Isoform specific function of calpain 2 in regulating membrane protrusion.
Previous studies have demonstrated a role for calpains in cell migration through their capacity to regulate focal adhesion dynamics and rear retraction. In this study, we provide evidence that calpains also modulate membrane protrusion activity in fibroblasts. We find that an immortalized Capn4(-/-) fibroblast line displays an altered morphology, characterized by numerous thin membrane projections and increased transient membrane activity. Furthermore, we show that protrusion kinetics of lamellipodia at the leading edge are improperly regulated in Capn4(-/-) cells, leading to impaired net forward lamellipodial extension. To address the isoform specific functions of calpain 1 and calpain 2 during cell protrusion, we stably introduced small interfering RNAs (siRNAs) targeting each isoform into a fibroblast cell line. Despite a loss in calpain 1 activity, calpain 1 knockdown cells show normal morphology and membrane protrusion dynamics. However, cells in which calpain 2 is knocked down are characterized by a protrusive morphology, increased transient membrane activity and altered protrusion kinetics, similar to the Capn4(-/-) fibroblasts. Additionally, we find that calpain 2, but not calpain 1, is required for proteolysis of the cytoskeletal and focal adhesion proteins FAK, paxillin, spectrin, and talin. Together, our findings support a novel role for calpain 2 in limiting membrane protrusions and in regulating lamellipodial dynamics at the leading edge of migrating cells.
Cellular and Molecular Biology Program, University of Wisconsin-Madison, Madison, WI 53706, USA.
S
S
Franco
B
B
Perrin
A
A
Huttenlocher
D000818:Animals; D002154:Calpain / Q000378:metabolism*; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000502:physiology*; D022081:Cell Surface Extensions / Q000378:metabolism* / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism / Q000648:ultrastructure; D034622:RNA Interference; D034741:RNA, Small Interfering; D013049:Spectrin / Q000378:metabolism; D016608:Talin / Q000378:metabolism
D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D020033:Protein Isoforms; C496062:Pxn protein, mouse; D034741:RNA, Small Interfering; D016608:Talin; D013049:Spectrin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse; D002154:Calpain