Calcium-dependent plasma membrane repair requires m- or mu-calpain, but not calpain-3, the proteasome, or caspases.
Mechanically damaged plasma membrane undergoes rapid calcium-dependent resealing that appears to depend, at least in part, on calpain-mediated cortical cytoskeletal remodeling. Cells null for Capns1, the non-catalytic small subunit present in both m- and mu-calpains, do not undergo calcium-mediated resealing. However, it is not known which of these calpains is needed for repair, or whether other major cytosolic proteinases may participate. Utilizing isozyme-selective siRNAs to decrease expression of Capn1 or Capn2, catalytic subunits of mu- and m-calpains, respectively, in a mouse embryonic fibroblast cell line, we now show that substantial loss of both activities is required to compromise calcium-mediated survival after cell scrape-damage. Using skeletal myotubes derived from Capn3-null mice, we were unable to demonstrate loss of sarcolemma resealing after needle scratch or laser damage. Isolated muscle fibers from Capn3 knockout mice also efficiently repaired laser damage. Employing either a cell line expressing a temperature sensitive E1 ubiquitin ligase, or lactacystin, a specific proteasome inhibitor, it was not possible to demonstrate an effect of the proteasome on calcium-mediated survival after injury. Moreover, several cell-permeant caspase inhibitors were incapable of significantly decreasing survival or inhibiting membrane repair. Taken together with previous studies, the results show that m- or mu-calpain can facilitate repair of damaged plasma membrane. While there was no evidence for the involvement of calpain-3, the proteasome or caspases in early events of plasma membrane repair, our studies do not rule out their participation in downstream events that may link plasma membrane repair to adaptive remodeling after injury.
Department of Physiology and Pharmacology, University of Toledo College of Medicine, Toledo, OH, USA. ronald.mellgren@utoledo.edu
Ronald L
RL
Mellgren
Katsuya
K
Miyake
Irina
I
Kramerova
Melissa J
MJ
Spencer
Nathalie
N
Bourg
Marc
M
Bartoli
Isabelle
I
Richard
Peter A
PA
Greer
Paul L
PL
McNeil
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D009124:Muscle Proteins; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*
D009124:Muscle Proteins; D002154:Calpain; C491373:Capn3 protein, mouse; D020169:Caspases; C449000:m-calpain; D046988:Proteasome Endopeptidase Complex; D002118:Calcium