Contraction due to microtubule disruption is associated with increased phosphorylation of myosin regulatory light chain.
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 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 (LC20). 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% fetal bovine serum. The contraction following nocodazole treatment was associated with a 2- to 4-fold increase in LC20 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.
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002642:Chick Embryo; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts; D007537:Isometric Contraction / Q000187:drug effects; D007700:Kinetics; D008870:Microtubules / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D009132:Muscles / Q000187:drug effects / Q000502:physiology*; D018994:Myosin Light Chains / Q000302:isolation & purification / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation; D013997:Time Factors
D003470:Culture Media; D018994:Myosin Light Chains; D017239:Paclitaxel; D015739:Nocodazole