Contraction due to microtubule disruption is associated with increased phosphorylation of myosin regulatory light chain.

Abstract

Microtubules have been proposed to function as rigid struts which oppose cellular contraction. Consistent with this hypothesis, microtubule disruption strengthens the contractile force exerted by many cell types. We have investigated an alternative explanation for the mechanical effects of microtubule disruption: that microtubules modulate the mechanochemical activity of myosin by influencing phosphorylation of the myosin regulatory light chain (LC2o). We measured the force produced by a population of fibroblasts within a collagen lattice attached to an isometric force transducer. Treatment of cells with nocodazole, an inhibitor of microtubule polymerization, stimulated an isometric contraction that reached its peak level within 30 min and was typically 30-45% of the force increase following maximal stimulation with 30%o fetal bovine serum. The contraction following nocodazole treatment was associated with a 2-to 4-fold increase in LC2. phosphorylation. The increases in both force and LC20 phosphorylation, after addition of nocodazole, could be blocked or reversed by stabilizing the microtubules with paclitaxel (former generic name, taxol). Increasing force and LC20 phosphorylation by pretreatment with fetal bovine serum decreased the subsequent additional contraction upon microtubule disruption, a finding that appears inconsistent with a load-shifting mechanism. Our results suggest that phosphorylation of LC20 is a common mechanism for the contractions stimulated both by microtubule poisons and receptor-mediated agonists. The modulation of myosin activity by alterations in microtubule assembly may coordinate the physiological functions of these cytoskeletal components.

Whole cell mechanics of contractile fibroblasts: relations between effective cellular and extracellular matrix moduli

“…One model is the 'tensegrity' approach, in which microtubules act as internal equilibrators of tensile forces provided by actin cables (Ingber 1993). While our and other experiments agree that actin stress fibres exert tensile forces Zahalak et al 2000;Kumar et al 2006), the disruption of microtubules results in an increasing force exerted by cells (Kolodney & Elson 1995). This indicates either that microtubules act in parallel with stress fibres to directly oppose contraction or that actin stress fibres do not rely on the compressive resistance from microtubules for their function (note that the increase in contraction is explained by the release of Ca 2+ stores associated with microtubule disruption and the associated increased phosphorylation of the myosin regulatory light chain).…”

Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

“…As nocodazole was washed out, microtubules reformed, cells regenerated their processes, and the matrix returned to a more relaxed state. Kolodney et al previously used an isometric force transducer to measure the tension exerted by chick embryo fibroblasts within collagen matrices (Kolodney and Elson, 1995;Kolodney and Wysolmerski, 1992). Disrupting microtubules induced a 2-3 fold increase in force which correlated temporally with increased phosphorylation of the myosin regulatory light chain.…”

“…Kolodney et al used an isometric force transducer to quantitatively monitor the tension exerted by a dense population of chick embryo fibroblasts within collagen matrices (Kolodney and Elson, 1995;Kolodney and Wysolmerski, 1992). Disrupting microtubules induced a 2-3 fold increase in force over several minutes and reached maximum at about 30 minutes.…”

Dendritic Fibroblasts in Three-dimensional Collagen Matrices

“…Also, disrupting microtubules of bipolar chick embryo fibroblasts in collagen matrices was reported to induce abnormal pseudopodia (Tomasek and Hay, 1984). Loss of fibroblast polarity caused by disrupting microtubules typically results in an increase in cellular isometric tension and actin stress fibers (Danowski, 1989;Kolodney and Elson, 1995). For fibroblasts in floating collagen matrices, however, rather than formation of stress fibers, the consequence of disrupting microtubules was complete loss of the dendritic network.…”