A NUMB-EFA6B-ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility.
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ABSTRACT:
SUBMITTER: Zobel M
PROVIDER: S-EPMC6123001 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Invasion of the colon wall by Entamoeba histolytica during amoebic dysentery entails migration of trophozoites through tissue layers that are rich in extracellular matrix. Transcriptional silencing of the E. histolytica surface metalloprotease EhMSP-1 produces hyperadherent less-motile trophozoites that are deficient in forming invadosomes. Reversible protein phosphorylation is often implicated in regulation of cell motility and invadosome formation. To identify such intermediaries of the EhMSP-1-silenced phenotype, here we compared the phosphoproteomes of EhMSP-1-silenced and vector control trophozoites by using quantitative tandem mass spectrometry-based proteomics. Six proteins were found to be differentially phosphorylated in EhMSP-1-silenced and control cells, including EhCoactosin, a member of the ADF/cofilin family of actin-binding proteins, which was more frequently phosphorylated at serine 147. Regulated overexpression of wild-type, phosphomimetic, and nonphosphorylatable EhCoactosin variants was used to test if phosphorylation functions in control of E. histolytica actin dynamics. Each of the overexpressed proteins colocalized with F-actin during E. histolytica phagocytosis. Nonetheless, trophozoites overexpressing an EhCoactosin phosphomimetic mutant formed more and poorly coordinated cell membrane protrusions compared to those in control or cells expressing a nonphosphorylatable mutant, while trophozoites overexpressing nonphosphorylatable EhCoactosin were significantly more motile within a model of mammalian extracellular matrix. Therefore, although EhCoactosin's actin-binding ability appeared unaffected by phosphorylation, EhCoactosin phosphorylation helps to regulate amoebic motility. These data help to understand the mechanisms underlying altered adherence and motility in EhMSP-1-silenced trophozoites and lay the groundwork for identifying kinases and phosphatases critical for control of amoebic invasiveness.IMPORTANCE Invasive amoebiasis, caused by the intestinal parasite Entamoeba histolytica, causes life-threatening diarrhea and liver abscesses, but, for unknown reasons, only approximately 10% of E. histolytica infections become symptomatic. A key requirement of invasion is the ability of the parasite to migrate through tissue layers. Here, we systematically looked for differences in protein phosphorylation between control parasites and a previously identified hyperadherent E. histolytica cell line that has reduced motility. We identified EhCoactosin, an actin-binding protein not previously known to be phosphoregulated, as one of the differentially phosphorylated proteins in E. histolytica and demonstrated that EhCoactosin phosphorylation functions in control of cell membrane dynamics and amoebic motility. This and the additional differentially phosphorylated proteins reported lay the groundwork for identifying kinases and phosphatases that regulate tissue invasiveness.
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Project description:PKCepsilon controls the transport of endocytosed beta1-integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCepsilon target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCepsilon dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCepsilon/integrin positive vesicles. Finally, introduction of ectopic wild-type vimentin is shown to promote cell motility in a PKCepsilon-dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCepsilon null cells. Our results indicate that PKC-mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.
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Project description:Epithelial-to-mesenchymal transitions (EMTs) are crucial for morphogenesis and carcinoma metastasis, yet mechanisms controlling the underlying cell behaviors are poorly understood. RhoGTPase signaling has been implicated in EMT; however, previous studies have yielded conflicting results regarding Rho function, and its role in EMT remains poorly understood. Elucidation of precise Rho functions has been challenging because Rho signaling is highly context dependent and its activity is tightly regulated spatiotemporally within the cell. To date, few studies have examined how Rho affects cell motility in intact organisms, and the pattern of Rho activity during motile cell behaviors of EMT has not been determined in any system. Here, we image endogenous active Rho during EMT in vivo, and analyze effects of Rho and Rho-kinase (ROCK) manipulation on cell motility in vivo. We show that Rho is activated in a discrete apical region of premigratory neural crest cells during EMT, and Rho-ROCK signaling is essential for apical detachment and generation of motility within the neuroepithelium, a process that has been poorly understood. Furthermore, we find that Arhgap1 restricts Rho activation to apical areas, and this restriction is necessary for detachment. Our results provide new insight into mechanisms controlling local Rho activation and how it affects dynamic cell behaviors and actomyosin contraction during key steps of EMT in an intact living organism.
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Project description:p27(kip1) (p27) is an inhibitor of cyclin/cyclin-dependent kinase complexes, whose nuclear loss indicates a poor prognosis in various solid tumors. When located in the cytoplasm, p27 binds Op18/stathmin (stathmin), a microtubule (MT)-destabilizing protein, and restrains its activity. This leads to MT stabilization, which negatively affects cell migration. Here, we demonstrate that this p27 function also influences morphology and motility of cells immersed in three-dimensional (3D)matrices. Cells lacking p27 display a decrease in MT stability, a rounded shape when immersed in 3D environments, and a mesenchymal-amoeboid conversion in their motility mode. Upon cell contact to extracellular matrix, the decreased MT stability observed in p27 null cells results in accelerated lipid raft trafficking and increased RhoA activity. Importantly, cell morphology, motility, MT network composition, and distribution of p27 null cells were rescued by the concomitant genetic ablation of Stathmin, implicating that the balanced expression of p27 and stathmin represents a crucial determinant for cytoskeletal organization and cellular behavior in 3D contexts.
