Expression Of Rab7 Research Articles

Expression profile of bio-defense genes in Penaeus monodon gills in response to formalin inactivated white spot syndrome virus vaccine

White spot syndrome virus (WSSV) is the most devastating pathogen of penaeid shrimp. While developing technology to vaccinate shrimp against WSSV, it is imperative to look into the immune response of the animal at molecular level. However, very little information has been generated in this direction. The present study is an attempt to understand the expression of bio-defense genes in gill tissues of Penaeus monodon in response to formalin inactivated WSSV. A WSSV vaccine with a viral titer of 1×109 DNA copies was prepared and orally administered to P. monodon at a rate of 1.75×106 DNA copies of inactivated virus preparation (IVP) day−1 for 7days. The animals were challenged with WSSV on 1st and 5th day post vaccination, and temporal expression of bio-defense genes in gill tissues was studied. Survival of 100% and 50% were observed respectively on 1st and 5th day post vaccination challenge. The humoral immune genes prophenoloxidase (proPO), alpha 2-macroglobulin (α2M), crustin and PmRACK, and the cell mediated immune genes caspase and Rab7 were up regulated in gill tissue upon vaccination and challenge. The expression of humoral gene crustin and cellular gene Rab7 was related to survival in IVP administered shrimp. Results of the study suggest that these genes have roles in protecting shrimp from WSSV on vaccination.

PKCι interacts with Rab14 and modulates epithelial barrier function through regulation of claudin-2 levels

PKCι is essential for the establishment of epithelial polarity and the normal assembly of tight junctions. We find that PKCι knockdown does not compromise the steady-state distribution of most tight junction proteins but results in increased transepithelial resistance (TER) and decreased paracellular permeability. Analysis of the levels of tight junction components demonstrates that claudin-2 protein levels are decreased. However, other tight junction proteins, such as claudin-1, ZO-1, and occludin, are unchanged. Incubation with an aPKC pseudosubstrate recapitulates the phenotype of PKCι knockdown, including increased TER and decreased levels of claudin-2. In addition, overexpression of PKCι results in increased claudin-2 levels. ELISA and coimmunoprecipitation show that the TGN/endosomal small GTPase Rab14 and PKCι interact directly. Immunolabeling shows that PKCι and Rab14 colocalize in both intracellular puncta and at the plasma membrane and that Rab14 expression is required for normal PKCι distribution in cysts in 3D culture. We showed previously that knockdown of Rab14 results in increased TER and decreased claudin-2. Our results suggest that Rab14 and aPKC interact to regulate trafficking of claudin-2 out of the lysosome-directed pathway.

Rab25 upregulation correlates with the proliferation, migration, and invasion of renal cell carcinoma

Renal cell carcinoma (RCC) is a common urological cancer with a poor prognosis. A recent cohort study revealed that the median survival of RCC patients was only 1.5 years and that <10% of the patients in the study survived up to 5 years. In tumor development, Rab GTPase are known to play potential roles such as regulation of cell proliferation, migration, invasion, communication, and drug resistance in multiple tumors. However, the correlation between Rabs expression and the occurrence, development, and metastasis of RCC remains unclear. In this study, we analyzed the transcriptional levels of 52 Rab GTPases in RCC patients. Our results showed that high levels of Rab25 expression were significantly correlated with RCC invasion classification (P < 0.01), lymph-node metastasis (P < 0.001), and pathological stage (P < 0.01). Conversely, in 786-O and A-498 cells, knocking down Rab25 protein expression inhibited cell proliferation, migration, and invasion. Our results also demonstrated that Rab25 is a target gene of let-7d, and further suggested that Rab25 upregulation in RCC is due to diminished expression of let-7d. These findings indicate that Rab25 might be a novel candidate molecule involved in RCC development, thus identifying a potential biological therapeutic target for RCC.

Expression of Ras-related protein 25 predicts chemotherapy resistance and prognosis in advanced non-small cell lung cancer.

