in RAB18, a member of small G protein, cause Warburg micro syndrome (WARBM), whose clinical features include vision impairment, postnatal microcephaly, and lower limb spasticity. Previously, our Rab18 -/- mice exhibited hind limb weakness and spasticity as well as signs of axonal degeneration in the spinal cord and lumbar spinal nerves. However, the cellular and molecular function of RAB18 and its roles in the pathogenesis of WARBM are still not fully understood. Using immunofluorescence staining and expression of Rab18 and organelle markers, we find that Rab18 associates with lysosomes and actively traffics along neurites in cultured neurons. Interestingly, Rab18 -/- neurons exhibit impaired lysosomal transport. Using autophagosome marker LC3-II, we show that Rab18 dysfunction leads to aberrant autophagy activities in neurons. Electron microscopy further reveals accumulation of lipofuscin-like granules in the dorsal root ganglion of Rab18 -/- mice. Surprisingly, Rab18 colocalizes, cofractionates, and coprecipitates with the lysosomal regulator Rab7, mutations of which cause Charcot-Marie-Tooth (CMT) neuropathy type 2B. Moreover, Rab7 is upregulated in Rab18-deficient neurons, suggesting a compensatory effect. Together, our results suggest that the functions of RAB18 and RAB7 in lysosomal and autophagic activities may constitute an overlapping mechanism underlying WARBM and CMT pathogenesis in the nervous system.

The objective of this study is to determine whether Rab7 could delay intervertebral disc degeneration and study the mechanism. We chose old mice to established intervertebral disc degeneration model and study the effect of Rab7 on intervertebral disc by western blotting, quantitative real-time PCR, immunohistochemistry et al. Nucleus pulposus cells were cultured to study the exact mechanism. Expression of Rab7 was decreased in degenerative human nucleus pulposus tissues. With the aging of mice, the degree of intervertebral disc degeneration was aggravated and the expression of Rab7 was decreased. In addition, overexpression of Rab7 can reduce the degeneration of nucleus pulposus cells and reduce the levels of p38 and p-p38. Rab7 is a protective factor for intervertebral discs and could delays intervertebral disc degeneration through the inhibition of the p38MAPK pathway., (Copyright © 2019. Published by Elsevier Inc.)

Increasing evidence suggests that Ras-related in brain 7 (Rab7), an endosome-localized small GTPase contributes to cerebral ischemic brain injury. In the present study, we investigated the role of Rab7 in ischemic stroke-induced formation of astrogliosis and glial scar. Rats were subjected to transient middle cerebral artery occlusion (tMCAO); the rats were injected with the Rab7 receptor antagonist CID1067700 (CID). Primary astrocytes were subjected to an oxygen and glucose deprivation and reoxygenation (OGD/Re) procedure; CID was added to the cell culture media. We found that Rab7 was significantly elevated over time in both the in vivo and in vitro astrocytic injury models, and administration of CID significantly down-regulated the glial scar markers such as glial fibillary acidic protein (GFAP), neurocan and phosphacan. Moreover, administration of CID significantly attenuated the brain atrophy and improved neurologic deficits in tMCAO rats, and protected astrocytes against OGD/Re-induced injury. Further, CID downregulated the protein levels of Lamp1 and active cathepsin B in astrocytes after OGD/Re or tMCAO injury; CID inhibited the co-localization of cathepsin B and Rab7, Lamp1 and Rab7; CID decreased OGD/Re-induced increase in lysosomal membrane permeability and blocked OGD/Re-induced release of cathepsin B from the lysosome into the cytoplasm in astrocytes. Taken together, these results suggest that Rab7 is involved in ischemic stroke-induced formation of astrogliosis and glial scar. CID administration attenuates brain atrophy and improves neurologic deficits and inhibits astrogliosis and glial scar formation after ischemic stroke via reducing the activation and release of cathepsin B from the lysosome into the cytoplasm.

