Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin.
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.
Larsen
Anna K
AK
Department of Food Science, Faculty of Life Sciences, Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark. akl@life.ku.dk
Lametsch
René
R
Elce
John
J
Larsen
Jørgen K
JK
Thomsen
Bo
B
Larsen
Martin R
MR
Lawson
Moira A
MA
Greer
Peter A
PA
Ertbjerg
Per
P
D000818:Animals; D002154:Calpain / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D051338:Cofilin 1 / Q000378:metabolism*; D005347:Fibroblasts; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C495811:Cfl1 protein, mouse; D051338:Cofilin 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D021122:Protein Subunits; D014335:Tropomyosin; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D002154:Calpain