Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin

TY - JOUR

T1 - Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin

AU - Larsen, Anna K

AU - Lametsch, René

AU - Elce, John S

AU - Larsen, Jørgen K

AU - Thomsen, Bo

AU - Larsen, Martin R

AU - Lawson, Moira A

AU - Greer, Peter A

AU - Ertbjerg, Per

PY - 2008

Y1 - 2008

N2 - Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels of tropomyosin in calpain-knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dynamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild-type cells. These results suggest a regulatory connection between calpain and RhoGDI-1 in promoting formation of membrane blebs

AB - Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels of tropomyosin in calpain-knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dynamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild-type cells. These results suggest a regulatory connection between calpain and RhoGDI-1 in promoting formation of membrane blebs

KW - cofilin

KW - green fluorescent protein (GFP)

KW - m-calpain

KW - proteomics

KW - Rho GDP-dissociation inhibitor (RhoGDI-1)

KW - tropomyosin

U2 - 10.1042/BJ20070522

DO - 10.1042/BJ20070522

M3 - Journal article

C2 - 18076376

SN - 0264-6021

VL - 411

SP - 657

EP - 666

JO - Biochemical Journal

JF - Biochemical Journal

ER -