Amit Bhatt

PURPOSE: Our group has previously published the Graded Prognostic Assessment (GPA), a prognostic index for patients with brain metastases. Updates have been published with refinements to create diagnosis-specific Graded Prognostic Assessment indices. The purpose of this report is to present the updated diagnosis-specific GPA indices in a single, unified, user-friendly report to allow ease of access and use by treating physicians. METHODS: A multi-institutional retrospective (1985 to 2007) database of 3,940 patients with newly diagnosed brain metastases underwent univariate and multivariate analyses of prognostic factors associated with outcomes by primary site and treatment. Significant prognostic factors were used to define the diagnosis-specific GPA prognostic indices. A GPA of 4.0 correlates with the best prognosis, whereas a GPA of 0.0 corresponds with the worst prognosis. RESULTS: Significant prognostic factors varied by diagnosis. For lung cancer, prognostic factors were Karnofsky performance score, age, presence of extracranial metastases, and number of brain metastases, confirming the original Lung-GPA. For melanoma and renal cell cancer, prognostic factors were Karnofsky performance score and the number of brain metastases. For breast cancer, prognostic factors were tumor subtype, Karnofsky performance score, and age. For GI cancer, the only prognostic factor was the Karnofsky performance score. The median survival times by GPA score and diagnosis were determined. CONCLUSION: Prognostic factors for patients with brain metastases vary by diagnosis, and for each diagnosis, a robust separation into different GPA scores was discerned, implying considerable heterogeneity in outcome, even within a single tumor type. In summary, these indices and related worksheet provide an accurate and facile diagnosis-specific tool to estimate survival, potentially select appropriate treatment, and stratify clinical trials for patients with brain metastases.

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. 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. focal adhesion kinase embryonic day (e.g. E9.5) SV40 large T antigen charge-coupled device embryonic stem cells Calpains have been implicated in a large number of physiological processes including cell spreading and migration (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 2Potter D.A. Tirnauer J.S. Janssen R. Croall D.E. Hughes C.N. Fiacco K.A. Mier J.W. Maki M. Herman I.M. J. Cell Biol. 1998; 141: 647-662Crossref PubMed Scopus (229) Google Scholar, 3Kulkarni S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), myoblast fusion (4Balcerzak D. Poussard S. Brustis J.J. Elamrani N. Soriano M. Cottin P. Ducastaing A. J. Cell Sci. 1995; 108: 2077-2082PubMed Google Scholar, 5Dourdin N. Balcerzak D. Brustis J.J. Poussard S. Cottin P. Ducastaing A. Exp. Cell Res. 1999; 246: 433-442Crossref PubMed Scopus (53) Google Scholar), cell cycle control (6Mellgren R.L. Zhang W. Lu Q. Lane R.D. Wang K.K.W. Yuen P.W. Calpain: Pharmacology and Toxicology of Calcium-dependent Protease. Taylor & Francis, Philadelphia1999: 161-178Google Scholar) and apoptosis (7McGinnis K.M. Gnegy M.E. Park Y.H. Mukerjee N. Wang K.K. Biochem. Biophys. Res. Commun. 1999; 263: 94-99Crossref PubMed Scopus (165) Google Scholar, 8Ruiz-Vela A. Gonzalez de Buitrago G. Martinez A.C. EMBO J. 1999; 18: 4988-4998Crossref PubMed Scopus (156) Google Scholar, 9Wolf B.B. Goldstein J.C. Stennicke H.R. Beere H. Amarante-Mendes G.P. Salvesen G.S. Green D.R. Blood. 1999; 94: 1683-1692Crossref PubMed Google Scholar). Various forms of calpains have also been implicated in pathological processes such as ischemia-induced damage, Alzheimer’s disease, muscular dystrophy, and, more recently, diabetes (10Turner P.R Westwood T. Regen C.M. Steinhardt R.A. Nature. 1988; 335: 735-738Crossref PubMed Scopus (358) Google Scholar, 11Lee K.S. Frank S. Vanderklish P. Arai A. Lynch G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7233-7237Crossref PubMed Scopus (333) Google Scholar, 12Hiramatsu K. Kassell N.F. Lee K.S. Stroke. 