Ras-related protein 25 (Rab25) is involved in many human malignancies. However, its role in chemotherapy response and prognosis in advanced non-small cell lung cancer (NSCLC) remains unknown. Therefore, we investigated the relationship between Rab25 and chemotherapy sensitivity and prognosis in NSCLC. Rab25 expression was assessed using immunohistochemistry in 324 advanced NSCLC patients. Its correlations with clinical features were analyzed. Sensitivity to cisplatin (DDP) was compared between DDP-sensitive A549 and DDP-resistant A549/DDP cells. Furthermore, small interfering RNA (siRNA) targeting Rab25 was used for in vitro experiments. Patients with positive Rab25 expression had a significantly lower chemotherapy response rate (P = 0.004) and poorer overall survival (OS, P = 0.0012) than those with negative Rab25 expression. Multivariate analysis indicated that Rab25 expression was an independent prognostic factor for OS (P = 0.016). Moreover, Rab25 expression was significantly higher in A549/DDP cells than in A549 cells. Knockdown of Rab25 by siRNA suppressed cell migration and invasion. Cisplatin resistance in A549/DDP cells was also partially reversed by Rab25 silencing. Rab25 expression is a potential prognostic index for advanced NSCLC patients and its inhibition may improve chemosensitization in NSCLC treatment.

Inactivation of Rab23 inhibits the invasion and motility of pancreatic duct adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is charac-terized by a poor prognosis and high mortality rate. In this study, we investigated the expression of Rab23 in non-tumor pancreatic tissues and PDACs via immunohistochemistry. Rab23 was found in 39 of 58 (67.2%) and in 11 of 30 (36.7%) of the PDAC and non-tumor pan-creatic tissue samples (P = 0.0073), respectively. There were signifi-cant correlations between Rab23 expression and unfavorable variables, including cancer differentiation level (P = 0.0089), lymph nodal (P = 0.0099), and distant metastases (P = 0.0173). Inactivation with small interfering RNA against Rab23 in the human pancreatic cancer cell line Panc-1 inhibited the migration and invasive potential of the cells. Our data provide new insight into the essential role of Rab23 in PDAC inva-sion and metastasis and suggest that Rab23 expression is a useful indi-cator of metastatic potential; hence, it may be a new therapeutic target for this common malignancy.

Molecular cloning, characterization, and expression of Rab5B, Rab6A, and Rab7 from Litopenaeus vannamei (Penaeidae).

The Rab protein family belongs to a superfamily of ras-like GTP-binding proteins. Rab proteins regulate many steps of membrane trafficking. In this study, three Rab family members, Rab5B, Rab6A, and Rab7, designated LvRab5B, LvRab6A, and LvRab7, were cloned from Litopenaeus vannamei. The full-length cDNA sequences of LvRab5B, LvRab6A, and LvRab7 were 1383, 873, and 767 nucleotides in length and they encoded proteins of 211, 212, and 205 amino acids, respectively. Using qRT-PCR, the mRNA expression levels of the three proteins were determined in the hepatopancreas of L. vannamei at different stages after infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus challenge. The results indicated that the mRNA expression levels of LvRab5B, LvRab6A, and LvRab7 were all significantly up-regulated after virus injection, suggesting that these genes may play essential roles in the immune response to viral infection in shrimp.

Claudin-7 expression induces mesenchymal to epithelial transformation (MET) to inhibit colon tumorigenesis.

