Rab11 Family-interacting Protein Research Articles

Myosin Va-dependent Transport of NMDA Receptors in Hippocampal Neurons.

N-methyl-D-aspartate receptor (NMDAR) trafficking is a key process in the regulation of synaptic efficacy and brain function. However, the molecular mechanism underlying the surface transport of NMDARs is largely unknown. Here we identified myosin Va (MyoVa) as the specific motor protein that traffics NMDARs in hippocampal neurons. We found that MyoVa associates with NMDARs through its cargo binding domain. This association was increased during NMDAR surface transport. Knockdown of MyoVa suppressed NMDAR transport. We further demonstrated that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates NMDAR transport through its direct interaction with MyoVa. Furthermore, MyoVa employed Rab11 family-interacting protein 3 (Rab11/FIP3) as the adaptor proteins to couple themselves with NMDARs during their transport. Accordingly, the knockdown of FIP3 impairs hippocampal memory. Together, we conclude that in hippocampal neurons, MyoVa conducts active transport of NMDARs in a CaMKII-dependent manner.

IKZF4/NONO-RAB11FIP3 axis promotes immune evasion in gastric cancer via facilitating PD-L1 endosome recycling

As an immune checkpoint protein expressed by diverse cancer cells, programmed death ligand 1 (PD-L1) facilitates immune evasion by interacting with programmed cell death-1 (PD-1) on T cells. Despite the clinical benefits observed in various cancer types, strategies targeting PD-1/PD-L1 have demonstrated limited efficacy in gastric cancer (GC). Furthermore, the regulation of PD-L1, especially at post-translational modification levels, remains largely unknown. Therefore, it is crucial to elucidate the mechanisms governing PD-L1 expression to enhance anti-tumor immunity. In this study, we have identified that IKAROS family zinc finger 4 (IKZF4) and Non-POU domain-containing octamer-binding (NONO) synergistically regulate and enhance the expression of RAB11 family-interacting protein 3 (RAB11FIP3) in GC. The IKZF4/NONO-RAB11FIP3 axis facilitates the endosomal recycling of PD-L1, particularly on the cell membrane of GC cells. Moreover, overexpression of RAB11FIP3 mitigates the hypo-expression of PD-L1 protein resulting from IKZF4 or NONO deletion. Functionally, the silencing of RAB11FIP3 or IKZF4 promotes T cell proliferation, and enhances T-cell cytotoxicity towards GC cells in vitro, which further inhibits tumor immune evasion in mice via increasing the infiltration of CD8+ T cells into the tumor microenvironment (TME) to suppress GC progression. Our study suggests that the IKZF4/NONO-RAB11FIP3 axis promotes immune evasion by facilitating PD-L1 endosome recycling, thus presenting a potential therapeutic target for GC treatment.

Biological Function of Rab11-Family Interacting Protein 4 in the Development of Preeclampsia and Their Underlying Mechanism

To elucidate the biological function of Rab11-FIP4 in the development of preeclampsia, as well as its clinical significance and underlying mechanism. Relative levels of Rab11-FIP4, IL-6 and TNF-α in placental tissues of PE pregnant women and healthy pregnant women were detected by qRT-PCR. The correlation between Rab11-FIP4 and IL-6 or TNF-α was assessed by Pearson correlation test. After constructing overexpression lentivirus targeting Rab11-FIP4, its influences on biological characteristics of HTR-8/SVneo cells and expression levels of inflammatory cytokines were examined. Rab11-FIP4 was highly expressed in placental tissues of PE pregnant women. Overexpression of Rab11-FIP4 enhanced migratory and invasive capacities of trophoblasts, and upregulated inflammatory proteins. Rab11-FIP4 aggravates the development of PE by upregulating inflammatory proteins, and stimulating migratory and invasive capacities of trophoblasts.

Rab11FIP1-deficient mice develop spontaneous inflammation and show increased susceptibility to colon damage.

