Morphological study of fibroblasts treated with cytochalasin D and colchicine using a confocal laser scanning microscopy.

The role of actin filaments and microtubules in 3D cell morphology was investigated using confocal laser scanning microscopy and image analysis based on a region-growing method. Fibroblasts were treated with cytochalasin D or colchicine to disrupt the actin filaments or microtubules, respectively, and the structure and distribution of these cytoskeletal filaments were observed using a confocal laser scanning microscope. From the 3D reconstructed fluorescence images of the cytoskeleton, morphological parameters such as volume, adhesion area, height, and volume ratio of individual cells were determined. The volume ratio was defined as the ratio of the partial volume for every 10% of the height to the total cell volume. The cell volume decreased slightly after the disruption of actin filaments and microtubules, but the change was not significant. The cell adhesion area was significantly decreased after the disruption of actin filaments and microtubules, and was significantly smaller in actin filament-disrupted cells than in microtubule-disrupted cells. Cell height increased significantly after actin filament disruption, whereas it remained almost unchanged after microtubule disruption. Analysis of the volume ratio revealed that the cell shape changed from a cone to a hemisphere after disruption of actin filaments and slightly shifted toward a hemisphere-like shape after microtubule disruption. These results suggest that actin filaments are the major component responsible for the maintenance of global cell shape and that the contribution of microtubules to global cell morphology is much less than that of actin filaments.