1993; 24: 1725-1728Crossref PubMed Scopus (29) Google Scholar, 13Saito K. Elce J.S. Hamos J.E. Nixon R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, R.L. A. J. Stroke. PubMed Scopus Google Scholar, G. S. Kassell N.F. Lee K.S. Stroke. PubMed Scopus Google Scholar, T.C. H. Suzuki K. J. PubMed Scopus Google Scholar, Croall D.E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. H. S. K.S. S. P. N. J. PubMed Scopus Google Scholar). However, the physiological functions of calpain and substrates in remain understood. is partly of the of of used calpain (6Mellgren R.L. Zhang W. Lu Q. Lane R.D. Wang K.K.W. Yuen P.W. Calpain: Pharmacology and Toxicology of Calcium-dependent Protease. Taylor & Francis, Philadelphia1999: 161-178Google Scholar). the role of calpain during migration been 4−/− embryonic cells a the role of calpain in a of processes the of calpain calpains and consist of a μ- or m-80-kDa large subunit (genes Capn 1 and Capn and a common 28-kDa small subunit (Capn 4). been but that Capn 4−/− at Elce J.S. K. Biol. PubMed Scopus Google Scholar). The Capn displayed a in suggesting a role calpain in cell migration. Interestingly, μ- or activity is by casein zymography in Capn 4−/− cells and embryonic these cells The not the of the calpain the observed Interestingly, of the Capn 1 and loss of had in the of been observed K. Croall D.E. Full Text PDF PubMed Scopus Google the that calpain was focal adhesions M. A. Biol. PubMed Scopus Google Scholar), a role calpain in the actin cell and migration. also been and focal adhesion proteins, including focal adhesion kinase and talin R. J. Cell Biol. 1999; PubMed Scopus Google Scholar, M. Suzuki H. S. Saido T.C. M. Biochem. Biophys. 1999; PubMed Scopus Google Scholar, K. S. D.E. Saido T.C. Fox J.E. J. Cell Biol. PubMed Scopus Google Scholar, D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). also cell spreading and migration by the of including not only but also kinase and the of However, the role calpain in migration is have of cell spreading and calpain D.A. Tirnauer J.S. Janssen R. Croall D.E. Hughes C.N. Fiacco K.A. Mier J.W. Maki M. Herman I.M. J. Cell Biol. 1998; 141: 647-662Crossref PubMed Scopus (229) Google Scholar, 3Kulkarni S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), have a role calpain in cell and focal complex (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, R. J. Cell Biol. 1999; PubMed Scopus Google assess the role of calpain during cell migration, we of focal and of focal adhesion 4+/+ and Capn The in observed in theCapn 4−/− cells were partially restored the wild-type by transfection 4−/− cells with a vector expressing the rat calpain small subunit. The results that calpain is during demonstrate unequivocally that calpain a critical role during cell migration and in the organization of the actin Capn 4−/− cells migration and a loss of central focal adhesions and stress fibers. The on cell and the organization of the actin cytoskeleton were in a cell that the rat 28-kDa small calpain in the observed Interestingly, the of calpain activity in the Capn 4−/− cell was and the cell only levels of suggesting that of the subunit not a the the critical of calpain as a regulator of the actin cytoskeleton and cell Capn 4−/− cells were 4+/+ cells, with a loss of central focal adhesions and stress fibers. is with calpain and that cell spreading is a D.A. Tirnauer J.S. Janssen R. Croall D.E. Hughes C.N. Fiacco K.A. Mier J.W. Maki M. Herman I.M. J. Cell Biol. 1998; 141: 647-662Crossref PubMed Scopus (229) Google Scholar, 3Kulkarni S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). these the Capn 4−/− cells were more and Capn at 4−/− cells plated on of prominent with thin projections and prominent retraction in control cells. have that small in have on the of S. A. Biol. Scopus Google Scholar). that calpain cell and migration in a by the activity of the of S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) in that calpain of and is with in Capn 4+/+ cells the Capn 4−/− cells had prominent membrane and that delayed retraction and the migration suggesting that calpain is important of cell during