Organization of stress fibers in cultured fibroblasts after extraction of actin with bovine brain gelsolin-like protein.
Mouse and quail embryo fibroblasts were extracted with Triton X-100 and the resulting cytoskeletons were treated with gelsolin-like actin-capping protein (the 90-kDa protein-actin complex isolated from bovine brain). Staining of cells with rhodamine-conjugated phalloin or an antibody to actin did not reveal any actin-containing structures after treatment with the 90-kDa protein-actin complex. Extraction of actin was confirmed by SDS-gel electrophoresis. Immunofluorescence microscopy showed that vinculin and alpha-actinin were released from the cytoskeletons together with actin. However, myosin remained associated with the cytoskeleton after treatment with the 90-kDa protein-actin complex. The distribution of myosin in treated cells showed no significant difference from that in control cells: in both cases myosin was localized mainly in the stress fibers. Double-fluorescence staining showed the absence of actin in myosin-containing stress fibers of treated cells. The ultrastructural organization of actin-depleted stress fibers was studied by transmission electron microscopy of platinum replicas. On electron micrographs these fibers appeared as bundles of filaments containing clusters of globular material. It is concluded that myosin localization in stress fibers does not depend on actin.
Verkhovsky
A B
AB
A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Surgucheva
I G
IG
Svitkina
T M
TM
Tint
I S
IS
Gelfand
V I
VI
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000502:physiology*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D003370:Coturnix; D003599:Cytoskeleton / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D018260:Gelsolin; D007150:Immunohistochemistry; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D009124:Muscle Proteins / Q000378:metabolism; D009218:Myosins / Q000502:physiology*; D012995:Solubility; D016596:Vinculin
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D009124:Muscle Proteins; D000185:Actinin; D016596:Vinculin; D009218:Myosins