Functional dissection of human protease mu-calpain in cell migration using RNAi.
Calpains are a family of calcium-dependent cysteine proteases involved in a variety of cellular functions. Two isoforms, m-calpain and mu-calpain, have been implicated in cell migration. However, since conventional inhibitors used for the studies of the functions of these enzymes lack specificity, the individual physiological function and biochemical mechanism of these two isoforms, especially mu-calpain, are not clear. In contrast, RNA interference has the potential to allow a sequence-specific destruction of target RNA for functional assay of gene of interest. In the present study, we found that small interfering RNAs-mediated knockdown of mu-calpain expression in MCF-7 cells that do not express m-Calpain led to a reduction of cell migration. This isoform-specific function of mu-calpain was further confirmed by the rescue experiment as overexpression of mu-calpain but not m-calpain could restore the cell migration rate. Knockdown of mu-calpain also altered cell morphology with increased filopodial projections and a highly elongated tail that seemed to prevent cell spreading and migration with reduced rear detachment ability. Furthermore, knockdown of mu-calpain decreased the proteolytic products of filamin and talin, which were specifically rescued by overexpression of mu-calpain but not m-calpain, suggesting that their proteolysis could be one of the key mechanisms by which mu-calpain regulates cell migration.
Wu
Meiqun
M
Mammalian Cell Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Que., Canada H4P 2R2.
Yu
Zhenbao
Z
Fan
Jinjiang
J
Caron
Antoine
A
Whiteway
Malcolm
M
Shen
Shi-Hsiang
SH
D002154:Calpain / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D003285:Contractile Proteins / Q000378:metabolism; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D016608:Talin / Q000378:metabolism
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; D016608:Talin; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain