We compared spreading of Vero fibroblasts when microtubules were depolymerized or stabilized. After initial attachment, cells start blebbing, which continues for different times and abruptly transfers into spreading. After spreading initiation, most cells spread in an anisotropic way through stochastic formation of lamellipodia. A second mode that occurs in 15% of cells was rapid, isotropic spreading via formation of circular lamella. The rate of spreading was maximal at the beginning and decreased during the first hour according to a logarithmic law. After 60 min, many cells formed stable edges and started to migrate on the substrate. However, the cell area slowly continued to increase. Actin bundles were formed 20 min after cell attachment. They first run along the cell boundary. This system disassembled within 20-40 min and was substituted with stress fibers crossing the cell. In isotropically spread cells, no actin bundles were seen. Microtubules in the spreading cells enter into large blebs and all nascent lamellas; later, they form a radial array. When MTs have been depolymerized or stabilized blebbing started, before cells attach to the substrate and continue much longer than in control cells. After both treatments, the initial rate of spreading decreases several-fold and remains constant for many hours. After 24 h, the mean area occupied by cells with an altered MT system was the same as in control. Alteration of the MT system had a moderate effect on the actin system: formation of actin cables occurred at the same time as in control (within 20 min upon cell attachment); however, they started growing even in cells undergoing prolonged blebbing. Actin cables running along the cell margin were similar to those in control cells, but they did not disappear for up to 1 h. When stabilized, MTs form a chaotic array: they do not enter blebs and, in spread cells, run parallel to the cell margin at a distance of 3-5 μm. We conclude that dynamic MTs speed up completion of blebbing and promote early stages of fibroblast spreading.
ASJC Scopus subject areas
- Cell Biology