Reduced Cell Migration and Disruption of the Actin Cytoskeleton in Calpain-deficient Embryonic Fibroblasts

• Published in: The Journal of biological chemistry 2001-12, Vol.276 (51), p.48382-48388
• Main Authors: Dourdin, Nathalie, Bhatt, Amit K., Dutt, Previn, Greer, Peter A., Arthur, J. Simon C., Elce, John S., Huttenlocher, Anna
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Animals; Antigens, Polyomavirus Transforming - physiology; Calpain - genetics; Calpain - physiology; Capn4 gene; Cell Line; Cell Line, Transformed; Cell Movement - physiology; Cytoskeleton - metabolism; Embryo, Mammalian - cytology; Embryo, Mammalian - metabolism; Fibroblasts - cytology; Fibroblasts - metabolism; Hydrolysis; Mice; Rats; talin; Talin - metabolism; vinculin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M108893200

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The μ- and m-calpains consist of a μ- or m-80-kDa large subunit (genes Capn 1 and Capn 2), and a common 28-kDa small subunit (Capn 4). To assess the role of calpain in migration, we used fibroblasts obtained fromCapn 4−/− 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. Capn 4−/− 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 Capn 4−/− cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, α-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn 4+/+ and Capn 4−/− cells. FAK, α-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 theCapn 4−/− 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 Capn 4−/− 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.