J. Cell Biol., Volume 141, Number 3, May 4, 1998 647-662

Calpain Regulates Actin Remodeling during Cell Spreading

David A. Potter,^* Jennifer S. Tirnauer,^* Richard Janssen,^* Dorothy E. Croall,^§ Christina N. Hughes,^* Kerry A. Fiacco,^* James W. Mier,^* Masatoshi Maki, and Ira M. Herman^¶

^* Division of Hematology and Oncology, Tupper Research Institute, Department of Medicine, New England Medical Center, Boston, Massachusetts;  Department of Biochemistry, ^¶ Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111; ^§ Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono, Maine 04469-5735; and  Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, 464-8601 Nagoya, Japan

Previous studies suggest that the Ca²⁺-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.

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