In normal colon, claudin-7 is one of the highly expressed claudin proteins and its knockdown in mice results in altered epithelial cell homeostasis and neonatal death. Notably, dysregulation of the epithelial homeostasis potentiates oncogenic transformation and growth. However, the role of claudin-7 in the regulation of colon tumorigenesis remains poorly understood. Using a large colorectal cancer (CRC) patient database and mouse models of colon cancer, we found claudin-7 expression to be significantly downregulated in cancer samples. Most notably, forced claudin-7 expression in poorly differentiated and highly metastatic SW620 colon cancer cells induced epithelial characteristics and inhibited their growth in soft agar and tumor growth in vivo. By contrast, knockdown of claudin-7 in HT-29 or DLD-1 cells induced epithelial-to-mesenchymal transition (EMT), colony formation, xenograft-tumor growth in athymic mice and invasion. Importantly, a claudin-7 signature gene profile generated by overlapping the DEGs (differentially expressed genes in a high-throughput transcriptome analysis using claudin-7-manipulated cells) with human claudin-7 signature genes identified high-risk CRC patients. Furthermore, Rab25, a colon cancer suppressor and regulator of the polarized cell trafficking constituted one of the highly upregulated DEGs in claudin-7 overexpressing cells. Notably, silencing of Rab25 expression counteracted the effects of claudin-7 expression and not only increased proliferation and cell invasion but also increased the expression of p-Src and mitogen-activated protein kinase-extracellular signal-regulated kinase 1/2 that were suppressed upon claudin-7 overexpression. Of interest, CRC cell lines, which exhibited decreased claudin-7 expression, also exhibited promoter DNA hypermethylation, a modification associated with transcriptional silencing. Taken together, our data demonstrate a previously undescribed role of claudin-7 as a colon cancer suppressor and suggest that loss of claudin-7 potentiates EMT to promote colon cancer, in a manner dependent on Rab25.

The microRNA-367 inhibits the invasion and metastasis of gastric cancer by directly repressing Rab23.

Dysregulated expression of microRNAs is often found in gastric cancer, and it contributes to the pathogenesis of gastric cancer via regulation of the cell cycle, proliferation, apoptosis, migration, and invasion. In this study, we aimed to investigate the role of miR-367 in the invasion and metastasis of gastric cancer. The correlation between the expression level of miR-367 and the clinicopathologic features of 37 patients with gastric cancer was analyzed by using real-time polymerase chain reaction (RT-PCR). In addition, we investigated the effect of miR-367 on the invasion and migration of the gastric cancer cell lines HS746T and SGC-7901 using transwell and scratch-wound assays, and the target gene of miR-367 was predicated and demonstrated by the bioinformatics method and luciferase reporter system, respectively. The results showed that the expression of miR-367 was significantly reduced in the gastric cancer tissues compared with the paraneoplastic tissues, and significantly correlated with the differentiation level, tumor-node-metastasis (TNM) stage, and metastasis of gastric cancer. Notably, the overexpression of miR-367 in gastric cancer cells inhibited the cellular migration and invasion. Furthermore, the luciferase reporter system demonstrated that Rab23 was a target gene of miR-367, and ectopic expression of Rab23 could reverse the invasion and migration inhibitory activity of miR-367. Our study shows that miR-367 is a key negative regulator of the invasion and metastasis of gastric cancer and establishes a strong rationale for developing miR-367 as a novel therapeutic agent against gastric cancer.

ASSA14-03-20 Cellular repressor of E1A-stimulated genes protects against angiotensin II-induced vascular remodelling via degradation of angiotensin II type 1 receptor

