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Borg5/Cdc42EP curbs Septin dependent stress fiber formation by antagonizing alpha Actinin 4 and Myosin IIA

ABSTRACT: The Borg5/Cdc42EP family comprises septin binding proteins, which are known to participate in septin-dependent stress fiber formation. We show here that epithelial Borg5/Cdc42Ep1 restrains stress fibers instead. Borg5 decorates septin filament-aligned thin F-actin filaments under the nucleus of MDCK cells, which in Borg5 depleted cells are replaced by thick stellate stress fibers. These associate with either cell-substratum- or cell-cell- adhesions, increasing tension on E-cadherin based junctions and causing MDCK cells to undergo collective streaming. We identified two known septin partners, Myosin-IIA and alpha Actinin 4 (ACTN4), among proteins that co isolated with Borg5 from MDCK lysates. We found that Borg5 binds Myosin-IIA and ACTN4 directly and negatively regulates their F-actin association. Since both septin partners along with Septins 2 and 9 were furthermore required for the Borg5 depletion-induced F actin phenotype, we propose that Borg5 prevents stellate stress fiber formation by counteracting septin dependent ACTN4 and Myosin IIA activities. The mass spectrometry raw files and search results for Borg 5 affinity purifications are deposited here.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Canis Lupus Familiaris (ncbitaxon:9615)

SUBMITTER: Beatrix Ueberheide

PROVIDER: MSV000092958 | MassIVE |

SECONDARY ACCESSION(S): PXD045675

REPOSITORIES: MassIVE

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Project description:Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated contractile activity, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active RhoA (RhoAV14). Organ cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 revealed strong contractile cell morphology, increased actin stress fibers and focal adhesions, along with increased levels of phosphorylated myosin II, and collagen IV, fibronectin and laminin. cDNA microarray analysis of RNA extracted from RhoAV14 expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of myosin II, paxillin and focal adhesion kinase, and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the contractile force, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells. Keywords: Gene Expression Two condition experiment: Human trabecular mesh work cells infected with Adenivirus expressing GFP Vs Adenovirus expressing GFP and constitutively active RhoAV14

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Borg5/Cdc42EP curbs Septin dependent stress fiber formation by antagonizing alpha Actinin 4 and Myosin IIA

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