The small GTPase, Rab11a, regulates vesicle trafficking and cell polarity in epithelial cells through interaction with Rab11 family-interacting proteins (Rab11-FIPs). We hypothesized that deficiency of Rab11-FIP1 would affect mucosal integrity in the intestine. Global Rab11FIP1 knockout (KO) mice were generated by deletion of the second exon. Pathology of intestinal tissues was analyzed by immunostaining of colonic sections and RNA-sequencing of isolated colonic epithelial cells. A low concentration of dextran sodium sulfate (DSS, 2%) was added to drinking water for 5 days, and injury score was compared between Rab11FIP1 KO, Rab11FIP2 KO, and heterozygous littermates. Rab11FIP1 KO mice showed normal fertility and body weight gain. More frequent lymphoid patches and infiltration of macrophages and neutrophils were identified in Rab11FIP1 KO mice before the development of rectal prolapse compared with control mice. The population of trefoil factor 3 (TFF3)-positive goblet cells was significantly lower, and the ratio of proliferative to nonproliferative cells was higher in Rab11FIP1 KO colons. Transcription signatures indicated that Rab11FIP1 deletion downregulated genes that mediate stress tolerance response, whereas genes mediating the response to infection were significantly upregulated, consistent with the inflammatory responses in the steady state. Lack of Rab11FIP1 also resulted in abnormal accumulation of subapical vesicles in colonocytes and the internalization of transmembrane mucin, MUC13, with Rab14. After DSS treatment, Rab11FIP1 KO mice showed greater body weight loss and more severe mucosal damage than those in heterozygous littermates. These findings suggest that Rab11FIP1 is important for cytoprotection mechanisms and for the maintenance of colonic mucosal integrity.NEW & NOTEWORTHY Although Rab11FIP1 is important in membrane trafficking in epithelial cells, the gastrointestinal phenotype of Rab11FIP1 knockout (KO) mice had never been reported. This study demonstrated that Rab11FIP1 loss induces mistrafficking of Rab14 and MUC13 and decreases in colonic goblet cells, resulting in impaired mucosal integrity.

Rab11‐FIP1 Deficient Mice Develops Spontaneous Inflammation and Show DSS Susceptibility in the Colon

Background and Aims A small GTPase, Rab11a regulates vesicle trafficking and cell polarity in epithelial cells through interacting with Rab11 family-interacting proteins (Rab11-FIPs). Intestine-specific Rab11a knockout mice demonstrate epithelial hyperplasia, dysplasia, and immune cell infiltration, implicating that Rab11a-mediated protein trafficking is important to maintain mucosal homeostasis. Nevertheless, the specific phenotype for loss of Rab11a-binding protein family members, FIPs, had not been fully characterized. We hypothesize that the deficiency of Rab11-FIP1, also known as RCP, affects mucosal integrity in the intestine. Methods Global Rab11-FIP1 knockout (KO) mice were generated by deletion of the exon 2. The immunoreactivity for Rab11-FIP1 antibody in the intestinal epithelial cells was lost in Rab11-FIP1 KO mouse tissues. Pathology of intestinal tissues were analyzed by immunostaining of intestinal sections and RNA-sequencing of isolated colonic epithelial cells. Low concentration of sodium dextran sulfate (DSS, 2%) was added into drinking water for 5 days and colitis score was compared between Rab11-FIP1 KO and heterozygous littermates. Results Birth rate and body weight gain were normal in the Rab11-FIP1 KO mouse line and no gross phenotype was found by the age of 8 weeks. At the age of 8–12 weeks, some Rab11-FIP1 KO mice spontaneously developed rectal prolapse along with chronic inflammation. The morphology and frequency of trefoil factor (TFF)3+ goblet cells in the colon were altered by Rab11-FIP1 loss. The number of lymphoid patches and neutrophil infiltration, which was quantified by Ly6B.2+ cell numbers, in the colon were significantly increased by Rab11-FIP1 loss before the development of rectal prolapse compared to wild type and Rab11-FIP1 heterozygous mice. The frequency of CD3+ intraepithelial lymphocytes and F4/80+ macrophages in colonic lamina propria were also higher in Rab11-FIP1 KO mice. The crypt height and the ratio of Ki67+ proliferating cells were significantly higher in Rab11-FIP1 KO colon. After DSS treatment followed by a 24-hour-recovery period, Rab11-FIP1 KO mice showed greater body weight loss and more severe colonic mucosal damage than in heterozygous littermates. mRNA signatures indicated that Rab11-FIP1 deletion downregulates genes that mediate stress tolerance response, such as Hsp members, Reg4, Nox1, and Ces1g. Genes that mediate the response to infection, including Pla2g2a, Duox2, Abca13, reg3g, Pecam1, and Optn, were significantly upregulated in Rab11-FIP1 KO colon, consistent with spontaneous neutrophil infiltration. Conclusion Loss of Rab11-FIP1 increases spontaneous and DSS-induced inflammatory response, suggesting a decrease in cytoprotection mechanisms in the colon.