migration (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, S.P. A. Horwitz A.F. Lauffenburger D.A. J. Cell Sci. 1998; PubMed Google Scholar, H. A. K. A. J. Cell Biol. 1999; PubMed Google Scholar). The of the is not but the in or a in focal adhesion at the of the The of focal complexes observed calpain (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) and in theCapn 4−/− cells that calpain a role in focal complex R. J. Cell Biol. 1999; PubMed Scopus Google Scholar) by that focal complex in a that calpain activity implicated in the of focal adhesions during cell migration the cleavage of or more focal adhesion such as α-actinin, FAK, spectrin, talin, and vinculin have been as calpain substrates M. A. Biol. PubMed Scopus Google Scholar, R. J. Cell Biol. 1999; PubMed Scopus Google Scholar, T. K. Nature. PubMed Scopus Google Scholar, Nature. PubMed Scopus Google Scholar, R. Maki M. M. H. PubMed Scopus Google Scholar). results that spectrin and talin were cleaved in Capn but not in Capn 4−/− cells and that and vinculin were not cleaved in either cell type when plated on fibronectin. the levels of of of these were in Capn 4+/+ and Capn 4−/− cells. proteolysis of been focal adhesion D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), but in the same as and D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the and of in Capn 4+/+ and Capn 4−/− cells. that of not migration of embryonic fibroblasts the of The of of these calpain substrates and the role of calpain in these in theCapn 4−/− talin, a calpain been with the at adhesion M. A. Biol. PubMed Scopus Google Scholar). was in focal adhesion in cells but not in cells H. S. A. D. D. D.R. J. Cell Biol. 1998; PubMed Scopus Google Scholar). Moreover, calpain talin at in of and a of M. A. Biol. PubMed Scopus Google Scholar, T. K. Nature. PubMed Scopus Google Scholar, Nature. PubMed Scopus Google Scholar, R. Maki M. M. H. PubMed Scopus Google Scholar). also been that proteolysis of talin focal adhesions of cells, of a role calpain was Exp. Cell Res. 1995; PubMed Scopus Google Scholar). of talin in 4+/+ fibroblasts the of a of talin, was not detected 4−/− cells. the of talin cleavage in the Capn 4−/− cells of the calpain 28-kDa subunit that talin breakdown is calpain that calpain the cleavage of talin the adhesion and talin is with the of the of the A. K. K. Nature. PubMed Scopus Google Scholar, K. J. Cell Biol. PubMed Scopus Google Scholar) and also with actin Biochem. PubMed Google Scholar, S. J. Cell Biol. PubMed Scopus Google Scholar, M. S. T. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar), is that the of cleavage of at in the abnormal organization of the actin cytoskeleton and the migration observed in Capn 4−/− remain calpain cell migration and the organization of the actin results that talin proteolysis by calpain is cell migration, is that calpain substrates the focal the these substrates and more calpain the cytoskeleton and cell migration. 4−/− cells important the of these results that role calpain is cell migration by the actin cell and Calpains have been implicated in a large number of physiological processes including cell spreading and migration (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 2Potter D.A. Tirnauer J.S. Janssen R. Croall D.E. Hughes C.N. Fiacco K.A. Mier J.W. Maki M. Herman I.M. J. Cell Biol. 1998; 141: 647-662Crossref PubMed Scopus (229) Google Scholar, 3Kulkarni S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), myoblast fusion (4Balcerzak D. Poussard S. Brustis J.J. Elamrani N. Soriano M. Cottin P. Ducastaing A. J. Cell Sci. 1995; 108: 2077-2082PubMed Google Scholar, 5Dourdin N. Balcerzak D. Brustis J.J. Poussard S. Cottin P. Ducastaing A. Exp. Cell Res. 1999; 246: 433-442Crossref PubMed Scopus (53) Google Scholar), cell cycle control (6Mellgren R.L. Zhang W. Lu Q. Lane R.D. Wang K.K.W. Yuen P.W. Calpain: Pharmacology and Toxicology of Calcium-dependent Protease. Taylor & Francis, Philadelphia1999: 161-178Google Scholar) and apoptosis (7McGinnis K.M. Gnegy M.E. Park Y.H. Mukerjee N. Wang K.K. Biochem. Biophys. Res. Commun. 