Change of the Topography of Ventral Cell Surface during Spreading of Fibroblasts as Revealed by Evanescent Wave-Excited Fluorescence Microscopy: Effect of Contractility and Microtubule Integrity

Abstract

Evanescent wave-excited fluorescence microscopy, which selectively probes the ventral membranes of cells adhered to glass substrate, was utilized to observe the change in the topography of the ventral plasma membranes of Swiss 3T3 fibroblasts during spreading. In the initial stage of the spreading (up to 2 hours after seeding), the ventral membrane was close (<100nm) to the substrate in the peripheral and the central regions. About 4 hrs after seeding, the ventral surface assumed a flat topography for a short period and then gradually became uneven, displaying streak pattern of cell-to-substrate contact (6-8 hours after seeding). By 24 hours after seeding, cells gained polygonal shape and most regions except for the focal adhesions were separated from the substrate. Within these well-spread cells actin stress fibers were found to emanate obliquely from the focal adhesions, as previously reported. When cells were grown in the presence of 2, 3-butanedione monoxime (BDM), an inhibitor of actomyosin-based contraction of stress fibers and the cell, the ventral membranes in majority of the cells displayed flat topography, and the tilt of the stress fibers decreased. Cells grown in the presence of colchicine, a microtubule-depolymerizing agent also possessed flat ventral membrane and less tilted stress fibers. These results suggest that the contraction of stress fibers and integrity of microtubules are important in the formation of the uneven topography of ventral membrane and the tilt of stress fibers.