Phospho-Form Specific Substrates of Protein Kinase B (AKT1).

Protein kinase B (AKT1) is hyper-activated in diverse human tumors. AKT1 is activated by phosphorylation at two key regulatory sites, Thr308 and Ser473. Active AKT1 phosphorylates many, perhaps hundreds, of downstream cellular targets in the cytosol and nucleus. AKT1 is well-known for phosphorylating proteins that regulate cell survival and apoptosis, however, the full catalog of AKT1 substrates remains unknown. Using peptide arrays, we recently discovered that each phosphorylated form of AKT1 (pAKT1S473, pAKT1T308, and ppAKT1S473,T308) has a distinct substrate specificity, and these data were used to predict potential new AKT1 substrates. To test the high-confidence predictions, we synthesized target peptides representing putative AKT1 substrates. Peptides substrates were synthesized by solid phase synthesis and their purity was confirmed by mass spectrometry. Most of the predicted peptides showed phosphate accepting activity similar to or greater than that observed with a peptide derived from a well-established AKT1 substrate, glycogen synthase kinase 3β (GSK-3β). Among the novel substrates, AKT1 was most active with peptides representing PIP3-binding protein Rab11 family-interacting protein 2 and cysteinyl leukotriene receptor 1, indicating their potential role in AKT1-dependent cellular signaling. The ppAKT1S473,T308 enzyme was highly selective for peptides containing a patch of basic residues at −5, −4, −3 and aromatic residues (Phe/Tyr) at +1 positions from the phosphorylation site. The pAKT1S473 variant preferred more acidic peptides, Ser or Pro at +4, and was agnostic to the residue at −5. The data further support our hypothesis that Ser473 phosphorylation plays a key role in modulating AKT1 substrate selectivity.

Crystal structure of the Rab-binding domain of Rab11 family-interacting protein 2.

The small GTPases Rab11, Rab14 and Rab25 regulate membrane trafficking through the recruitment of Rab11 family-interacting proteins (FIPs) to endocytic compartments. FIPs are multi-domain effector proteins that have a highly conserved Rab-binding domain (RBD) at their C-termini. Several structures of complexes of Rab11 with RBDs have previously been determined, including those of Rab11-FIP2 and Rab11-FIP3. In addition, the structures of the Rab14-FIP1 and Rab25-FIP2 complexes have been determined. All of the RBD structures contain a central parallel coiled coil in the RBD that binds to the switch 1 and switch 2 regions of the Rab. Here, the crystal structure of the uncomplexed RBD of FIP2 is presented at 2.3 Å resolution. The structure reveals antiparallel α-helices that associate through polar interactions. These include a remarkable stack of arginine residues within a four-helix bundle in the crystal lattice.

A Circular RNA Molecule, circRAB11FIP1, Is Associated with TP53 Mutations and Is of Potential Prognostic and Functional Significance in Mantle Cell Lymphoma: Data from the Nordic MCL2 and MCL3 Studies

