Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin

Abstract

SUMMARYDuring morphogenesis, forces generated by cells are coordinated and channeled by the viscoelastic properties of the embryo. Microtubules and F-actin are considered to be two of the most important structural elements within living cells accounting for both force production and mechanical stiffness. In this paper, we investigate the contribution of microtubules to the stiffness of converging and extending dorsal tissues in Xenopus laevis embryos using cell biological, biophysical and embryological techniques. Surprisingly, we discovered that depolymerizing microtubules stiffens embryonic tissues by three-to fourfold. We attribute tissue stiffening to Xlfc, a previously identified RhoGEF, which binds microtubules and regulates the actomyosin cytoskeleton. Combining drug treatments and Xlfc activation and knockdown lead us to the conclusion that mechanical properties of tissues such as viscoelasticity can be regulated through RhoGTPase pathways and rule out a direct contribution of microtubules to tissue stiffness in the frog embryo. We can rescue nocodazole-induced stiffening with drugs that reduce actomyosin contractility and can partially rescue morphogenetic defects that affect stiffened embryos. We support these conclusions with a multi-scale analysis of cytoskeletal dynamics, tissue-scale traction and measurements of tissue stiffness to separate the role of microtubules from RhoGEF activation. These findings suggest a re-evaluation of the effects of nocodazole and increased focus on the role of Rho family GTPases as regulators of the mechanical properties of cells and their mechanical interactions with surrounding tissues. , 2000). Disruption of microtubules can also increase cellular tension (Danowski, 1989;Dennerll et al., 1988; Kolodney and Elson, 1995;Stamenovic et al., 2002b;Wang et al., 2001) by activating actomyosin contraction; but it is unknown whether activation of actomyosin in the absence of microtubules is achieved through mechanical interactions or via signaling pathways (Birukova et al., 2004;Chang et al., 2008; Kolodney and Elson, 1995;Verin et al., 2001). Microtubules can signal to actomyosin through Rho family GTPases. RhoGTPases are key elements in the regulation of the actomyosin cytoskeleton in both cultured cells and during morphogenesis (Hall, 2005;Settleman, 2001). The Rho family members are well known to control cellular processes such as actin assembly, as well as the organization of myosin II-mediated contractility into lamellipodia and filopodia to guide cell migration and cell contractility. RhoGTPases can also regulate processes such as assembly of ECM (Dzamba et al., 2009) and the bulk elasticity of tissues through control of cortical actomyosin contractility and cell traction forces (Paszek et al., 2005). Thus, disrupting microtubules with drugs such as nocodazole may have inadvertently been testing not the role of microtubules but the role of RhoGTPases in development.In this paper, we first investigate the contribution of microtubules to tissue stiffness and how mic...

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Microtubule depolymerization induces traction force increase through two distinct pathways

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