In this study, GST Pull-down and mass spectrometry was applied to the precipitation and identification of the small GTP-binding protein (Rab7) interacting protein in hemocyte of Litopenaeus vannamei. According to the search in GenBank with the peptide mass fingerprint, the 45 kDa protein which was pulled down with the GST-tagged Rab7 (GST-Rab7, GTP bound form) was identified to be β-actin with 28% coverage of amino acid sequences. The interaction of Rab7 with β-actin was verified by both GST Pull-down and ELISA in vitro. Meanwhile, confocal microscopic observation showed that Rab7 could be co-localized with β-actin in hemocytes at 12 h post white spot syndrome virus (WSSV) infection (hpi). GST Pull-down and western blotting were used to analyze the cross-interaction between WSSV VP28, Rab7 and β-actin. The results showed that the GST-VP28, His-tagged Rab7 (His-Rab7) and His-β-actin formed a tripartite complex. At 12 hpi, confocal microscopic observation showed that WSSV could be co-localized with Rab7 and β-actin in hemocytes respectively. Furthermore, based on the in vivo neutralization assay, recombinant His-β-actin accelerated the infection of WSSV, conversely, recombinant His-Rab7 delayed WSSV infection in shrimp. These results suggested the interaction of Rab7 with β-actin and this interaction was involved in WSSV infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Missing in metastasis (MIM), an inverse Bin-Amphiphysin-Rvs (I-BAR) domain protein, promotes endocytosis of C-X-C chemokine receptor 4 (CXCR4) in mammalian cells. In response to the CXCR4 ligand stromal cell-derived factor 1 (SDF-1 or CXCL12), MIM associates with RAS-related GTP-binding protein 7 (RAB7) 30 min after stimulation. However, RAB7's role in MIM function remains undefined. Here we show that RNAi-mediated suppression of RAB7 expression in human HeLa cells has little effect on the binding of MIM to RAB5 and on the recruitment of CXCR4 to early endosomes but effectively abolishes MIM-mediated CXCR4 degradation, chemotactic response, and sorting into late endosomes and lysosomes. To determine whether I-BAR domain proteins interact with RAB7, we examined cells expressing insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein bearing an Src homology 3 (SH3) domain. We observed that both MIM and IRTKS interact with RAB5 at an early response to SDF-1 and that IRTKS binds poorly to RAB7 but strongly to RAB11 at a later time point. Moreover, IRTKS overexpression reduced CXCR4 internalization and enhanced the chemotactic response to SDF-1. Interestingly, deletion of the SH3 domain in IRTKS abolished the IRTKS-RAB11 interaction and promoted CXCR4 degradation. Furthermore, the SH3 domain was required for selective targeting of MIM-IRTKS fusion proteins by both RAB7 and RAB11. Hence, to the best of our knowledge, our results provide first evidence that the SH3 domain is critical in the regulation of specific endocytic pathways by I-BAR domain proteins., (© 2019 Li et al.)

The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP-bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7-positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P 2 causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome-lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affected by acute PI(4,5)P 2 production. Deletion of PI4K2A greatly reduces PIP5Kγ-mediated PI(4,5)P 2 production in Rab7-positive endosomes leading to impaired Rab7 inactivation and increased number of LC3-positive structures with defective autophagosome-lysosome fusion. These results reveal a late endosomal PI4P-PI(4,5)P 2 -dependent regulatory loop that impacts autophagosome flux by affecting Rab7 cycling and PLEKHM1 association., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Osteoclast lysosomes secrete lytic enzymes into bone resorption lacunae, and sort the lysosomal proton pumping vacuolar-type ATPase (V-ATPase) to the plasma membrane to form the acidic environment required for bone digestion. The a3 isoform of V-ATPase is essential for outward trafficking of the secretory lysosomes and interacts physically with Rab7, a small GTPase that regulates trafficking of late endosomes and lysosomes, to recruit it to lysosomes. However, it is unclear whether organelle acidification by V-ATPase is required for the lysosome trafficking. Here, we showed that incubation of osteoclasts with the V-ATPase inhibitor bafilomycin A1 abolished the osteoclast-characteristic peripheral localization of secretory lysosomes, Rab7, and α-tubulin. Although bafilomycin A1 had little or no effect on Rab7 activation and its interaction with a3, treatment with the inhibitor significantly reduced the lysosomal localization of Rab7. Even constitutively active Rab7 did not localize to lysosomes in the presence of the inhibitor. These results suggest that organelle acidification by V-ATPase is required for localization of activated Rab7 to lysosomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The small GTPase, Rab7a, and the regulators of its GDP/GTP-binding status were shown to have roles in both endocytic membrane traffic and autophagy. Classically known to regulate endosomal retrograde transport and late endosome-lysosome fusion, earlier work has indicated a role for Rab7a in autophagosome-lysosome fusion as well as autolysosome maturation. However, as suggested by recent findings on PTEN-induced kinase 1 (PINK1)-Parkin-mediated mitophagy, Rab7a and its regulators are critical for the correct targeting of Atg9a-bearing vesicles to effect autophagosome formation around damaged mitochondria. This mitophagosome formation role for Rab7a is dependent on an intact Rab cycling process mediated by the Rab7a-specific guanine nucleotide exchange factor (GEF) and GTPase activating proteins (GAPs). Rab7a activity in this regard is also dependent on the retromer complex, as well as phosphorylation by the TRAF family-associated NF-κB activator binding kinase 1 (TBK1). Here, we discuss these recent findings and broadened perspectives on the role of the Rab7a network in PINK1-Parkin mediated mitophagy.

