Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains.
The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced mu- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed.
Mazères
Germain
G
Laboratoire Biosciences de l'Aliment, ISTAB USC-INRA 2009, Talence Cedex, France.
Leloup
Ludovic
L
Daury
Laetitia
L
Cottin
Patrick
P
Brustis
Jean-Jacques
JJ
D000199:Actins / Q000502:physiology; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000378:metabolism / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects*; D002459:Cell Fusion; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal; D008870:Microtubules / Q000502:physiology; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D040901:Proteomics; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D000199:Actins; D002135:Calcium-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; C033668:calpastatin; D002154:Calpain