Background Cellular repressor of E1A-stimulated genes (CREG), a new cardiovascular homeostasis regulator, has been proposed to reduce the neointimal formation after vascular injury via the maintenance of the quiescent mature vascular smooth muscle cell (VSMC) phenotype. We hypothesised that CREG is a negative regulator of angiotensin (Ang) II-mediated vascular remodelling. Methods Ten-week-old male heterozygous CREG-deficient (CREG +/- ) mice and their littermate wild-type (WT) mice were used. An osmotic minipump was implanted subcutaneously to infuse a dose of Ang II (1.5 mg/kg · d) or saline (vehicle) for 14 days. Ang II-infused WT mice were then treated with placebo or recombinant human CREG (rhCREG; 300 μg/kg · d). Systolic blood pressure (SBP); extent of vascular remodelling; protein level of collagen type I/III, angiotensin-converting enzyme-2 (ACE2), angiotensin receptor type 1 (AT1R) and Rab7 were evaluated. Primary VSMC culture was performed to assess AT1R and collagen protein expression before and after CREG gene transfection and knockdown. Results CREG levels are high in vascular media under basal conditions but rapidly decrease in response to Ang II. Ang II infusion for 14 days resulted that levels of SBP, intima medial thickness and vascular remodelling of the aorta and mesenteric artery were significantly greater in CREG +/- mice compared with the WT controls. Vascular gene expression level of CREG was lower in Ang II-treated CREG +/- mice than in WT mice. However, daily treatment of Ang II-infused WT mice with rhCREG blunted the increase of SBP. Ang II-mediated vascular remodelling and expression of collagen type I/III were also suppressed by rhCREG. Moreover, rhCREG treatment inhibited Ang II-mediated up-regulation of AT1R expression and down-regulation of ACE2 expression. Ang II-induced vascular remodelling was inhibited by rhCREG in association with reduced plasma Ang II and increased plasma Ang 1–7 levels. In adult VSMCs, CREG over-expression increased the expression of Rab7 by a regulatory mechanism of ubiquitination. Importantly, Ang II-mediated collagen production and AT1R expression was inhibited by CREG over-expression and augmented by CREG knock-down in a rab7-dependent manner. Conclusions Elevated Ang II induced hypertension and vascular remodelling, which were exacerbated by CREG deficiency, whereas rhCREG attenuated Ang II-induced adverse vascular remodelling. Hence, CREG is an important negative regulator of Ang II-induced vascular disease and suppresses adverse vascular remodelling.

MIR144 and MIR451 regulate human erythropoiesis via RAB14.

Expression levels of MIR144 and MIR451 increase during erythropoiesis, a pattern that is conserved from zebrafish to humans. As these two miRs are expressed from the same polycistronic transcript, we manipulated MIR144 and MIR451 in human erythroid cells individually and together to investigate their effects on human erythropoiesis. Inhibition of endogenous human MIR451 resulted in decreased numbers of erythroid (CD71(hi) CD235a(hi) CD34(-) ) cells, consistent with prior studies in zebrafish and mice. In addition, inhibition of MIR144 impaired human erythroid differentiation, unlike in zebrafish and mouse studies where the functional effect of MIR144 on erythropoiesis was minimal. In this study, we found RAB14 is a direct target of both MIR144 and MIR451. As MIR144 and MIR451 expression increased during human erythropoiesis, RAB14 protein expression decreased. Enforced RAB14 expression phenocopied the effect of MIR144 and/or MIR451 depletion, whereas shRNA-mediated RAB14 knockdown protected cells from MIR144 and/or MIR451 depletion-mediated erythropoietic inhibition. RAB14 knockdown increased the frequency and number of erythroid cells, increased β-haemoglobin expression, and decreased CBFA2T3 expression during human erythropoiesis. In summary, we utilized MIR144 and MIR451 to identify RAB14 as a novel physiological inhibitor of human erythropoiesis.

Epstein-barr virus blocks the autophagic flux and appropriates the autophagic machinery to enhance viral replication.