Background: Magnetic nanoparticles (MNPs) have been widely used in biomedical applications. Proper control of the duration of MNPs in circulation promises to improve further their applications, in particularly drug delivery. It is known that the uptake of tissue-associated MNPs is mainly carried out by macrophages. Yet, the molecular mechanism to control MNPs internalization in macrophages remains to be elusive. Missing-in-metastasis (MIM) is a scaffolding protein that is highly expressed in macrophages and regulates receptor-mediated endocytosis. We hypothesize that uptake of MNPs may also involve the function of MIM., Methods: We investigated the effect of MIM expression on the intracellular trafficking of MNPs by transmission electronic microscopy, flow cytometry, o-phenanthroline photometric analysis, Perl's staining, immunofluorescence microscopy and co-immunoprecipitation. To explore the molecular events in MIM-mediated MNPs uptake, we examined the effect of MNPs on the interaction of MIM with clathrin, Rab5 and Rab7., Results: Uptake of MNPs was significantly enhanced in cells overexpressing MIM. Upon exposure to MNPs, MIM was associated with clathrin light chain in endocytic vesicles and Rab7, a protein that regulates late endosomes. However, MNPs caused dissociation of MIM with Rab5, an early endosome-associated protein., Conclusions: MIM regulates internalization of MNPs via promoting their trafficking from plasma membrane to late endosomes., General Significance: Our data unveiled a novel pathway which MNPs internalization and intracellular trafficking in macrophages. This new pathway may allow us to control the uptake of MNPs within cells by targeting MIM, thereby improving their medical applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Salmonella Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, S. Typhimurium resides intracellularly in a specialized vesicle, called the Salmonella -containing vacuole (SCV) that utilizes the host endocytic vesicular transport pathway (VTP). Here, we probed the possible role of SUMOylation, a post-translation modification pathway, in SCV biology. Proteome analysis by complex mass-spectrometry (MS/MS) revealed a dramatically altered SUMO-proteome (SUMOylome) in S. Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, in vitro SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7 K175R ) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by S. Typhimurium ensures availability of long-lived but functionally compromised RAB7, which was beneficial to the pathogen. Overall, this SUMOylation-dependent switch of RAB7 controlled by S. Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond Salmonella -host crosstalk. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Most patients with chronic kidney disease (CKD) present with proteinuria and extracellular matrix (ECM) deposition in the interstitium. Matrix metalloproteinase-2 (MMP-2) is important for maintaining ECM metabolism and it affects the formation and development of CKD. Autophagy has been reported to be protective against renal tubular injury, but the role of autophagy related to ECM metabolism is unclear. Rab7 is a shared molecule of endocytosis and autophagy. The aim of this study is to explore the role of autophagy in regulating MMP-2 activity and to determine whether Rab7 functions in regulating MMP-2 activity and injury in albumin-overloaded TECs. In this study, abovine serum albumin (BSA)-overload rat model was first established and collagen deposition and deficient autophagic response were observed in vivo, and stimulation with albumin nanoparticles resulted in MMP-2 overactivation and obstructed autophagic flux induced by lysosomal dysfunction in vitro. Furthermore, overactivation of MMP-2 was mediated by its related regulatory molecules such as membrane-type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinase-2 (TIMP-2) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) on the membrane of TECs (HK-2 cellline). After up-regulating Rab7, albumin-induced MMP-2 overactivation was attenuated, which was reversed by chloroquine (CQ; an endocytosis inhibitor). In addition, our data indicated that up-regulation of Rab7 relieved epithelial-mesenchymal transition (EMT) and apoptosis in albumin-treated TECs. Taken together, our study demonstrated that autophagy regulates MMP-2 activity in a Rab7-dependent manner. Thus, Rab7 is a newly developed target for protecting TECs from albumin-induced injury., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Endosomal transport represents the primary mode for intracellular trafficking and signaling transduction and thus has to be tightly controlled. The molecular processes controlling the endosomal positioning utilize several large protein complexes, one of which contains the small GTPase Rab7, Rab-interacting lysosomal protein (RILP), and oxysterol-binding protein-related protein 1 (ORP1L). Rab7 is known to interact with RILP through a canonical binding site termed the effector-interacting switch region, but it is not clear how Rab7 interacts with ORP1L, limiting our understanding of the overall process. Here, we report structural and biochemical investigation of the Rab7-ORP1L interaction. We found that, contrary to prior studies, the interaction between Rab7 and the N-terminal ankyrin repeat domain (ARD N ) of ORP1L is independent of Rab7's GTP- or GDP-binding state. Moreover, we show that Rab7 interacts with ORP1L ARD N via a unique region consisting of helix3 (α3) and 3 10 -helix 2 (η2). This architecture leaves the canonical effector-interacting switch regions available for RILP binding and thus allows formation of the ORP1L-Rab7-RILP tripartite complex. Mutational disruption of the interacting interface between ORP1L and Rab7 compromised the ability of ORP1L-Rab7-RILP to regulate the late endosome positioning. Collectively, our results again manifested the versatility in the interaction between GTPase and its effector., (© 2018 Ma et al.)

