TY - JOUR
T1 - Reduced Cell Migration and Disruption of the Actin Cytoskeleton in Calpain-deficient Embryonic Fibroblasts
AU - Dourdin, Nathalie
AU - Bhatt, Amit K.
AU - Dutt, Previn
AU - Greer, Peter A.
AU - Arthur, J. Simon C.
AU - Elce, John S.
AU - Huttenlocher, Anna
PY - 2001/12/21
Y1 - 2001/12/21
N2 - 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 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, α-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4+/+ and Capn4-/- 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 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.
AB - 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 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, α-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4+/+ and Capn4-/- 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 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.
UR - http://www.scopus.com/inward/record.url?scp=0035930548&partnerID=8YFLogxK
U2 - 10.1074/jbc.M108893200
DO - 10.1074/jbc.M108893200
M3 - Article
C2 - 11602605
AN - SCOPUS:0035930548
SN - 0021-9258
VL - 276
SP - 48382
EP - 48388
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 51
ER -