Autophagy is a catabolic pathway that helps cells to survive under stressful conditions. Cells also use autophagy to clear microbiological infections, but microbes have learned how to manipulate the autophagic pathway for their own benefit. The experimental evidence obtained in this study suggests that the autophagic flux is blocked at the final steps during the reactivation of Epstein-Barr virus (EBV) from latency. This is indicated by the level of the lipidated form of LC3 that does not increase in the presence of bafilomycin and by the lack of colocalization of autophagosomes with lysosomes, which correlates with reduced Rab7 expression. Since the inhibition of the early phases of autophagy impaired EBV replication and viral particles were observed in autophagic vesicles in the cytoplasm of producing cells, we suggest that EBV exploits the autophagic machinery for its transportation in order to enhance viral production. The autophagic block was not mediated by ZEBRA, an immediate-early EBV lytic gene, whose transfection in Ramos, Akata, and 293 cells promoted a complete autophagic flux. The block occurred only when the complete set of EBV lytic genes was expressed. We suggest that the inhibition of the early autophagic steps or finding strategies to overcome the autophagic block, allowing viral degradation into the lysosomes, can be exploited to manipulate EBV replication. This study shows, for the first time, that autophagy is blocked at the final degradative steps during EBV replication in several cell types. Through this block, EBV hijacks the autophagic vesicles for its intracellular transportation and enhances viral production. A better understanding of virus-host interactions could help in the design of new therapeutic approaches against EBV-associated malignancies.

Rab25 expression predicts poor prognosis in clear cell renal cell carcinoma.

Rab25 has been implicated in a number of types of cancer. However, its expression status and clinical implications in clear cell renal cell carcinoma (ccRCC) remain to be investigated. The purpose of this study was to investigate the significance of Rab25 status in patients with ccRCC. Rab25 expression was determined by western blot analysis in 30 fresh ccRCC samples. Immunohistochemistry was performed on the ccRCC samples and paired adjacent noncancerous tissues from 107 patients with ccRCC who had undergone surgery. The prognostic role and correlations with other clinicopathological factors were evaluated. Rab25 expression was upregulated in ccRCC tissues compared with that in paired adjacent noncancerous tissues. A high expression of Rab25 protein was significantly correlated with the primary tumor stage; lymph node metastasis; distant metastasis; tumor, node and metastasis stage and histological grade. A Kaplan-Meier survival analysis by log-rank test demonstrated that elevated Rab25 expression predicted lower overall survival time in patients with ccRCC. Notably, multivariate analyses revealed that expression of Rab25 was an independent prognostic factor in ccRCC (hazard ratio, 3.43; 95% confidence interval, 1.13–10.38; P=0.023). In conclusion, Rab25 is a potential prognostic biomarker in ccRCC.

The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages.

The cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrPC in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrPC in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrPC promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrPC suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrPC as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

Rab31 is expressed in neural progenitor cells and plays a role in their differentiation

Rab31 is expressed in both GFAP- and nestin- positive fibres in regions of neurogenic potential in the adult mouse brain. To investigate the role of Rab31 in neural progenitor cells (NPCs), we cultured NPCs and found significant levels of Rab31 expression in these cells. Rab31 levels showed a sharp initial decrease and then reappeared gradually in a subpopulation of astrocytes when NPCs were induced to differentiate. Silencing of Rab31 hindered, while overexpression enhanced, the differentiation of NPCs to astrocytes. Our results suggest a previously unrecognised role for Rab31 in influencing the differentiation and fate of NPCs.

CREG1 ameliorates myocardial fibrosis associated with autophagy activation and Rab7 expression