The retromer complex is a vital component of the endosomal protein sorting machinery necessary for sorting into both the endosome-to-Golgi retrieval pathway and also the endosome-to-cell-surface recycling pathway. Retromer mediates cargo selection through a trimeric complex comprising VPS35, VPS29 and VPS26, which is recruited to endosomes by binding to Rab7a and Snx3. Retromer function is linked to two distinct neurodegenerative diseases, Parkinson's disease and Alzheimer's disease and modulating retromer function has been proposed as an avenue to explore for a putative therapy in these conditions. We hypothesised that activating Rab7a to promote the recruitment of retromer to endosomes could positively modulate its activity. Here, we show that inhibition of the GTPase activating protein TBC1D5 can enhance Rab7a activation and lead to a gain of function for retromer., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Macroautophagy (simply called autophagy hereafter) is an intracellular degradation mechanism that is activated by nutrient starvation. Although it is well known that starvation induces autophagosome formation in an mTORC1-dependent manner, whether starvation also regulates autophagosome or autolysosome maturation was unclear. In the present study, we succeeded in demonstrating that starvation activates autolysosome maturation in mammalian cells. We found that knockout (KO) of Rab7 (herein referring to the Rab7a isoform) caused an accumulation of a massive number of LC3-positive autolysosomes under nutrient-rich conditions, indicating that Rab7 is dispensable for autophagosome-lysosome fusion. Intriguingly, the autolysosomes that had accumulated in Rab7-KO cells matured and disappeared after starvation for a brief period (∼10 min), and we identified glutamine as an essential nutrient for autolysosome maturation. In contrast, forced inactivation of mTORC1 through treatment with its inhibitor Torin2 failed to induce autolysosome maturation, suggesting that the process is controlled by an mTORC1-independent mechanism. Since starvation-induced autolysosome maturation was also observed in wild-type cells, the nutrient-starvation-induced maturation of autolysosomes is likely to be a generalized mechanism in the same manner as starvation-induced autophagosome formation. Such multistep regulatory mechanisms would enable efficient autophagic flux during starvation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson's disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles., Competing Interests: KY, CW, SS, CM, RK, NN, YH, MK, KT, NM No competing interests declared, WH, RY Reviewing editor, eLife, (© 2018, Yamano et al.)

