Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells

Grosheva, Inna; Vittitow, Jason L; Goichberg, Polina; Gabelt, B’Ann T.; Kaufman, Paul L.; Borrás, Terete; Geiger, Benjamin; Bershadsky, Alexander D.

doi:10.1016/j.exer.2006.01.006

Cited by 51 publications

(41 citation statements)

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“…High levels of GFPcaldesmon expression often abolished circumferential actin bundles and stress fibers (supplementary material Fig. S7), as in trabecular meshwork cells (Grosheva et al, 2006). The assembly of NMIIA was inhibited; however, no clear change in the size of NMIIB clusters was observed in cells expressing high levels of GFPcaldesmon (supplementary material Fig.…”

Section: Caldesmon Overexpression and Cali Of Mlc Further Reveals Thementioning

confidence: 99%

Cytoskeletal coherence requires myosin-IIA contractility

Cai

Rossier

Gauthier

et al. 2010

Journal of Cell Science

190

168

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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 ...

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Section: Caldesmon Overexpression and Cali Of Mlc Further Reveals Thementioning

confidence: 99%

Cytoskeletal coherence requires myosin-IIA contractility

Cai

Rossier

Gauthier

et al. 2010

Journal of Cell Science

190

168

View full text Add to dashboard Cite

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“…G-protein-coupled AR receptors are well known to change adenylyl cyclase activity and intracellular cAMP concentration, but it is now well established that other pathways, such as phospholipase C (PLC), Ca 2+ and mitogen-activated protein kinases, are relevant as well (Haas & Selbach, 2000;Jacobson & Gao, 2006). Studying the Ca 2+ transients is of particular interest because of calcium's role in modulating contractility (Stumpff et al, 1997) and the actin cytoskeleton (Grosheva et al, 2006) of TM cells. Changes in intracellular calcium due to AR activation may stem from activation of PLC with subsequent formation of inositol triphosphate and/ or modulation of membrane calcium-channel activity (Lorentzen & Schwabe, 2001).…”

Section: Discussionmentioning

confidence: 99%

Cell-specific differential modulation of human trabecular meshwork cells by selective adenosine receptor agonists

Karl

Peterson-Yantorno

Civan

2007

Experimental Eye Research

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Activation of A 1 and A 2A subtype adenosine receptors (AR) likely exert opposing effects on outflow of aqueous humor, and thereby, on intraocular pressure. Selective agonists of adenosine receptor (AR) subtypes have previously been applied to trabecular meshwork (TM) and Schlemm's canal (SC) cells to identify the site(s) of differential purinergic modulation. However, the apparent changes in volume monitored by previously measuring projected cell area might have partially reflected cell contraction and relaxation. In addition, whole-cell current responses of the TM cells previously described were highly variable following application of selective A 1 , A 2A and A 3 agonists. The complexity of the electrophysiologic responses may have reflected cell heterogeneity of the populations harvested from collagenase digestion of TM explants. We now report measurements of TM-cell volume using calcein fluorescence quenching, an approach independent of contractile state. Furthermore, we have applied selective AR agonists to a uniform population of human TM cells, the hTM5 cell line. A 1 , but not A 2A or A 3 , AR agonists triggered TM-cell shrinkage. Both A 1 and A 2A AR agonists produced reproducible increases in TM-cell whole-cell currents of similar magnitude.The results suggest that previous measurements of explant-derived TM cells may have reflected a range of responses from phenotypically different cell populations, and that the opposing effects of A 1 and A 2A agonists on outflow resistance are not likely to be mediated by actions on a single population of TM cells. These opposing effects might reflect AR responses by two or more subpopulations of TM cells, by TM and SC cells or by inner-wall SC cells, alone.

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“…155,156,171,185,[188][189][190][191][192] These interesting yet confusing results may have stemmed from (i) the difference of cell types used, (ii) different stages of cells and (iii) different levels of over-expression. Nevertheless, several observations are particularly noteworthy.…”

Section: Nonmuscle Cad and Its Roles During Cell Proliferation And MImentioning

confidence: 99%

“…Since Rho activation induces cell adhesion, it had been suggested that focal adhesion involves actomyosin-based contractility. 192 What was not addressed is how much CaD in the cell was phosphorylated. It was observed that phosphorylated, but not unphosphorylated, CaD colocalizes with vinculin.…”

Section: Nonmuscle Cad and Its Roles During Cell Proliferation And MImentioning

confidence: 99%

Caldesmon and the Regulation of Cytoskeletal Functions

Wang

2008

Advances in Experimental Medicine and Biology

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Caldesmon (CaD) is an extraordinary actin-binding protein, because in addition to actin, it also binds myosin, calmodulin and tropomyosin. As a component of the smooth muscle and nonmuscle contractile apparatus CaD inhibits the actomyosin ATPase activity and its inhibitory action is modulated by both Ca 2+ and phosphorylation. The multiplicity of binding partners and diverse biochemical properties suggest CaD is a potent and versatile regulatory protein both in contractility and cell motility. However, after decades of investigation in numerous laboratories, hard evidence is still lacking to unequivocally identify its in vivo functions, although indirect evidence is mounting to support an important role in connection with the actin cytoskeleton. This chapter reviews the highlights of the past findings and summarizes the current views on this protein, with emphasis of its interaction with tropomyosin.

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Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells

Cited by 51 publications

References 50 publications

Cytoskeletal coherence requires myosin-IIA contractility

Cytoskeletal coherence requires myosin-IIA contractility

Cell-specific differential modulation of human trabecular meshwork cells by selective adenosine receptor agonists

Caldesmon and the Regulation of Cytoskeletal Functions

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