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

Abstract

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The -and m-calpains consist of a -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 vinculincontaining focal complexes at the cell periphery. The levels of the focal adhesion proteins, ␣-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4 ؉/؉ and Capn4 ؊/؊ cells. FAK, ␣-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.

Podosome-type adhesions and focal adhesions, so alike yet so different

“…Indeed, Zaidel-Bar , 2007 et al recent studies have demonstrated that the calpain family has a regulatory function in cell motility, partly through the capacity to down regulate integrin-mediated adhesion complexes ( ; ; ; ). In that Glading , 2000et al Dourdin , 2001et al Bhatt , 2002et al Glading , 2002 context, talin seems to be one of major target of calpain leading to a rate-limiting step critical for FA disassembly ( ). It Franco , 2004 et al has also been shown that the expression of a calpain-resistent cortactin impaired cell migration and increased transient membrane…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…Thus, microtubules may direct kinesin motors bound to disassembly factors to focal sites to promote adhesion turn over. The molecular nature of factors that are delivered to focal adhesions by microtubules have remained elusive [134,137,139] but might include the class of calcium activated proteases, calpains [140,141] (see below). Interestingly, Ezratty et.…”

“…One might speculate that proteolysis of FAK is a negative feedback event that prevents liberated FAK from destabilizing adjacent adhesions. However, it should be noted that calpain cleavage of FAK may be restricted to v-src-transformed cells [141,196].…”

Ceramide triggers an NF-κB-dependent survival pathway through calpain

“…26 To further assess the role played by calpain in NF-kB induction by C2 ceramide, we transiently transfected wild-type and Capn4À/À MEFs with a LUC expression plasmid driven by the human iNOS promoter, a target of NF-kB. After 24 h, C2 ceramide was added and LUC activity was measured 4 and 8 h later.…”

“…Calpains constitute a large family of cytosolic calciumdependent proteases which play an important role in the regulation of intracellular activities including cell spreading and migration, 25,26 myoblast fusion, programmed cell death and cell cycle progression. 27 The ubiquitous calpains, Calpain I (micro-calpain) and II (milli-calpain), consist of a large 80 kDa subunit encoded by the Capn1 and Capn2 genes, respectively, and a common 28 kDa small subunit encoded by Capn4.…”