Requirement for Annexin A1 in Plasma Membrane Repair

Abstract

Ca2؉ entering a cell through a torn or disrupted plasma membrane rapidly triggers a combination of homotypic and exocytotic membrane fusion events. These events serve to erect a reparative membrane patch and then anneal it to the defect site. Annexin A1 is a cytosolic protein that, when activated by micromolar Ca 2؉ , binds to membrane phospholipids, promoting membrane aggregation and fusion. We demonstrate here that an annexin A1 function-blocking antibody, a small peptide competitor, and a dominant-negative annexin A1 mutant protein incapable of Ca 2؉ binding all inhibit resealing. Moreover, we show that, coincident with a resealing event, annexin A1 becomes concentrated at disruption sites. We propose that Ca 2؉ entering through a disruption locally induces annexin A1 binding to membranes, initiating emergency fusion events whenever and wherever required.Plasma membrane disruption, a common form of cell injury, is provoked in many mammalian tissues by physiological levels of mechanical stress (1). Disease can result from a failure either to prevent or repair disruptions. In Duchenne muscular dystrophy, for example, normal muscle contractions result in a pathological level of disruption injury because of increased fiber fragility (2). In limb-girdle muscular dystrophy, on the other hand, the failure is in the repair of normally occurring disruptions (3). Indeed, membrane repair, or resealing, is the universal survival response that eukaryotic cells mount when confronted with a life-threatening disruption in the integrity of their plasma membrane.The mechanism of resealing is now well characterized at the cellular level. A large plasma membrane disruption (Ͼ1 m diameter) locally and rapidly (subsecond to second time scale) elicits Ca 2ϩ -activated, homotypic vesicle fusion (4, 5). The "patch" vesicle thus formed then fuses exocytotically with the plasma membrane surrounding the defect site, restoring barrier continuity (6, 7). Identification of the protein components of this process is under way, and members of the SNARE 2 family (membrane proteins thought to be required for many fusion events) are implicated in resealing (8). However, as an emergency response it must be rapid and Ca 2ϩ -responsive and yet capable of being activated with a great level of temporal and spatial flexibility. Therefore, it has been hypothesized that resealing-based fusion may utilize, in addition to canonical elements such as the SNAREs, a specialized subset of fusion components (9). Dysferlin, a Ca 2ϩ -activated membrane-binding protein that mediates exocytotic fusion events in nematodes (10), is one candidate emergency fusion component. Thus, it was recently shown that skeletal muscle cells from dysferlin null mice fail to reseal disruptions (3).One protein shown by immunolocalization and immunoprecipitation to associate with dysferlin in normal, undisturbed skeletal muscle is annexin A1 (11), a member of the annexin family of Ca 2ϩ -regulated membrane-binding proteins (12). This observation, as well as the following additional po...

The Final Conformation of the Complete Ectodomain of the HA2 Subunit of Influenza Hemagglutinin Can by Itself Drive Low pH-dependent Fusion

“…Thus, the present study is an independent confirmation of these more recent studies demonstrating that neither the transmembrane domain nor the cytosolic domain is required for an opening of a fusion pore. This conclusion is also in agreement with reports that transmembrane proteins are not required for fusion in autophagy (59), in nuclear membrane fusion in yeasts (60), in membrane repair (61), and in fusion at the early stages of nuclear envelope assembly in Xenopus oocytes (62,63). The fusogenic activity of the HA2 ectodomain is also consistent with the hypothesis that both fusion and fission of biological membranes are driven by membrane elastic stresses (64,65).…”

Blebbing confers resistance against cell lysis

“…Annexin A1 might also trigger a sealing of the lipid bilayer within the bleb-to-cell contact region by recruiting intracellular vesicles according to the 'classical' resealing pathway. 1,2 We tested these postulates either by inactivating annexin A1 with a blocking antibody 13 or by downregulating annexin A1 expression level with siRNA. In the first set of experiments, native HEK 293 cells expressing endogenous annexin A1 were permeabilized in the presence of the antibody, which penetrated the cells via the perforated plasma membrane.…”

“…2,12 It is thus hardly surprising that an elevation in [Ca 2 þ ] i is sensed as an 'immediate danger' signal by an injured cell. 2,13,14 In injured cells the influx of extracellular Ca 2 þ triggers a process of plasma membrane blebbing, which is often considered to be a harbinger of cell death. [15][16][17][18][19][20][21][22] Blebs are sprouted by cells whose plasma membrane has become locally detached from the actin cortex.…”

Intracellular Ca2+ operates a switch between repair and lysis of streptolysin O-perforated cells

“…19 Here, we show that annexin A1 might be involved in the inactivation of SLO pores. Annexin A1 has earlier been shown to be indispensable for plasma membrane repair 20 and to play a protective role in a variety of pathological conditions. 21 Some of the annexin A1-plugged SLO pores remained within the plasma membrane of recovering cells for prolonged periods (Figures 4 and 5; Supplementary video 3).…”