Characterization of Chinese hamster ovary cells with impaired spreading properties on fibronectin.
The development of receptor-defective or -deficient mutants can be applied to the investigation of cell-matrix interactions including cell adherence and spreading. In the present study we developed a series of ethyl methyl sulfonate (EMS)-induced Chinese hamster ovary (CHO) cell mutants, which adhere to fibronectin but have impaired spreading characteristics. Using morphometric analysis, a significant suppression in the degree of cell spreading between the wild-type and the mutant cells (P less than 0.001) was seen. This inability of the mutant cells to spread adequately on fibronectin also resulted in a decreased number and diameter of stress fibers as compared to wild-type cells. The decreased cell spreading of the mutant cells was not due to inherent differences in cell size or volume, as determined by fluorescence-activated cell sorter (FACS) analysis. Since integrins, specifically the fibronectin receptor (alpha FN/beta 1), are important in cell adhesion and cell spreading, we carried out a comparative immunochemical analysis, using a monoclonal antibody to the beta 1 subunit of integrin (7E2). Western blot analysis of cell extracts and cell membranes indicated that both wild-type and mutant cells expressed the alpha and beta 1 subunits of the fibronectin receptor; the mutant cells displayed reduced levels of the subunit. Immunohistochemical analysis indicated that, despite the presence of the receptor in both cell types, their patterns of localization and aggregation were different. The wild-type cells showed a needle-like distribution of the receptor, in contrast to the clumped appearance in the mutants.(ABSTRACT TRUNCATED AT 250 WORDS)
Joseph
L B
LB
Department of BioStructure and Function, University of Connecticut Health Center, Farmington 06032.
Kreutzer
D L
DL
Tanzer
M L
ML
D000199:Actins / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005020:Ethyl Methanesulfonate; D005260:Female; D005353:Fibronectins / Q000378:metabolism; D016023:Integrins / Q000378:metabolism; D009154:Mutation; D010053:Ovary; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism
D000199:Actins; D005353:Fibronectins; D016023:Integrins; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; D005020:Ethyl Methanesulfonate