Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain.
Ontology highlight
ABSTRACT:
SUBMITTER: Zaman R
PROVIDER: S-EPMC8185690 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Many unicellular tubes such as capillaries form lumens intracellularly, a process that is not well understood. Here we show that the cortical membrane organizer ERM-1 is required to expand the intracellular apical/lumenal membrane and its actin undercoat during single-cell Caenorhabditis elegans excretory canal morphogenesis. We characterize AQP-8, identified in an ERM-1-overexpression (ERM-1[++]) suppressor screen, as a canalicular aquaporin that interacts with ERM-1 in lumen extension in a mercury-sensitive manner, implicating water-channel activity. AQP-8 is transiently recruited to the lumen by ERM-1, co-localizing in peri-lumenal cuffs interspaced along expanding canals. An ERM-1[++]-mediated increase in the number of lumen-associated canaliculi is reversed by AQP-8 depletion. We propose that the ERM-1/AQP-8 interaction propels lumen extension by translumenal flux, suggesting a direct morphogenetic effect of water-channel-regulated fluid pressure.
| S-EPMC4091717 | biostudies-literature
Project description:Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Although the cell-intrinsic role of Pcdh7 in osteoclast differentiation has been demonstrated, the molecular mechanisms of Pcdh7 regulating osteoclast differentiation remain to be determined. Here, we demonstrate that Pcdh7 contributes to osteoclast differentiation by regulating small GTPases, RhoA and Rac1, through its SET oncoprotein binding domain. Pcdh7 is associated with SET along with RhoA and Rac1 during osteoclast differentiation. Pcdh7-deficient (Pcdh7-/-) cells showed abolished RANKL-induced RhoA and Rac1 activation, and impaired osteoclast differentiation. Impaired osteoclast differentiation in Pcdh7-/- cells was restored by retroviral transduction of full-length Pcdh7 but not by a Pcdh7 mutant that lacks SET binding domain. The direct crosslink of the Pcdh7 intracellular region induced the activation of RhoA and Rac1, which was not observed when Pcdh7 lacks the SET binding domain. Additionally, retroviral transduction of the constitutively active form of RhoA and Rac1 completely restored the impaired osteoclast differentiation in Pcdh7-/- cells. Collectively, these results demonstrate that Pcdh7 controls osteoclast differentiation by regulating RhoA and Rac1 activation through the SET binding domain.
| S-EPMC8658210 | biostudies-literature
Project description:Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.
| S-EPMC1855021 | biostudies-literature
Project description:Adhesion to the extracellular matrix regulates numerous changes in the actin cytoskeleton by regulating the activity of the Rho family of small GTPases. Here, we report that adhesion and the associated changes in cell shape and cytoskeletal tension are all required for GTP-bound RhoA to activate its downstream effector, ROCK. Using an in vitro kinase assay for endogenous ROCK, we found that cells in suspension, attached on substrates coated with low density fibronectin, or on spreading-restrictive micropatterned islands all exhibited low ROCK activity and correspondingly low myosin light chain phosphorylation, in the face of high levels of GTP-bound RhoA. In contrast, allowing cells to spread against substrates rescued ROCK and myosin activity. Interestingly, inhibition of tension with cytochalasin D or blebbistatin also inhibited ROCK activity within 20 min. The abrogation of ROCK activity by cell detachment or inhibition of tension could not be rescued by constitutively active RhoA-V14. These results suggest the existence of a feedback loop between cytoskeletal tension, adhesion maturation, and ROCK signaling that likely contributes to numerous mechanochemical processes.
| S-EPMC2064860 | biostudies-literature
Project description:The invasive properties of cancer cells are intimately linked to their mechanical phenotype, which can be regulated by intracellular biochemical signalling. Cell contractility, induced by mechanotransduction of a stiff fibrotic matrix, and the epithelial-mesenchymal transition (EMT) promote invasion. Metastasis involves cells pushing through the basement membrane into the stroma-both of which are altered in composition with cancer progression. Agonists of the G protein-coupled oestrogen receptor (GPER), such as tamoxifen, have been largely used in the clinic, and interest in GPER, which is abundantly expressed in tissues, has greatly increased despite a lack of understanding regarding the mechanisms which promote its multiple effects. Here, we show that specific activation of GPER inhibits EMT, mechanotransduction and cell contractility in cancer cells via the GTPase Ras homolog family member A (RhoA). We further show that GPER activation inhibits invasion through an in vitro basement membrane mimic, similar in structure to the pancreatic basement membrane that we reveal as an asymmetric bilayer, which differs in composition between healthy and cancer patients.
| S-EPMC7073197 | biostudies-literature
Project description:Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1β and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1β/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1β/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1β activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1β generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1β/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague.