1999; 263: 94-99Crossref PubMed Scopus (165) Google Scholar, 8Ruiz-Vela A. Gonzalez de Buitrago G. Martinez A.C. EMBO J. 1999; 18: 4988-4998Crossref PubMed Scopus (156) Google Scholar, 9Wolf B.B. Goldstein J.C. Stennicke H.R. Beere H. Amarante-Mendes G.P. Salvesen G.S. Green D.R. Blood. 1999; 94: 1683-1692Crossref PubMed Google Scholar). Various forms of calpains have also been implicated in pathological processes such as ischemia-induced damage, Alzheimer’s disease, muscular dystrophy, and, more recently, diabetes (10Turner P.R Westwood T. Regen C.M. Steinhardt R.A. Nature. 1988; 335: 735-738Crossref PubMed Scopus (358) Google Scholar, 11Lee K.S. Frank S. Vanderklish P. Arai A. Lynch G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7233-7237Crossref PubMed Scopus (333) Google Scholar, 12Hiramatsu K. Kassell N.F. Lee K.S. Stroke. 1993; 24: 1725-1728Crossref PubMed Scopus (29) Google Scholar, 13Saito K. Elce J.S. Hamos J.E. Nixon R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, R.L. A. J. Stroke. PubMed Scopus Google Scholar, G. S. Kassell N.F. Lee K.S. Stroke. PubMed Scopus Google Scholar, T.C. H. Suzuki K. J. PubMed Scopus Google Scholar, Croall D.E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. H. S. K.S. S. P. N. J. PubMed Scopus Google Scholar). However, the physiological functions of calpain and substrates in remain understood. is partly of the of of used calpain (6Mellgren R.L. Zhang W. Lu Q. Lane R.D. Wang K.K.W. Yuen P.W. Calpain: Pharmacology and Toxicology of Calcium-dependent Protease. Taylor & Francis, Philadelphia1999: 161-178Google Scholar). the role of calpain during migration been 4−/− embryonic cells a the role of calpain in a of processes the of calpain The calpains and consist of a μ- or m-80-kDa large subunit (genes Capn 1 and Capn and a common 28-kDa small subunit (Capn 4). been but that Capn 4−/− at Elce J.S. K. Biol. PubMed Scopus Google Scholar). The Capn displayed a in suggesting a role calpain in cell migration. Interestingly, μ- or activity is by casein zymography in Capn 4−/− cells and embryonic these cells The not the of the calpain the observed Interestingly, of the Capn 1 and loss of had in the of been observed K. Croall D.E. Full Text PDF PubMed Scopus Google Scholar). the that calpain was focal adhesions M. A. Biol. PubMed Scopus Google Scholar), a role calpain in the actin cell and migration. also been and focal adhesion proteins, including focal adhesion kinase and talin R. J. Cell Biol. 1999; PubMed Scopus Google Scholar, M. Suzuki H. S. Saido T.C. M. Biochem. Biophys. 1999; PubMed Scopus Google Scholar, K. S. D.E. Saido T.C. Fox J.E. J. Cell Biol. PubMed Scopus Google Scholar, D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). also cell spreading and migration by the of including not only but also kinase and the of However, the role calpain in migration is have of cell spreading and calpain D.A. Tirnauer J.S. Janssen R. Croall D.E. Hughes C.N. Fiacco K.A. Mier J.W. Maki M. Herman I.M. J. Cell Biol. 1998; 141: 647-662Crossref PubMed Scopus (229) Google Scholar, 3Kulkarni S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), have a role calpain in cell and focal complex (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, R. J. Cell Biol. 1999; PubMed Scopus Google Scholar). To assess the role of calpain during cell migration, we of focal and of focal adhesion 4+/+ and Capn The in observed in theCapn 4−/− cells were partially restored the wild-type by transfection 4−/− cells with a vector expressing the rat calpain small subunit. The results that calpain is during migration. demonstrate unequivocally that calpain a critical role during cell migration and in the organization of the actin Capn 4−/− cells migration and a loss of central focal adhesions and stress fibers. The on cell and the organization of the actin cytoskeleton were in a cell that the rat 28-kDa small calpain in the observed Interestingly, the of calpain activity in the Capn 4−/− cell was and the cell only levels of suggesting that of the subunit not a the the critical of calpain as a regulator of the actin cytoskeleton and cell Capn 4−/− cells were 