A Plasma Membrane Wound Proteome

Abstract

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, thiolreactive 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 wildtype 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.Cells in tissues of multicellular organisms are often subjected to mechanical stresses that result in PM 2 damage (1-4). It is therefore not surprising that mechanisms have evolved to rapidly repair breaches in the PM that would otherwise rapidly lead to cell death. The currently accepted model posits calcium-dependent homotypic fusion of intracellular vesicles to provide a large membrane "patch" that rapidly (within seconds) seals the opening in the PM, maintaining cell viability. The nature of the intracellular membrane component(s) required for repair is a matter of speculation (5, 6). Few other details of the PM repair process are known, although some proteins, including membrane fusion proteins (5), myosin motor proteins (5), dysferlin (7), annexin A1 (8), calpains (9), and MG53 (10), have been shown to participate in repair in various model systems. To my knowledge, a systematic search for a "plasma membrane repairome," i.e. proteins required for repair of mechanically damaged membrane, has only been carried out in marine oocytes. Several high molecular weight yolk granule proteins were found to comprise a complex that facilitated vesicle patch formation (11).The entire chronologic sequence of repair and subsequent remodeling of the wound site is another area that requires further exploration, because an expeditious, full resolution of PM damage must be essential in tis...

Wound Repair: Toward Understanding and Integration of Single-Cell and Multicellular Wound Responses

“…On the basis of a variety of considerations, we think it likely that most of the membranous compartments near the wound site would be likely to fuse with the plasma membrane upon exposure to the millimolar level of calcium in the extracellular medium (Bement et al 2007). Consistent with this expectation, proteomic analysis of plasma membrane wounds has revealed several components of the endoplasmic reticulum (Mellgren 2010) in membrane patches.…”

Protein disulfide isomerase-associated 6 is an ATF6-inducible ER stress response protein that protects cardiac myocytes from ischemia/reperfusion-mediated cell death

“…Interestingly, in the same study, cell-surface PDIA6 was found to associate with integrins, suggesting that PDIA6 may contribute to platelet activation by affecting the configuration of disulfide bonds of several integrins. These intriguing effects were extended further when it was shown that PDIA6 was one of several other ER proteins that relocated to the cell surface upon plasma membrane wounding [50]. Cell-surface PDIA6 has also been shown to promote tumor immune evasion through the shedding of tumor associated ligands [51].…”

Cytoskeleton responses in wound repair

“…In fibroblasts, calpain mediates the rapid fragmentation of talin and vimentin after wounding, as a first step towards the removal of damaged cytoskeleton [8]. Calpain has also been shown to rapidly cleave caldesmon, a calmodulin dependent actin-linked protein involved in actin filament stabilization and actomyosin contraction, suggesting an alternative mechanism facilitating the rapid disassembly and clearance of the local cytoskeleton at the wound site [9]. …”