Retromer is an endosomal multi-protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans -Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer-associated RAB7-specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer-dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin-mediated mitophagy., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Since the lack of targeted treatment, triple-negative breast cancer (TNBC) has poor outcomes. Histone deacetylase inhibitors (HDACi) blocking the activity of specific HDACs have emerged as cancer therapeutic agents. However, the therapeutic efficiency is still not satisfactory for patients with solid tumor. We thus performed screening for the synergistic agents of Vorinostat (SAHA). The resulting candidate Simvastatin was obtained. The efficacy and mechanism of combination have been studied in TNBC cells. The synergism of SAHA and Simvastatin was evaluated by IC 50 of proliferation and combination index (CI). The antitumor activities of combination were further evaluated in TNBC cells. The pro-apoptotic effects were determined by flow cytometry and Western blot. Autophagosome-lysosome fusion was monitored using confocal microscope. The underlying mechanism was further studied by over-expressing of wild-type or inactive (C205S/C207S) Rab7 in compounds treated cells. The in vivo efficacy was also evaluated in mice. The combination of SAHA and Simvastatin had potent synergism in apoptosis of TNBC cells. It exerted pro-apoptosis effect by compromising the fusion between autophagosome and lysosome. Over-expressing of wild-type, but not inactive Rab7 rescued cells from apoptosis induced by the combinatory treatments. Mevalonate supplementation also decreased the combinatory treatment-induced apoptosis. These results indicate that the combinatory treatment enhances the apoptosis of TNBC cells by interrupting Rab7 prenylation and obstructing autophagosome-lysosome fusion. Combination between SAHA and Simvastatin could also significantly decrease the tumor growth in xenografted mice by inducing apoptosis and inhibiting Rab7 prenylation. Rab7 is a potential target for the combined effects of Simvastatin and SAHA., (Copyright © 2017. Published by Elsevier B.V.)

Macroautophagy is an evolutionally conserved membrane trafficking pathway that delivers intracellular materials to lysosomes for degradation and recycling. Rab7, as a member of the Rab GTPase superfamily, has a unique role in the regulation of macroautophagy, especially in modulating autophagy flux. The functional states of Rab7 generally switch between GTP-bound and GDP-bound states under the control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Activated GTP-Rab7 is capable of regulating autophagosome formation, autophagosome transportation along microtubules, endosome and autophagosome maturation, as well as lysosome biogenesis via interacting with its effector molecules. Rab7-mediated macroautophagy is closely associated with various pathological processes of several neurologic diseases, such as Parkinson's disease, Huntington's disease, Alzheimer's disease, Charcot-Marie-Tooth type 2B disease, and cerebral ischemic diseases. Considering that macroautophagy can be the prime therapeutic target in certain nervous system diseases, in-depth study of Rab7 in the regulation of macroautophagy may be helpful to identify novel strategies for the treatment of autophagy-related neurologic diseases. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: Alterations and mutations of endo-lysosomal trafficking proteins have been associated with cancer progression. Identification and characterization of endo-lysosomal trafficking proteins in invasive cholangiocarcinoma (CCA) cells may benefit prognosis and drug design for CCA., Objective: To identify and characterize endo-lysosomal trafficking proteins in invasive CCA., Methods: A lysosomal-enriched fraction was isolated from a TNF-α induced invasive CCA cell line (KKU-100) and uninduced control cells and protein identification was performed with nano-LC MS/MS. Novel lysosomal proteins that were upregulated in invasive CCA cells were validated by real-time RT-PCR. We selected Rab7 for further studies of protein level using western blotting and subcellular localization using immunofluorescence. The role of Rab7 in CCA invasion was determined by siRNA gene knockdown and matrigel transwell assay., Results: Rab7 mRNA and protein were upregulated in invasive CCA cells compared with non-treated controls. Immunofluorescence studies demonstrated that Rab7 was expressed predominantly in invasive CCA cells and was localized in the cytoplasm and lysosomes. Suppression of Rab7 translation significantly inhibited TNF-α-induced cell invasion compared to non-treated control (p= 0.044)., Conclusions: Overexpression of Rab7 in CCA cells was associated with cell invasion, supporting Rab7 as a novel candidate for the development of diagnostic and therapeutic strategies for CCA.