In cardiomyocytes subjected to stress, autophagy activation is a critical survival mechanism that preserves cellular energy status while degrading damaged proteins and organelles. However, little is known about the mechanisms that govern this autophagic response. Cellular repressor of E1A genes (CREG1) is an evolutionarily conserved lysosomal protein, and an important new factor in regulating tissue homeostasis that has been shown to antagonize injury of tissues or cells. In the present study, we aimed to investigate the regulatory role of CREG1 in cardiac autophagy, and to clarify autophagy activation mechanisms. First, we generated a CREG1 haploinsufficiency (Creg1+/−) mouse model, and identified that CREG1 deficiency aggravates myocardial fibrosis in response to aging or angiotensin II (Ang II). Conversely, exogenous infusion of recombinant CREG1 protein completely reversed cardiac damage. CREG1 deficiency in Creg1+/− mouse heart showed a marked accumulation of autophagosome that acquired LC3II and beclin-1, and a decrease in autophagic flux clearance as indicated by upregulating the level of p62. Inversely, restoration of CREG1 activates cardiac autophagy. Furthermore, chloroquine, an inhibitor of lysosomal acidification, was used to confirm that CREG1 protected the heart tissue against Ang II-induced fibrosis by activating autophagy. Using adenoviral infection of primary cardiomyocytes, overexpression of CREG1 with concurrent resveratrol treatment significantly increased autophagy, while silencing CREG1 blocked the resveratrol-induced autophagy. These results suggest that CREG1-induced autophagy is required to maintain heart function in the face of stress-induced myocardiac damage. Both in vitro and in vivo studies identified that CREG1 deficiency influenced the maturation of lysosomes and reduced the expression of Rab7, which might be involved in CREG1-induced cardiomyocyte autophagy. These findings suggest that autophagy activation via CREG1 may be a viable therapeutic strategy for improving cardiac performance under pathologic conditions. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice.

Autophagic dysfunction is observed in diabetes mellitus. Resveratrol has a beneficial effect on diabetic cardiomyopathy. Whether the resveratrol-induced improvement in cardiac function in diabetes is via regulating autophagy remains unclear. We investigated the mechanisms underlying resveratrol-mediated protection against heart failure in diabetic mice, with a focus on the role of sirtuin 1 (SIRT1) in regulating autophagic flux. Diabetic cardiomyopathy in mice was induced by streptozotocin (STZ). Long-term resveratrol treatment improved cardiac function, ameliorated oxidative injury and reduced apoptosis in the diabetic mouse heart. Western blot analysis revealed that resveratrol decreased p62 protein expression and promoted SIRT1 activity and Rab7 expression. Inhibiting autophagic flux with bafilomycin A1 increased diabetic mouse mortality and attenuated resveratrol-induced down-regulation of p62, but not SIRT1 activity or Rab7 expression in diabetic mouse hearts. In cultured H9C2 cells, redundant or overactive H2O2 increased p62 and cleaved caspase 3 expression as well as acetylated forkhead box protein O1 (FOXO1) and inhibited SIRT1 expression. Sirtinol, SIRT1 and Rab7 siRNA impaired the resveratrol amelioration of dysfunctional autophagic flux and reduced apoptosis under oxidative conditions. Furthermore, resveratrol enhanced FOXO1 DNA binding at the Rab7 promoter region through a SIRT1-dependent pathway. These results highlight the role of the SIRT1/FOXO1/Rab7 axis in the effect of resveratrol on autophagic flux in vivo and in vitro, which suggests a therapeutic strategy for diabetic cardiomyopathy.

Molecular Mechanisms of TLR2-Mediated Antigen Cross-Presentation in Dendritic Cells

Cross-presentation is a key function of dendritic cells (DCs), which present exogenous Ags on MHC class I molecules to prime CTL responses. The effects of TLR triggering on the cross-presentation of exogenous Ags by DCs remain unclear. In this study, we used synthetic dipalmitoylated peptides and TLR2 agonist-conjugated peptides as models to elucidate the mechanisms of TLR2-mediated cross-presentation. We observed that the internalization of dipalmitoylated peptides by bone marrow-derived DCs was facilitated by TLR2 via clathrin-mediated endocytosis. The administration of these dipalmitoylated peptide-pulsed bone marrow-derived DCs eliminated established tumors through TLR2 signaling. We further demonstrated that the induction of Ag-specific CTL responses and tumor regression by dipalmitoylated peptides was TAP independent. In addition, presentation of dipalmitoylated peptides by MHC class I molecules was blocked in the presence of an endosomal acidification inhibitor (chloroquine) or a lysosomal degradation inhibitor (Z-FL-COCHO). The endocytosed dipalmitoylated peptide also passed rapidly from early endosome Ag-1-positive endosomes to RAS-related GTP-binding protein 7 (Rab7)-associated late endosomes compared with their nonlipidated counterparts. Furthermore, we found that dipalmitoylated peptide-upregulated Rab7 expression correlated with Ag presentation via the TLR2/MyD88 pathway. Both JNK and ERK signaling pathways are required for upregulation of Rab7. In summary, our data suggest that TLR2-mediated cross-presentation occurs through the upregulation of Rab7 and a TAP-independent pathway that prime CTL responses.