4+/+ cells, with a loss of central focal adhesions and stress fibers. is with calpain and that cell spreading is a D.A. Tirnauer J.S. Janssen R. Croall D.E. Hughes C.N. Fiacco K.A. Mier J.W. Maki M. Herman I.M. J. Cell Biol. 1998; 141: 647-662Crossref PubMed Scopus (229) Google Scholar, 3Kulkarni S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). these the Capn 4−/− cells were more and Capn at 4−/− cells plated on of prominent with thin projections and prominent retraction in control cells. have that small in have on the of S. A. Biol. Scopus Google Scholar). that calpain cell and migration in a by the activity of the of S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) in that calpain of and is with in Capn 4+/+ cells the Capn 4−/− cells had prominent membrane and that delayed retraction and the migration suggesting that calpain is important of cell during migration (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, S.P. A. Horwitz A.F. Lauffenburger D.A. J. Cell Sci. 1998; PubMed Google Scholar, H. A. K. A. J. Cell Biol. 1999; PubMed Google Scholar). The of the is not but the in or a in focal adhesion at the of the The of focal complexes observed calpain (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) and in theCapn 4−/− cells that calpain a role in focal complex R. J. Cell Biol. 1999; PubMed Scopus Google Scholar) by that focal complex in a that calpain activity implicated in the of focal adhesions during cell migration the cleavage of or more focal adhesion such as α-actinin, FAK, spectrin, talin, and vinculin have been as calpain substrates M. A. Biol. PubMed Scopus Google Scholar, R. J. Cell Biol. 1999; PubMed Scopus Google Scholar, T. K. Nature. PubMed Scopus Google Scholar, Nature. PubMed Scopus Google Scholar, R. Maki M. M. H. PubMed Scopus Google Scholar). results that spectrin and talin were cleaved in Capn but not in Capn 4−/− cells and that and vinculin were not cleaved in either cell type when plated on fibronectin. the levels of of of these were in Capn 4+/+ and Capn 4−/− cells. proteolysis of been focal adhesion D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), but in the same as and D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the and of in Capn 4+/+ and Capn 4−/− cells. that of not migration of embryonic fibroblasts the of The of of these calpain substrates and the role of calpain in these in theCapn 4−/− talin, a calpain been with the at adhesion M. A. Biol. PubMed Scopus Google Scholar). was in focal adhesion in cells but not in cells H. S. A. D. D. D.R. J. Cell Biol. 1998; PubMed Scopus Google Scholar). Moreover, calpain talin at in of and a of M. A. Biol. PubMed Scopus Google Scholar, T. K. Nature. PubMed Scopus Google Scholar, Nature. PubMed Scopus Google Scholar, R. Maki M. M. H. PubMed Scopus Google Scholar). also been that proteolysis of talin focal adhesions of cells, of a role calpain was Exp. Cell Res. 1995; PubMed Scopus Google Scholar). of talin in 4+/+ fibroblasts the of a of talin, was not detected 4−/− cells. the of talin cleavage in the Capn 4−/− cells of the calpain 28-kDa subunit that talin breakdown is calpain that calpain the cleavage of talin the adhesion and talin is with the of the of the A. K. K. Nature. PubMed Scopus Google Scholar, K. J. Cell Biol. PubMed Scopus Google Scholar) and also with actin Biochem. PubMed Google Scholar, S. J. Cell Biol. PubMed Scopus Google Scholar, M. S. T. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar), is that the of cleavage of at in the abnormal organization of the actin cytoskeleton and the migration observed in Capn 4−/− remain calpain cell migration and the organization of the actin results that talin proteolysis by calpain is cell migration, is that calpain substrates the focal the these substrates and more calpain the cytoskeleton and cell migration. 