Retromer is a multimeric protein complex that mediates endosome-to-trans-Golgi network (TGN) and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's disease and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 (also known as RAB7A) to coordinate endosome-to-TGN trafficking of cargo receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins, such as the epidermal growth factor receptor (EGFR). We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 colocalizes efficiently with and has a higher propensity to interact with retromer than nonpalmitoylatable Rab7. Rescue of Rab7 knockout cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with nonpalmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of EGFR or for its interaction with its effector, Rab-interacting lysosomal protein (RILP). Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN trafficking of the lysosomal sorting receptors., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The small molecular weight GTPase Rab7 is a key regulator for late endosomal/lysosomal membrane trafficking, it was known that Rab7 is phosphorylated, but the corresponding kinase and the functional regulation of Rab7 phosphorylation remain unclear. We provide evidence here that Rab7 is a substrate of Src kinase, and is tyrosine-phosphorylated by Src, withY183 residue of Rab7 being the optimal phosphorylation site for Src. Further investigations demonstrated that the tyrosine phosphorylation of Rab7 depends on the guanine nucleotide binding activity of Rab7 and the activity of Src kinase. The tyrosine phosphorylation of Rab7 is physiologically induced by EGF, and impairs the interaction of Rab7 with RILP, consequently inhibiting EGFR degradation and sustaining Akt signaling. These results suggest that the tyrosine phosphorylation of Rab7 may be involved in coordinating membrane trafficking and cell signaling., (Copyright © 2017. Published by Elsevier Inc.)

Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID 50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P 2 , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1 Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1 ; however, deleting both genes did not rescue the pah1 Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Charcot-Marie-Tooth 2B peripheral sensory neuropathy (CMT2B) is a debilitating autosomal dominant hereditary sensory neuropathy. Patients with this disease lose pain sensation and frequently need amputation. Axonal dysfunction and degeneration of peripheral sensory neurons is a major clinical manifestation of CMT2B. However, the cellular and molecular pathogenic mechanisms remain undefined. CMT2B is caused by missense point mutations (L129F, K157N, N161T/I, V162M) in Rab7 GTPase. Strong evidence suggests that the Rab7 mutation(s) enhances the cellular levels of activated Rab7 proteins, thus resulting in increased lysosomal activity and autophagy. As a consequence, trafficking and signaling of neurotrophic factors such as nerve growth factor (NGF) in the long axons of peripheral sensory neurons are particularly vulnerable to premature degradation. A "gain of toxicity" model has, thus, been proposed based on these observations. However, studies of fly photo-sensory neurons indicate that the Rab7 mutation(s) causes a "loss of function", resulting in haploinsufficiency. In the review, we summarize experimental evidence for both hypotheses. We argue that better models (rodent animals and human neurons) of CMT2B are needed to precisely define the disease mechanisms.

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34 -/- mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein-lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Endocytosis is a multistep process engaged in extracellular molecules internalization. Several proteins including the Rab GTPases family coordinate the endocytic pathway. The small GTPase Rab7 is present in late endosome (LE) compartments being a marker of endosome maturation. The Rab interacting lysosomal protein (RILP) is a downstream effector of Rab7 that recruits the functional dynein/dynactin motor complex to late compartments. In the present study, we have found Rab24 as a component of the endosome-lysosome degradative pathway. Rab24 is an atypical protein of the Rab GTPase family, which has been attributed a function in vesicle trafficking and autophagosome maturation. Using a model of transiently expressed proteins in K562 cells, we found that Rab24 co-localizes in vesicular structures labeled with Rab7 and LAMP1. Moreover, using a dominant negative mutant of Rab24 or a siRNA-Rab24 we showed that the distribution of Rab7 in vesicles depends on a functional Rab24 to allow DQ-BSA protein degradation. Additionally, by immunoprecipitation and pull down assays, we have demonstrated that Rab24 interacts with Rab7 and RILP. Interestingly, overexpression of the Vps41 subunit from the homotypic fusion and protein-sorting (HOPS) complex hampered the co-localization of Rab24 with RILP or with the lysosomal GTPase Arl8b, suggesting that Vps41 would affect the Rab24/RILP association. In summary, our data strongly support the hypothesis that Rab24 forms a complex with Rab7 and RILP on the membranes of late compartments. Our work provides new insights into the molecular function of Rab24 in the last steps of the endosomal degradative pathway., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)