Ultrastructural cytoskeleton alterations and modification of actin expression in the NIH/3T3 cell line after transformation with Ha-ras-activated oncogene.
Cytoskeleton alterations of NIH/3T3 fibroblast monolayers transfected with Ha-ras-activated oncogene were studied by immunofluorescence, immunoelectron microscopy, and immunoelectrophoretic analysis of actin isoforms. Transformation foci were found to consist of cells with a round shape and rare stress fibers that spread sparsely, forming rare focal contacts and fibronexuses. The loss of stress fibers in transformed cells was confirmed by staining with rhodamine-phalloidin and with a fluorescinated anti-non-muscle cell actin antibody. The transformed cells were anchored to the substrate prominently by filaments that contained fibronectin, as showed by immunoelectron microscopy. A down-regulation of alpha-actin isoform was observed by immunofluorescence and immunoblotting analysis using a specific monoclonal antibody. The diffuse distribution of alpha-actin, lacking a specific association with stress fibers, challenges the hypothesis of a connection between alpha-actin down-regulation and stress fiber loss.
Division of Experimental Oncology A, University of Milan, Italy.
L
L
Lombardi
D
D
Ballinari
I
I
Bongarzone
M
M
Migliari
P
P
Mondellini
C
C
Traversari
S
S
Modina
D000199:Actins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002461:Cell Line, Transformed; D003599:Cytoskeleton / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras); D012235:Rhodamines; D013194:Staining and Labeling
D000199:Actins; D011518:Proto-Oncogene Proteins; D012235:Rhodamines; D016283:Proto-Oncogene Proteins p21(ras)