INTRODUCTION: Circular RNAs (circRNAs) are covalently closed endogenous RNA molecules with tissue- and disease specific expression patterns, that are emerging as potential diagnostic and prognostic biomarkers. These molecules exert diverse regulatory functions, and several studies have reported prognostic relevance and functional significance of circRNAs in cancer. However, no studies have yet examined the role of circRNAs in Mantle Cell Lymphoma (MCL). We have previously shown that the Nanostring nCounter technology enables specific, sensitive and accurate quantification of circRNAs in formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients (Dahl et al, 2018). Here, we quantified circRNA expression in MCL tumors to address whether certain circRNAs could serve as prognostic markers in MCL. Samples were obtained from patients treated in the two Nordic clinical trials MCL2 and MCL3, all treated with immuno-chemotherapy and autologous stem cell transplant, which due to the improved prognosis observed with this regimen, remains the current standard of care. MATERIALS AND METHODS: We profiled the genome-wide circRNA expression in MCL using high-throughput RNA-sequencing (RNA-seq) of 14 diagnostic MCL samples, from which high-quality RNA from lymph nodes with MCL tumor infiltration was available. Based on these data, we designed assays for analyses of 41 differentially expressed circRNAs and quantified the expression using the NanoString nCounter technology. We examined the prognostic potential of individual circRNAs in a training cohort of 75 patients (MCL2) and confirmed these results in an independent, but similarly treated, validation cohort of 90 patients (MCL3). RESULTS: The total cohort consisted of 165 previously untreated MCL patients <66 years (median=58 [28-65]). There were no differences in baseline characteristics between the two cohorts, but significantly more patients in MCL2 were assigned to high risk groups according to MIPI-C than in MCL3. After a median follow-up of 11.4 years for MCL2 and 7.5 years for MCL3, the median time to progression (TTP) was 8.5 and 7.7 years, respectively. In our training cohort we identified seven circRNAs that were significantly associated with both progression-free survival (PFS), TTP, overall survival (OS) and lymphoma specific survival (LSS). We dichotomized expression values of each individual circRNA and examined the prognostic value in the validation cohort MCL3. Only one (circRAB11FIP1) of the seven circRNAs identified in MCL2 retained prognostic value in MCL3. As shown in Figure 1, univariate analysis revealed that low circRAB11FIP1 expression was significantly associated with TTP in MCL2 (HR=5.1, [2.5;10.4], p<0.0001), while the effect in MCL3 was less pronounced, albeit significant; TTP (HR=2.8, [1.4;5.8], p<0.01). The same results were shown for LSS and PFS in both cohorts, but low circRAB11FIP1 was only significantly associated with OS in MCL2, not in MCL3. Finally, we performed a multivariate cox regression analysis of the entire cohort and found that circRAB11FIP1 was an independent prognostic factor for TTP, even when adjusting for MIPI and ki67-index (HR=2.36[1.3;4.4], p<0.01). Interestingly however, we found an association between circRAB11FIP1 expression and presence of TP53 mutations (unpaired t-test, p<0.01). In patients without TP53 mutations, circRAB11FIP1 levels were significantly higher (mean=378.9[353.2;404.6] than in patients with mutations (mean=297.9[246.8;349.0]). When incorporating presence of TP53 mutations into the multivariate analysis, low circRAB11FIP1 was no longer an independent prognostic factor for TTP (HR=1.48[0.8;2.9], p=0.26). CONCLUSION: Our study showed that low circRAB11FIP1 was significantly associated with shorter TTP, even when adjusting for known prognostic factors, indicating that circRAB11FIP1 could play a specific role in the pathogenesis of MCL. CircRAB11FIP1 originates from exon 2 of the host gene RAB11-Family Interacting Protein 1 located on chromosome 8, and no studies have yet examined the functional role of this circRNA. Interestingly, we found that low circRAB11FIP1 was significantly associated with the presence of TP53 mutations. This warrants future studies to examine whether there is a functional link between circRAB11FIP1 and TP53 in MCL, which may help explain why patients with TP53 mutations have an exceedingly poor prognosis. Figure Disclosures Kolstad: Merck: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerkeman:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

SorCS1-mediated sorting in dendrites maintains neurexin axonal surface polarization required for synaptic function.

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.

Knockdown Rab11-FIP2 inhibits migration and invasion of nasopharyngeal carcinoma via suppressing Rho GTPase signaling.

