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The role of crosstalk between cytoskeleton components in cell adhesion and spreading remains largely elusive. We employ highthroughput analysis to time the spreading of cells with mesenchymal motility type and observe consistent changes after the asynchronous cell behaviors are put into population synchrony. Spreading kinetics for Vero, 3T3 and MEF fibroblasts is substrate and celltype independent and can be divided into fast radial spreading phase (5 to 15 minutes) that can be either isotropic or anisotropic, and subsequent slow spreading phase (1 to 3 hours). Addressing the question of fast spreading phase regulation, we propose that dynamic microtubules (MT) are the key inductors of fast spreading that occurs through temporal myosin II relaxation. In accord with this supposition we show that inhibition of myosin II phosphorylation with blebbistatin or Y27632 had no effect on the fast spreading phase, while alter slow spreading phase. To test the hypothesis on the role of MTs, we analyzed cell spreading morphology and kinetics after treatment with MT inhibitors (nocodazole and paclitaxel) together with myosin II phosphorylation inhibitors (blebbistatin and Y27632). All treatments led to the loss of isotropically spread cell subpopulation. MT depolymerization and stabilization significantly decelerated the fast cell spreading phase (10fold compared to untreated cells). Besides, depolymerization of MTs dramatically altered cell morphology inhibiting the formation of thin lamellae. In cells with compromised MTs myosin II is hyperphosphorylated near the cell margin within 5 to 20 minutes after initial attachment. Using combined treatment we show that MT stabilization and complete depolymerization in the presence of myosin inhibitors had minor effects on the fast spreading phase. We propose that microtubules’ rapid growth towards the cell margin during the fast cell spreading phase temporarily inhibits myosin II phosphorylation and thus “permits” fast spreading