Morphological and biochemical analyses of contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal and transformed cells.
The expression and intracellular distribution of four contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal fibroblasts and their transformed counterparts by Rous or avian sarcoma virus were compared. By analyzing the isoformal expression of actin, caldesmon and tropomyosin using two-dimensional gel electrophoresis, only tropomyosin showed significant alteration in its isoformal expression accompanied by transformation. Morphological study revealed that in normal cells, myosin, caldesmon and tropomyosin were distributed periodically along stress fibers, but were excluded from focal adhesions (adhesion plaques), at which stress fibers terminate. By contrast, the contractile proteins were concentrated within the protrusions of the ventral cell surface of transformed cells, which are cell-adhesive structures with high motility (podosomes). Regional analysis indicated that the contractile proteins do not show diffuse distribution within podosomes. Myosin, some caldesmon and tropomyosin in association with F-actin were localized in the region surrounding the core domains of podosomes. A major part of the caldesmon was, however, located in the core domain with short F-actin bundles. In order to compare the stability and the molecular organization of stress fibers with that of the short F-actin bundles within podosomes, the dorsal plasma membranes of the cells were removed by lysis and squirting. Then, the ruptured cells were treated with various buffers containing high salt, ATP or Ca2+/calmodulin. Myosin, caldesmon and tropomyosin were strongly associated with stress fibers of the ruptured normal fibroblasts even in a buffer containing high salt or Ca2+/calmodulin. On the other hand, myosin and tropomyosin within podosomes were easily extracted by lysis and squirting. And, the remaining caldesmon in podosomes was separated from the short F-actin bundles with high salt or Ca2+/calmodulin buffer. The present findings suggest that the high motility of podosomes from transformed cells is based on the actomyosin system, and that the stable adherence of focal adhesions of normal cells is due to a lack of this system. The accumulation of contractile proteins and their dynamic association within podosomes might be the cause of the short half-life of the structure. In relation to its localization in the core domain of podosomes without myosin and tropomyosin, the function of caldesmon has been discussed.
Department of Neurochemistry and Neuropharmacology, Osaka University Medical School, Japan.
J
J
Tanaka
T
T
Watanabe
N
N
Nakamura
K
K
Sobue
D000199:Actins / Q000032:analysis*; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000032:analysis*; D002448:Cell Adhesion; D002460:Cell Line; D002461:Cell Line, Transformed; D002465:Cell Movement; D003599:Cytoskeleton / Q000737:chemistry*; D009218:Myosins / Q000032:analysis*; D051381:Rats; D011916:Rats, Inbred F344; D014335:Tropomyosin / Q000032:analysis*
D000199:Actins; D002148:Calmodulin-Binding Proteins; D014335:Tropomyosin; D009218:Myosins