Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts.
The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.
Dourdin
N
N
Department of Biochemistry and the Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L3N6, Canada.
Bhatt
A K
AK
Dutt
P
P
Greer
P A
PA
Arthur
J S
JS
Elce
J S
JS
Huttenlocher
A
A
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology; D002154:Calpain / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006868:Hydrolysis; D051379:Mice; D051381:Rats; D016608:Talin / Q000378:metabolism
D000199:Actins; D000952:Antigens, Polyomavirus Transforming; D016608:Talin; D002154:Calpain