Rab11 family interacting protein 2 (Rab11-FIP2) is a conserved protein and effector molecule for the small GTPase Rab11. By interacting with Rab11 and MYO5B, Rab11-FIP2 regulates endosome trafficking of plasma membrane proteins, promoting cellular motility. The endosomal trafficking system in nasopharyngeal carcinoma (NPC) remains unclear. Here, an outlier analysis using the Oncomine database suggested that Rab11-FIP2 but not Rab11 and MYO5B was overexpressed in NPC. We confirmed that the transcription of Rab11-FIP2 was upregulated in NPC cell lines and primary tumor tissues as compared with a normal nasopharyngeal epithelial cell line and normal nasopharynx tissues. We further confirmed the elevated protein expression level of Rab11-FIP2 in NPC biopsies. Instead of regulating the epithelial-mesenchymal transition or Akt signaling pathway, knockdown of Rab11-FIP2 inhibited the migration and invasion ability of NPC cell lines by decreasing the expression of Rac and Cdc42. In summary, Rab11-FIP2 could be an oncogene in NPC, mainly contributing to metastatic capacity by activating Rho GTPase signaling.

A transient helix in the disordered region of dynein light intermediate chain links the motor to structurally diverse adaptors for cargo transport.

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.

FIP4/Arfophilin-2 plays overlapping but distinct roles from FIP3/Arfophilin-1 in neuronal migration during cortical layer formation.

The class II Rab11 family-interacting proteins, FIP3 and FIP4, also termed Arfophilin-1 and Arfophilin-2, respectively, are endosomal proteins that function as dual effector proteins for Rab11 and ADP ribosylation factor (Arf) small GTPases. In the present study, we examined the expression and role of FIP4 in neuronal migration during cerebral layer formation. FIP4 mRNA was first weakly detected in post-mitotic migrating neurons in the upper intermediate zone, and expression was markedly increased in the cortical layer. Exogenously expressed FIP4 protein was localized to subpopulations of EEA1- and syntaxin 12-positive endosomes in migrating neurons, and was partially colocalized with FIP3. Knockdown of FIP4 by in utero electroporation significantly stalled transfected neurons in the lower cortical layer and decreased the speed of neuronal migration in the upper intermediate zone and in the cortical plate compared with control small hairpin RNA (shRNA)-transfected neurons. Furthermore, co-transfection of shRNA-resistant wild-type FIP4, but not wild type FIP3 or FIP4 mutants lacking the binding region for Rab11 or Arf, significantly improved the disturbed cortical layer formation caused by FIP4 knockdown. Collectively, our findings suggest that FIP4 and FIP3 play overlapping but distinct roles in neuronal migration downstream of Arf and Rab11 during cortical layer formation.

Rab11 family expression in the human placenta: Localization at the maternal-fetal interface.

Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human placenta, with novel localization at the maternal-fetal interface.

Proteomics Screen Identifies Class I Rab11 Family Interacting Proteins as Key Regulators of Cytokinesis.

The 14-3-3 protein family orchestrates a complex network of molecular interactions that regulates various biological processes. Owing to their role in regulating the cell cycle and protein trafficking, 14-3-3 proteins are prevalent in human diseases such as cancer, diabetes, and neurodegeneration. 14-3-3 proteins are expressed in all eukaryotic cells, suggesting that they mediate their biological functions through evolutionarily conserved protein interactions. To identify these core 14-3-3 client proteins, we used an affinity-based proteomics approach to characterize and compare the human and Drosophila 14-3-3 interactomes. Using this approach, we identified a group of Rab11 effector proteins, termed class I Rab11 family interacting proteins (Rab11-FIPs), or Rip11 in Drosophila We found that 14-3-3 binds to Rip11 in a phospho-dependent manner to ensure its proper subcellular distribution during cell division. Our results indicate that Rip11 plays an essential role in the regulation of cytokinesis and that this function requires its association with 14-3-3 but not with Rab11. Together, our results suggest an evolutionarily conserved role for 14-3-3 in controlling Rip11-dependent protein transport during cytokinesis.

ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin.

During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell–cell and cell–extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking.

Mycobacterium bovis BCG Interferes with miR-3619-5p Control of Cathepsin S in the Process of Autophagy.

