Plakophilin 4 controls the spatio-temporal activity of RhoA at adherens junctions to promote cortical actin ring formation and tissue tension
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1073891 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27888400_1.fastq.gz | Fastqsanger.gz | |||
| SRR27888400_2.fastq.gz | Fastqsanger.gz | |||
| SRR27888401_1.fastq.gz | Fastqsanger.gz | |||
| SRR27888401_2.fastq.gz | Fastqsanger.gz | |||
| SRR27888366_1.fastq.gz | Fastqsanger.gz |
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Project description:Plakophilin 4 (PKP4) is a component of cell-cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs is required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization.
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Project description:In acute critical illness the capillary barrier may break down resulting in hypovolemia, extracellular edema, tissue dysoxia and even death. Restoration of the tight junction dependent capillary barrier is regulated by small GTPases, the specific regulatory molecules most active in this setting are not well described. Transcriptional profiling of tight junction forming human dermal microvascular ECs (HDMECs) and adherens junctions forming human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is significantly differentially regulated in HDMECs before and after stimulation with tumor necrosis factor (TNF). HDMEC depleted of ArhGEF12 demonstrate significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of tight junction proteins claudin-5 and ZO-1. ArhGEF12 is a RhoA-GEF, an established finding which is inconsistent with our observed results. Pulldown activation assays from of HDMECs depleted of ArhGEF12 and treated with TNF show decreased Rap1A activity after four hours and paradoxically increased RhoA activity after 12 hours. In cell-free pulldown assays, immunoprecipitated ArhGEF12 effectively activates Rap1A and RhoA and not Rap2A-C, RhoB-C or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in tight junction forming EC, ArhGEF12 selectively activates Rap1A to promote capillary barrier restoration in a mechanism independent of traditionally described cAMP-mediated Epac1 activation. The unique dual in vitro specificity of ArhGEF12 for RhoA, Rap1A and not Rap1B cannot be explained by sequence identity and requires additional investigation.
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Project description:Rho family small GTPases serve as molecular switches in the regulation of diverse cellular functions including actin cytoskeleton remodeling, cell migration, gene transcription, and cell proliferation. Importantly, Rho overexpression is frequently seen in many carcinomas. However, published studies have almost invariably utilized immortal or tumorigenic cell lines to study Rho GTPase functions and there are no studies on the potential of Rho small GTPase to overcome senescence checkpoints and induce preneoplastic transformation of human mammary epithelial cells (hMECs). We found that ectopic expression of wild-type RhoA as well as a constitutively-active RhoA mutant (G14V) in primary hMEC strains led to their immortalization and preneoplastic transformation. Significantly, RhoA-T37A mutant, known to be incapable of interacting with many well known Rho-effectors ,was also capable of immortalizing hMECs.Our results demonstrate that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked cellular transformation and breast cancer. Through microarray analysis, we want to identify genes and pathways linked to RhoA induced hMECs immortalization. Experiment Overall Design: 4 samples, in triplicate analyses per sample.
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