4−/− cells important the of these results that role calpain is cell migration by the actin cell and demonstrate unequivocally that calpain a critical role during cell migration and in the organization of the actin Capn 4−/− cells migration and a loss of central focal adhesions and stress fibers. The on cell and the organization of the actin cytoskeleton were in a cell that the rat 28-kDa small calpain in the observed Interestingly, the of calpain activity in the Capn 4−/− cell was and the cell only levels of suggesting that of the subunit not a the the critical of calpain as a regulator of the actin cytoskeleton and cell migration. The Capn 4−/− cells were 4+/+ cells, with a loss of central focal adhesions and stress fibers. is with calpain and that cell spreading is a D.A. Tirnauer J.S. Janssen R. Croall D.E. Hughes C.N. Fiacco K.A. Mier J.W. Maki M. Herman I.M. J. Cell Biol. 1998; 141: 647-662Crossref PubMed Scopus (229) Google Scholar, 3Kulkarni S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). these the Capn 4−/− cells were more and Capn at 4−/− cells plated on of prominent with thin projections and prominent retraction in control cells. have that small in have on the of S. A. Biol. Scopus Google Scholar). that calpain cell and migration in a by the activity of the of S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) in that calpain of and is with in Capn 4+/+ cells the Capn 4−/− cells had prominent membrane and that delayed retraction and the migration suggesting that calpain is important of cell during migration (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, S.P. A. Horwitz A.F. Lauffenburger D.A. J. Cell Sci. 1998; PubMed Google Scholar, H. A. K. A. J. Cell Biol. 1999; PubMed Google Scholar). The of the is not but the in or a in focal adhesion at the of the The of focal complexes observed calpain (1Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) and in theCapn 4−/− cells that calpain a role in focal complex R. J. Cell Biol. 1999; PubMed Scopus Google Scholar) by that focal complex in a that calpain activity implicated in the of focal adhesions during cell migration the cleavage of or more focal adhesion such as α-actinin, FAK, spectrin, talin, and vinculin have been as calpain substrates M. A. Biol. PubMed Scopus Google Scholar, R. J. Cell Biol. 1999; PubMed Scopus Google Scholar, T. K. Nature. PubMed Scopus Google Scholar, Nature. PubMed Scopus Google Scholar, R. Maki M. M. H. PubMed Scopus Google Scholar). results that spectrin and talin were cleaved in Capn but not in Capn 4−/− cells and that and vinculin were not cleaved in either cell type when plated on fibronectin. the levels of of of these were in Capn 4+/+ and Capn 4−/− cells. proteolysis of been focal adhesion D. J. Biol. Chem. 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Cell migration requires the regulated and dynamic turnover of adhesive complexes. We have previously demonstrated that the calcium-dependent protease, calpain, regulates the organization of adhesive complexes and cell detachment during cell migration. Evidence is now provided that inhibiting calpain through over-expression of the endogenous inhibitor of calpain, calpastatin, and pharmacological inhibitors results in an inhibition of adhesive complex disassembly with stabilization of GFP-vinculin and GFP/RFP-zyxin at the cell periphery. Calpain was also required for the microtubule-mediated turnover of adhesive complex sites after nocodazole wash-out, suggesting that calpain may mediate focal complex disassembly downstream of microtubules. Using dual imaging of RFP-zyxin and GFP-alpha-actinin, we observed a temporal and spatial relationship between alpha-actinin localization to focal contacts and the subsequent disassembly or translocation of RFP-zyxin containing focal complexes in areas of cell retraction. Calpain inhibition disrupted alpha-actinin localization to zyxin-containing focal contacts and focal complex disassembly or translocation to the cell center. In addition, disrupting alpha-actinin localization to focal complexes through expression of the alpha-actinin rod domain, but not the head domain, resulted in inhibition of focal adhesion disassembly similar to calpain inhibition. Our studies suggest a novel mechanism of action whereby calpain may modulate alpha-actinin localization into focal complexes and their subsequent disassembly or translocation.