pp60v-src association with the cytoskeleton induces actin reorganization without affecting polymerization status.
The mechanism by which Rous sarcoma virus (RSV) induces a reorganization of actin and its associated proteins and a reduction in microfilament bundles is at present poorly understood. To examine the relationship between the organization of the microfilament system and the polymerization state of actin after transformation, we have investigated these changes in a Rat-1 cell line transformed by LA29, a temperature-sensitive (ts) mutant of RSV. Parallel immunofluorescence and biochemical analysis demonstrated that LA29 pp60v-src was ts for tyrosine kinase activity and cytoskeletal association. Changes in the distribution and organization of actin, alpha-actinin and vinculin were dependent on the association of a kinase-active pp60v-src molecule with the detergent-insoluble cytoskeleton. Whilst there was a transformation-dependent loss of microfilament bundles, biochemical quantitation demonstrated that the polymerization state of the actin in both detergent-soluble and insoluble fractions of these cells grown at temperatures either permissive or restrictive for transformation was quantitatively unchanged. These results indicate that the loss of microfilament bundles after transformation is not due to a net depolymerization of filamentous actin but rather to a reorganization of polymeric actin from microfilament bundles and stress fibers to other polymeric forms within the cell. The polymeric nature of the actin in these cells was confirmed by electron microscopy of cytoskeletons and substrate-adherent membranes.
Felice
G R
GR
Department of Biochemistry and Cell Biology, Hunterian Institute, Royal College of Surgeons of England, London, United Kingdom.
Eason
P
P
Nermut
M V
MV
Kellie
S
S
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000378:metabolism*; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003599:Cytoskeleton / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D008854:Microscopy, Electron; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D010766:Phosphorylation; D011108:Polymers; D051381:Rats
D000199:Actins; D011108:Polymers; D015688:Oncogene Protein pp60(v-src)