ARHI (DIRAS3) induces autophagy in ovarian cancer cells by downregulating the epidermal growth factor receptor, inhibiting PI3K and Ras/MAP signaling and activating the FOXo3a-mediated induction of Rab7.

The process of autophagy has been described in detail at the molecular level in normal cells, but less is known of its regulation in cancer cells. Aplasia Ras homolog member I (ARHI; DIRAS3) is an imprinted tumor suppressor gene that is downregulated in multiple malignancies including ovarian cancer. Re-expression of ARHI slows proliferation, inhibits motility, induces autophagy and produces tumor dormancy. Our previous studies have implicated autophagy in the survival of dormant ovarian cancer cells and have shown that ARHI is required for autophagy induced by starvation or rapamycin treatment. Re-expression of ARHI in ovarian cancer cells blocks signaling through the PI3K and Ras/MAP pathways, which, in turn, downregulates mTOR and initiates autophagy. Here we show that ARHI is required for autophagy-meditated cancer cell arrest and ARHI inhibits signaling through PI3K/AKT and Ras/MAP by enhancing internalization and degradation of the epidermal growth factor receptor. ARHI-mediated downregulation of PI3K/AKT and Ras/ERK signaling also decreases phosphorylation of FOXo3a, which sequesters this transcription factor in the nucleus. Nuclear retention of FOXo3a induces ATG4 and MAP-LC3-I, required for maturation of autophagosomes, and also increases the expression of Rab7, required for fusion of autophagosomes with lysosomes. Following the knockdown of FOXo3a or Rab7, autophagolysosome formation was observed but was markedly inhibited, resulting in numerous enlarged autophagosomes. ARHI expression correlates with LC3 expression and FOXo3a nuclear localization in surgical specimens of ovarian cancer. Thus, ARHI contributes to the induction of autophagy through multiple mechanisms in ovarian cancer cells.

Rab11 Endosomes Contribute to Mitotic Spindle Organization and Orientation

During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 endosomes in mitosis. Here, we show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. We found that mitotic recycling endosomes bind γ-TuRC components and associate with tubulin invitro. Rab11 depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores γ-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. We propose that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein.

Human Rab7 mutation mimics features of Charcot–Marie–Tooth neuropathy type 2B in Drosophila

Charcot–Marie–Tooth disease type 2B (CMT2B) is an inherited axonal peripheral neuropathy. It is characterised by prominent sensory loss, often complicated by severe ulcero-mutilations of toes or feet, and variable motor involvement. Missense mutations in RAB7A, the gene encoding the small GTPase Rab7, cause CMT2B and increase Rab7 activity. Rab7 is ubiquitously expressed and is involved in degradation through the lysosomal pathway. In the neurons, Rab7 plays a role in the long-range retrograde transport of signalling endosomes in the axons. Here we developed the first animal model of CMT2B, modelling one of the mutations (L129F) in Drosophila melanogaster. Behavioural assays show that this model recapitulates several hallmarks of the human disease. Upon expression of mutant Rab7 in the sensory neurons, larvae present with a reduction of temperature and pain perception. Furthermore, the larvae exhibit a crawling defect when the mutant protein is expressed in the motor neurons. Analysis of axonal transport of Rab7 positive vesicles in sensory neurons of Drosophila larvae and in neurites of mammalian neuroblastoma cells demonstrates that mutant vesicles pause less than their wild-type counterparts. This latter finding indicates that alterations in vesicle transport might contribute to the pathomechanism of CMT2B.