Main survival mechanism of pathogenic mycobacteria is to escape inimical phagolysosomal environment inside the macrophages. Many efforts have been made to unravel the molecular mechanisms behind this process. However, little is known about the involvement of microRNAs (miRNAs) in the regulation of phagolysosomal biosynthesis and maturation. Based on a bottom up approach, we searched for miRNAs that were involved in phagolysosomal processing events in the course of mycobacterial infection of macrophages. After infecting THP-1 derived macrophages with viable and heat killed Mycobacterium bovis BCG (BCG), early time points were identified after co-localization studies of the phagosomal marker protein LAMP1 and BCG. Differences in LAMP1 localization on the phagosomes of both groups were observed at 30 min and 4 h. After in silico based pre-selection of miRNAs, expression analysis at the identified time points revealed down-regulation of three miRNAs: miR-3619-5p, miR-637, and miR-324-3p. Consequently, most likely targets were predicted that were supposed to be mutually regulated by these three studied miRNAs. The lysosomal cysteine protease Cathepsin S (CTSS) and Rab11 family-interacting protein 4 (RAB11FIP4) were up-regulated and were considered to be connected to lysosomal trafficking and autophagy. Interaction studies verified the regulation of CTSS by miR-3619-5p. Down-regulation of CTSS by ectopic miR-3619-5p as well as its specific knockdown by siRNA affected the process of autophagy in THP-1 derived macrophages.

Rab11a Mediates Vascular Endothelial-Cadherin Recycling and Controls Endothelial Barrier Function.

Vascular endothelial (VE)-cadherin is the predominant component of endothelial adherens junctions essential for cell-cell adhesion and formation of the vascular barrier. Endocytic recycling is an important mechanism for maintaining the expression of cell surface membrane proteins. However, little is known about the molecular mechanism of VE-cadherin recycling and its role in maintenance of vascular integrity. Using calcium-switch assay, confocal imaging, cell surface biotinylation, and flow cytometry, we showed that VE-cadherin recycling required Ras-related proteins in brain (Rab)11a and Rab11 family-interacting protein 2. Yeast 2-hybrid assay and coimmunoprecipitation demonstrated that direct interaction of VE-cadherin with family-interacting protein 2 (at aa 453-484) formed a ternary complex with Rab11a in human endothelial cells. Silencing of Rab11a or Rab11 family-interacting protein 2 in endothelial cells prevented VE-cadherin recycling and VE-cadherin expression at endothelial plasma membrane. Furthermore, inactivation of Rab11a signaling blocked junctional reannealing after vascular inflammation. Selective knockdown of Rab11a in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide or polymicrobial sepsis. Rab11a/Rab11 family-interacting protein 2-mediated VE-cadherin recycling is required for formation of adherens junctions and restoration of VE barrier integrity and hence a potential target for clinical intervention in inflammatory disease.

Distinct patterns of phosphatidylserine localization within the Rab11a-containing recycling system.

The Rab11 GTPases and Rab11 family-interacting proteins (Rab11-FIPs) define integrated yet distinct compartments within the slow recycling pathway. The lipid content of these compartments is less well understood, although past studies have indicated phosphatidylserine (PS) is an integral component of recycling membranes. We sought to identify key differences in the presence of PS within Rab and Rab11-FIP containing membranes. We used live cell fluorescence microscopy and structured illumination microscopy to determine whether the previously published LactC2 probe for PS displays differential patterns of overlap with various Rab GTPases and Rab11-FIPs. Selective overlap was observed between the LactC2 probe and Rab GTPases when co-expressed in HeLa cells. Rab11-FIP1 proteins consistently overlapped with LactC2 along peripheral and pericentriolar compartments. The specificity of Rab11-FIP1 association with LactC2 was further confirmed by demonstrating that additional Rab11-FIPs (FIP2, FIP3, and FIP5) exhibited selective association with LactC2 containing compartments. Live cell dual expression studies of Rab11-FIPs with LactC2 indicated that PS is enriched along tubular compartments of the Rab11a-dependent recycling system. Additionally, we found that the removal of C2 domains from the Rab11-FIPs induced an accumulation of LactC2 probe in the pericentriolar region, suggesting that inhibition of trafficking through the recycling system can influence the distribution of PS within cells. Finally, we confirmed these findings using structured illumination microscopy suggesting that the overlapping fluorescent signals were on the same membranes. These results suggest distinct associations of Rab GTPases and Rab11-FIPs with PS-containing recycling system membrane domains.

A Functional Interplay between the Small GTPase Rab11a and Mitochondria-shaping Proteins Regulates Mitochondrial Positioning and Polarization of the Actin Cytoskeleton Downstream of Src Family Kinases

It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.

Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells

The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin-Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)-expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.