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Abstract
Dynamics of alterations of focal adhesions (FA) induced by a microtubule-depolymerizing drug, colcemid, was examined in several types of fibroblastic cells. Evolution of individual FA in cultured cells was monitored by interference-reflection microscopy (IRM); at the end of the monitoring period (3 hours) the cells were fixed and immunofluorescence microscopy of the same FA was performed with an antibody against vinculin. Control and colcemid-treated cells remained non-motile and did not show lamellipodial activity at the edges. During the incubation, formation of new FA or disappearance of pre-existing FA did not occur in either colcemid-treated or control cultures. However, FA in colcemid-treated cells significantly increased in size in the course of a 3 hour incubation. The growth of FA was centripetal and sometimes was accompanied by the fusion of several adjacent FA.
Immunofluorescence examination showed that colcemid-induced growth of FA was accompanied by accumulation of several proteins specific for these structures including vinculin, talin, paxillin and pp125FAK kinase. Immunoblotting with anti-vinculin antibody showed that incubation with colcemid considerably increased the amount of vinculin associated with the ventral membranes due to its partial redistribution from a soluble pool into the growing adhesions. A substantial increase in tyrosine phosphorylation of pp125FAK was also observed in colcemid-treated cells. In cells plated on elastic silicone rubber films, colcemid induced formation of wrinkles in the films and these wrinkles relaxed after treatment with cytochalasin D. These results confirm that microtubule depolymerization increases traction transmitted to the substratum by the actin cortex and shows that an increase in cortical tension accompanies maturation of FA.
Taken together, these data show that short-term incubation with colcemid does not affect the formation of initial FA. In contrast, microtubule depolymerization considerably stimulates the maturation FA, manifested by their centripetal growth. Maturation is proposed to be mediated by increased cortical tension, which is caused by microtubule depolymerization.