| S-EPMC5135138 | biostudies-literature
Project description:To identify novel proteins that control melanoma development and progression, we integrated high-throughput proteomics, mRNA and miRNAs datasets from isogenic primary (WM115) and metastatic (WM266-4) melanoma cell-lines. We focused on differentially expressed proteins that had non-differential mRNA levels and were putatively targeted by a high number of miRNAs. SNX18, a sub-family member of sorting-nexins that participates in endocytosis and autophagy was down-regulated by 6-fold in WM266-4, and predicted to be targeted by 17 miRNAs. Over-expression of SNX18 in several melanoma cell-lines significantly reduced migration rate, while silencing of SNX18 resulted in the opposite effect. Notably, progression tissue microarrays demonstrated a trend for SNX18 downregulation in early metastasis. Luciferase assays confirmed the direct regulation of SNX18 by multiple miRNAs. Indeed, over-expression of these miRNAs repressed endogenous SNX18 expression at the protein but not mRNA level. Further, protein but not mRNA expression levels correlated with better overall survival as determined in a cohort of 48 metastatic melanoma patients and in the TCGA database. Mechanistically, SNX18-mediated reduced migration coincided with RhoA activation and Cdc42 inactivation. Both play a role, as RhoA inhibitor and constitutively activated Cdc42 mutants abrogated this effect. Moreover, phosphorylation-defective SNX18 mutants (S233A,S233D) migrated faster, with S233A inactivating RhoA and activating Cdc42, while S233D activated both. The downstream phosphorylation of the p38>MAPKAPK2>HSP27 signaling pathway was concordant. Both mutants were more abundant in the plasma membrane. Taken together, phosphorylation of S233 is crucial for SNX18 activation, but de-phosphorylation dynamics, which may occur at the membrane, is required for functional effect. miRNA expression profile of primary (WM115) and metastatic (WM266-4) melanoma cell lines.
2021-09-17 | GSE120033 | GEO
Project description:To identify novel proteins that control melanoma development and progression, we integrated high-throughput proteomics, mRNA and miRNAs datasets from isogenic primary (WM115) and metastatic (WM266-4) melanoma cell-lines. We focused on differentially expressed proteins that had non-differential mRNA levels and were putatively targeted by a high number of miRNAs. SNX18, a sub-family member of sorting-nexins that participates in endocytosis and autophagy was down-regulated by 6-fold in WM266-4, and predicted to be targeted by 17 miRNAs. Over-expression of SNX18 in several melanoma cell-lines significantly reduced migration rate, while silencing of SNX18 resulted in the opposite effect. Notably, progression tissue microarrays demonstrated a trend for SNX18 downregulation in early metastasis. Luciferase assays confirmed the direct regulation of SNX18 by multiple miRNAs. Indeed, over-expression of these miRNAs repressed endogenous SNX18 expression at the protein but not mRNA level. Further, protein but not mRNA expression levels correlated with better overall survival as determined in a cohort of 48 metastatic melanoma patients and in the TCGA database. Mechanistically, SNX18-mediated reduced migration coincided with RhoA activation and Cdc42 inactivation. Both play a role, as RhoA inhibitor and constitutively activated Cdc42 mutants abrogated this effect. Moreover, phosphorylation-defective SNX18 mutants (S233A,S233D) migrated faster, with S233A inactivating RhoA and activating Cdc42, while S233D activated both. The downstream phosphorylation of the p38>MAPKAPK2>HSP27 signaling pathway was concordant. Both mutants were more abundant in the plasma membrane. Taken together, phosphorylation of S233 is crucial for SNX18 activation, but de-phosphorylation dynamics, which may occur at the membrane, is required for functional effect. In this study we compared the proteome, transcriptome and miRnome of primary and metastatic melanoma isogenic cell-lines and identified SNX18 as a novel inhibitor of melanoma migration.
2021-09-15 | GSE120029 | GEO
Project description:In human disease induced by Salmonella enterica serovar Typhimurium (S. Typhimurium), transepithelial migration of neutrophils rapidly follows attachment of the bacteria to the epithelial apical membrane. We have previously shown that during S. Typhimurium infection the multidrug resistance-associated protein 2 (MRP2) is highly expressed at the apical surface of the intestinal epithelia, and that it functions as an efflux pump for the potent neutrophil chemoattractant hepoxilin A(3) . However, the molecular mechanisms regulating its apical localization during active states of inflammation remain unknown. Thus, our objective was to determine the mechanistic basis for the translocation of MRP2 to the apical surface of intestinal epithelial cells during S. Typhimurium infection. We show that suppression of ezrin, through either RNAi or truncation of the C-terminus, results not only in a decrease in S. Typhimurium-induced neutrophil transmigration but also significantly attenuates the apical membrane expression of MRP2 during Salmonella infection. In addition, we determined that S. Typhimurium induces the activation of ezrin via a PKC-?-dependent pathway and that ezrin activation is coupled to apical localization of MRP2. Based on these results we propose that activation of ezrin is required for the apical localization of MRP2 during S. Typhimurium infection.
| S-EPMC3218228 | biostudies-literature
Project description:IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells.To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo.We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature.We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation.Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex-mediated diseases.
| S-EPMC4890651 | biostudies-literature