Cytoskeletal coherence requires myosin-IIA contractility

Abstract

SummaryMaintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in 'C' or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading. Adelstein, 2008;Vicente-Manzanares et al., 2009;Wylie and Chantler, 2008). NMIIA and NMIIB are the primary force generators in fibroblasts (Cai et al., 2006;Lo et al., 2004). Phosphorylation of myosin light chains (MLC), primarily by Rho kinases (ROCK) and MLC kinase (Totsukawa et al., 2004), regulates the NMII activity. ROCK has multiple protein targets including MLC, MLC phosphatase, adducin and moesin (Totsukawa et al., 2004). ROCK activates NMII by phosphorylating MLC and also by inactivating MLC phosphatase to inhibit MLC dephosphorylation (Totsukawa et al., 2004). Specific inhibitors have been developed for studying the functions of NMII, i.e. Y27632 inhibits ROCK and blebbistatin inhibits the ATPase activity of myosin-II. In addition to MLC phosphatase, some other proteins also negatively regulate NMII activity. For example, caldesmon interacts with actin, myosin-II and tropomyosin, and inhibits the ATPase activity of myosin-II (Marston et al., 1998). Caldesmon overexpression causes suppression of traction forces and focal adhesions (Helfman et al., 1999). Thus, there are a variety of ways to inhibit force generation on substrates.The plasma membrane limits the spreading of cells on substrates, and tension in the plasma membrane inhibits the ability of actin to polymerize at the periphery (Raucher and Sheetz, 2000). Although the tension in the membrane is typically very low (Sheetz, 2001), it can influence the behavior of cells (Keren et al., 2008) and the final shape of cells is heavily influenced by the final membrane area.We here demonstrate that NMIIA is crucial for the mechanical coherence of cytoplasm. Inhibition of NMII contractility or depletion of NMIIA causes cytoplasm to ...

Frustrated Phagocytic Spreading of J774A-1 Macrophages Ends in Myosin II-Dependent Contraction

“…The fragmentation of the cells implies that myosin is involved in maintaining cell shape. Similar results have been observed for spreading fibroblasts (44). The continued protrusion of isolated fronts also demonstrates that without the proper engagement of contractile machinery, the cell does not transition to late-stage phagocytosis.…”

Chemical tags: Applications in live cell fluorescence imaging

“…Chemical tags have been used to study the localization and dynamics of proteins in living cells, especially in experiments that can not be easily performed with fluorescent proteins. In the last few years, chemical tags have been used to label proteins with fluorophores suited for advanced imaging technologies such as super-resolution (SR) microscopy [57][58][59], Ca 2þ -imaging [60][61][62], pH sensing [63], hydrogen peroxide detection [64], chromophore assisted light inactivation [36,65,66], and multi-photon microscopy [19]. Recently, Kosaka et al demonstrated the use of the Halo-tag to perform in vivo imaging studies in live animals for the first time [67].…”

“…Fluorescein with a high singlet oxygen quantum yield is a good photosensitizer and, therefore, a suitable reagent for CALI experiments. Cai et al successfully used eDHFR-tag with TMP-fluorescein in CALI experiments to reveal that nonmuscle myosin II (NMII) is required to maintain cytoplasmic coherence [66]. The myosin light chain (MLC) is part of the NMII hexamer and can be labeled with TMP-fluorescein when expressed as a MLC-eDHFR fusion.…”

“…Laser inactivation of TMP-fluorescein labeled MLC-eDHFR fusion proteins by irradiation of a $7.0 mm diameter for 1 sec. (a-j) CALI of cytoplasmic myosin II locally destroyed the contractile actomyosin II network, revealed by the curvature of the actin cables close to the irradiated region marked with the dashed and dotted lines [66].…”

Cells test substrate rigidity by local contractions on submicrometer pillars

“…We also examined the distribution of phosphorylated myosin light chain (p-MLC). p-MLC is an indicator of active force-generating myosin II molecules (14), which exert traction forces and contribute to building a cohesive actomyosin network within the cytoplasm (15,16). P-MLC localized in small clusters at the edges of 2-μm pillars (Fig.…”