A new twist on plasma membrane repair

Cells in multicellular organisms are under constant mechanical stress, and often the plasma membrane (PM) is compromised. Fortunately, there is a vigorous repair mechanism that rapidly (within seconds) reseals the wound site by fusion with an internal membrane patch. Downstream events, remodeling of the injury site and forming replacement PM, must be carried out quickly (within minutes) if a cell is to survive multiple sequential injuries. The repertoire of proteins required to repair breaks (the PM repairome) is one of the major unknowns in this area of research. As an initial approach to defining the PM repairome, a cell surface biotinylation protocol was developed to identify intracellular proteins that become exposed at the site of reversible PM injury. It is likely that at least some of these proteins are important mediators of repair. These initial studies led to a surprising finding, namely the identification of some nuclear and endoplasmic reticulum resident proteins transiently exposed at the surface of cells that ultimately recovered from PM damage. Thus, in reversible mechanical damage to the PM, underlying cellular structures may also be injured, and will also require mechanisms for repair. Other proteins at wound sites were previously identified docking partners for pathogenic bacteria and viruses (vimentin and nucleolin), or found to be upregulated and exposed on the surface of cancer cells (nucleolin and nucleophosmin-1). The new information from these studies may lead to development of novel antimicrobial and antineoplastic drugs

Calcium-dependent plasma membrane repair requires m- or mu-calpain, but not calpain-3, the proteasome, or caspases

International audienceMechanically 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 noncatalytic 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 El 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 p-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. (C) 2009 Elsevier B.V. All rights reserved

A Plasma Membrane Wound Proteome: REVERSIBLE EXTERNALIZATION OF INTRACELLULAR PROTEINS FOLLOWING REPARABLE MECHANICAL DAMAGE

Cells in mechanically active tissues undergo constant plasma membrane damage that must be repaired to allow survival. To identify wound-associated proteins, a cell-impermeant, thiol-reactive biotinylation reagent was used to label and subsequently isolate intracellular proteins that become exposed on the surface of cultured cells after plasma membrane damage induced by scraping from substratum or crushing with glass beads. Scrape-damaged cells survived injury and were capable of forming viable colonies. Proteins that were exposed to the cell surface were degraded or internalized a few seconds to several minutes after damage, except for vimentin, which was detectable on the cell surface for at least an hour after injury. Seven major biotinylated protein bands were identified on SDS-PAGE gels. Mass spectrometric studies identified cytoskeletal proteins (caldesmon-1 and vimentin), endoplasmic reticulum proteins (ERp57, ERp5, and HSP47), and nuclear proteins (lamin C, heterogeneous nuclear ribonucleoprotein F, and nucleophosmin-1) as major proteins exposed after injury. Although caldesmon was a major wound-associated protein in calpain small subunit knock-out fibroblasts, it was rapidly degraded in wild-type cells, probably by calpains. Lamin C exposure after wounding was most likely the consequence of nuclear envelope damage. These studies document major intracellular proteins associated with the cell surface of reversibly damaged somatic cells. The studies also show that externalization of some proteins reported to have physiologic or pathologic roles on the cell surface can occur in cells undergoing plasma membrane damage and subsequent repair