Organization of the cytoskeleton in square fibroblasts

The relationship between the cytoskeleton, stress fiber formation, and cell shape has been difficult to determine in fibroblasts grown in tissue culture. Vagaries in cell shape are complicated, as well, by stochastic cell movements. We dictated the attachment sites and shape of fibroblasts by growing them on square adhesive substrates surrounded by nonadhesive substrates. Cytoskeletal models were made by treating the cells with buffered Triton X-100 and glycerol. The residues were then examined by scanning electron microscopy followed by light microscopy of the same cells. The cytoskeletons of randomly moving cells were examined with whole mount transmission microscopy to confirm images seen with scanning microscopy. The cells thus examined demonstrated definite relationships between ruffling activity and stress fiber terminations, which were limited to the more adhesive, palladium substrate. No stress fibers were seen to end on the lesser adhesive substrate, agarose, and ruffling did not occur across the agarose. Cells too small to fill an entire square tended to extend across one diagonal of the square, and the stress fibers ran parallel to the longest axis of these cells. Larger cells were able to completely fill their squares. The cytoskeletons of these cells were organized in a spatial relation to the square shape of the cells. The cortical meshwork was aligned circularly and diagonally within the cells. Stress fibers appeared to form from the microfilaments of the meshwork and were aligned diagonally across the cells. We conclude that the diagonal arrangement of the stress fibers and cortical meshwork is caused by the same mechanism by which smaller cells spread over the longest axis of a square. Regions of cells where the meshwork was absent or where stress fibers were tightly bundled were occupied by more randomly arranged cytoskeletal components. Regions of tightly bundled stress fibers did not seem to coincide with regions of cortical meshwork as seen by either whole mount transmission or scanning electron microscopy. Stress fibers were revealed in the light microscope to course beneath more randomly oriented cytoskeletal elements. These "lacework-like" elements were found frequently in square cells. Conspicuous structures in this random lacework were focal points of radially arranged filaments. Our observations suggest a continuity between stress fibers and the cortical microfilaments. The orientation of fibers and filaments was, in turn, dependent on cell shape for organization within the cell.