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Project description:Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival.
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Project description:BackgroundAdhesion molecules distributed on the cell-surface depends upon their dynamic trafficking that plays an important role during cancer progression. ADP-ribosylation factor 6 (Arf6) is a master regulator of membrane trafficking. CD147, a tumor-related adhesive protein, can promote the invasion of liver cancer. However, the role of Arf6 in CD147 trafficking and its contribution to liver cancer progression remain unclear.MethodsStable liver cancer cell lines with Arf6 silencing and over-expression were established. Confocal imaging, flow cytometry, biotinylation and endomembrane isolation were used to detect CD147 uptake and recycling. GST-pull down, gelatin zymography, immunofluorescence, cell adhesion, aggregation and tight junction formation, Transwell migration, and invasion assays were used to examine the cellular phenotypes. GEPIA bioinformatics, patient's specimens and electronic records collection, and immunohistochemistry were performed to obtain the clinical relevance for Arf6-CD147 signaling.ResultsWe found that the endocytic recycling of CD147 in liver cancer cells was controlled by Arf6 through concurrent Rab5 and Rab22 activation. Disruption of Arf6-mediated CD147 trafficking reduced the cell-matrix and cell-cell adhesion, weakened cell aggregation and junction stability, attenuated MMPs secretion and cytoskeleton reorganization, impaired HGF-stimulated Rac1 activation, and markedly decreased the migration and invasion of liver cancer cells. Moreover, high-expression of the Arf6-CD147 signaling components in HCC (hepatocellular carcinoma) was closely correlated with poor clinical outcome of patients.ConclusionsOur results revealed that Arf6-mediated CD147 endocytic recycling is required for the malignant phenotypes of liver cancer. The Arf6-driven signaling machinery provides excellent biomarkers or therapeutic targets for the prevention of liver cancer.
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Project description:Recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane. To determine how recycling endosomes perform their functions, searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers. However, it remains unclear whether all the conventional markers have identical localizations. Here we report finding that three well-known recycling endosome markers, i.e., Arf6, Rab11 and transferrin receptor (TfR), have different intracellular localizations in PC12 cells. The results of immunofluorescence analyses showed that the signals of endogenous Arf6, Rab11 and TfR in nerve growth factor-stimulated PC12 cells generally differed, although there was some overlapping. Our findings provide new information about recycling endosome markers, and they highlight the heterogeneity of recycling endosomes.
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Project description:Autophagic and endosomal dysfunctions are prominently observed at preclinical stages of Alzheimer's disease (AD) as well as in presenilin (PSEN) 1-deficient mice and neurons. In the latter, the defects relate to the γ-secretase-independent role of PSEN in lysosomal fusion and organelle turnover. While we demonstrated previously that the impaired capacity of lysosomal fusion is associated with a significant reduction in lysosomal calcium storage/release, the underlying mechanism remained unexplored. Here we demonstrate that PSEN-deficient cells are impaired in endosomal recycling of several cargo proteins reminiscent of clathrin-independent carriers and lipid rafts. This is accompanied by the accumulation of cholesterol in LAMP1-positive organelles. The small GTPase ARF6, an important regulator of lipid raft recycling, fully rescues the endo-lysosomal abnormalities in PSEN-/- cells, suggesting that defective recycling is upstream of lysosomal dysfunction. PSEN-/- cells and neurons present significantly reduced ARF6 expression levels. Importantly, similar decreased ARF6 levels are observed in aging murine neurons and brain and are even more pronounced in AD brains, suggesting a particular vulnerability of ARF6-mediated recycling in the early etiology of AD.
2017-12-15 | GSE44454 | GEO
Project description:Cell attachment to the extracellular matrix (ECM) requires a balance between integrin internalization and recycling to the surface that is mediated by numerous proteins, emphasizing the complexity of these processes. Upon ligand binding in various cells, the β1 integrin is internalized, traffics to early endosomes, and is returned to the plasma membrane through recycling endosomes. This trafficking process depends on the cyclical activation and inactivation of small guanosine triphosphatases (GTPases) by their specific guanine exchange factors (GEFs) and their GTPase-activating proteins (GAPs). In this study, we found that the cell surface antigen CD13, a multifunctional transmembrane molecule that regulates cell-cell adhesion and receptor-mediated endocytosis, also promoted cell migration and colocalized with β1 integrin at sites of cell adhesion and at the leading edge. A lack of CD13 resulted in aberrant trafficking of internalized β1 integrin to late endosomes and its ultimate degradation. Our data indicate that CD13 promoted ARF6 GTPase activity by positioning the ARF6-GEF EFA6 at the cell membrane. In migrating cells, a complex containing phosphorylated CD13, IQGAP1, GTP-bound (active) ARF6, and EFA6 at the leading edge promoted the ARF6 GTPase cycling and cell migration. Together, our findings uncover a role for CD13 in the fundamental cellular processes of receptor recycling, regulation of small GTPase activities, cell-ECM interactions, and cell migration.
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