Biology subjects

Balda, M.

Publications and source records attributed to Balda, M..

4 recordsLinked to original sources

bioRxiv2024

Inhibition of GEF-H1-RhoA signaling in inflammation with a stapled peptide mimicry of the RhoA67-78 helix

Guanine exchange factors (GEFs) are considered hard to drug with conventional small molecules, they lack conventional deep binding pockets and binding ligands are seldom reported. Here we report the design of a stapled peptide stP5 targeting the interaction between cytoskeletal regulator RhoA GTPase and its activator guanine exchange factor H1 (GEF-H1). StP5 is a modified RhoA mimic based on a previously identified bioactive -helical epitope to GEF-H1. StP5 effectively inhibits GEF-H1-induced morphological and transcriptional changes in cellular models for inflammation and does not affect the related GEF p114RhoGEF (ARHGEF18). StP5 peptide is approximately 100 fold more active in cellular assays than the unstapled P5 peptide. We provide a bioinformatic analysis of the stP5 bindings site in different GEFs, providing a basis for this selectivity. The GEF-H1 inhibitor stP5 represents a step towards fully drugging GEF-H1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/624118v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@d783b3org.highwire.dtl.DTLVardef@10798b2org.highwire.dtl.DTLVardef@1ba0817org.highwire.dtl.DTLVardef@693367_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

ZO-1 regulates Hippo-independent YAP activity and cell proliferation via a GEF-H1- and TBK1-regulated mechanosensitive signalling network

Tight junctions are a barrier-forming cell-cell adhesion complex and have been proposed to regulate cell proliferation. However, the underlying mechanisms are not well understood. Here, we used cells deficient in the junction scaffold ZO-1 alone or together with its paralog ZO-2, which disrupts the junctional barrier. We found that ZO-1 knockout increased cell proliferation, loss of cell density-dependent proliferation control, and promoted cell death. These phenotypes were enhanced by double ZO-1/ZO-2 knockout. Increased proliferation was dependent on YAP and ZONAB, two transcriptional regulators. ZO-1 knockout stimulated YAP nuclear translocation and activity without changes in Hippo-dependent phosphorylation. Knockout promoted TANK-binding Kinase 1 (TBK1) activation and increased expression of the RhoA activator GEF-H1. Knockdown of ZO-3, another paralog interacting with ZO1, was sufficient to induce GEF-H1 expression and YAP activity. GEF-H1, TBK1, and mechanotransduction at focal adhesions were required for YAP/TEAD activation in ZO-1-deficient cells. Thus, ZO-1 controls cell proliferation and Hippo-independent YAP activity by activating a GEF-H1- and TBK1-regulated mechanosensitive signalling network.

cell biology↗

bioRxiv2022

Reciprocal regulation between cell mechanics and ZO-1 guides tight junction assembly and epithelial morphogenesis

Formation and maintenance of tissue barriers require the coordination of cell mechanics and cell-cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 regulates cytoskeletal tension at cell-matrix and cell-cell interfaces in an ECM stiffness-regulated manner, possibly via differential organisation of the actin cytoskeleton. ZO-1 depletion inhibited junction assembly and disrupted morphogenesis in an ECM stiffness-dependent manner. Both processes were rescued by inhibition of cell contractility. Although ZO-1-deficient cells could assemble functional barriers at low tension, their tight junctions remained corrupted with strongly reduced and discontinuous recruitment of junctional components. Our results thus reveal that reciprocal regulation between ZO-1 and cell mechanics controls tight junction assembly and epithelial morphogenesis, and that tension-independent roles of ZO-1 control proper junction organisation.

cell biology↗

bioRxiv2021

Spatiotemporal Control of Actomyosin Contractility by MRCKβ Signaling Drives Phagocytosis

Phagocytosis requires myosin-generated contractile force to regulate actin dynamics. However, little is known about the molecular mechanisms that guide this complex morphodynamic process. Here we show that particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3 in the retinal pigment epithelium (RPE), triggering activation of MRCK{beta} and its co-effector N-WASP that cooperate to deform the membrane into cups. Continued MRCK{beta} activity then drives recruitment of a mechanosensing bridge enabling transmission of the cytoskeletal force required for cup closure and particle internalization. MRCK{beta} is also required for Fc receptor-mediated phagocytosis by macrophages. In vivo, MRCK{beta} is essential for RPE phagocytosis of photoreceptor debris and, hence, retinal integrity. MerTK-independent activation of MRCK{beta} signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. Thus, conserved MRCK{beta} signaling controls spatiotemporal regulation of actomyosin contractility to guide actomyosin dynamics-driven phagocytosis and represents the principle phagocytic effector pathway downstream of MerTK.

cell biology↗