The annexins, a family of Ca 2؉ -and lipid-binding proteins, are involved in a range of intracellular processes. Recent findings have implicated annexin A1 in the resealing of plasmalemmal injuries. Here, we demonstrate that another member of the annexin protein family, annexin A6, is also involved in the repair of plasmalemmal lesions induced by a bacterial pore-forming toxin, streptolysin O. An injury-induced elevation in the intra- 2؉ -sensitivities provide a cell with the means to react promptly to a limited injury in its early stages and, at the same time, to withstand a sustained injury accompanied by the continuous formation of plasmalemmal lesions.The annexins are a family of Ca 2ϩ -binding proteins expressed in most phyla and species (1-4). Twelve annexins are present in vertebrates (A1-A11 and A13) with different splice variants (1). Annexins share a common folding motif, the "annexin core," which harbors the Ca 2ϩ -and membrane-binding sites (2-4). In their Ca 2ϩ -bound form, the annexins translocate from the cytoplasm to the plasma membrane and associate with negatively charged phospholipids (2-4). The N-terminal region precedes the conserved core and is unique for a given member of the annexin family. It mediates interactions with protein ligands and regulates the annexin-membrane association (2-4). Different annexins have been shown to orchestrate a variety of intracellular processes, ranging from the regulation of membrane dynamics to cell migration, proliferation, and apoptosis (2-12). However, the intriguing question why the majority of cells express several annexins, which differ only slightly in their biochemical properties, remains unanswered.Recent findings have implicated annexin A1 in the resealing of plasmalemmal lesions following cell injury (13,14). An injury-induced rise in the local concentration of intracellular Ca 2ϩ (15) is sensed by annexin A1 and triggers its binding to the plasma membrane at the site of the injury (13,14). Subsequently, annexin A1 promotes fusion of the damaged membrane around the pore, forming sealed, lesion-containing structures: large, cytosol-containing blebs (14) or smaller, cytosol-free microvesicles (16). The microvesicles subsequently can be shed by the cell (16, 17).Here, we show that, similar to annexin A1, annexin A6 is directly involved in the repair of plasmalemmal lesions induced by streptolysin O (SLO).2 The shedding of microvesicles appears to be predominant in the elimination of pores by annexin A6-dependent repair. Annexin A6 requires lower [Ca 2ϩ ] i for its plasmalemmal binding and, thus, responds faster to an injury than annexin A1. Correspondingly, a plasmalemmal lesion can be repaired by annexin A6 even without involvement of annexin A1; however, the concerted action of both annexins is instrumental for the efficient repair of multiple, simultaneously occurring plasmalemmal lesions. EXPERIMENTAL PROCEDURESReagents-Monoclonal anti-annexin A6 and anti-annexin A1 antibodies were from BD Biosciences; an antiserum against SLO was from Bioacad...

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Pathogenic bacteria secrete pore-forming toxins that permeabilize the plasma membrane of host cells. Nucleated cells possess protective mechanisms that repair toxin-damaged plasmalemma. Currently two putative repair scenarios are debated: either the isolation of the damaged membrane regions and their subsequent expulsion as microvesicles (shedding) or lysosome-dependent repair might allow the cell to rid itself of its toxic cargo and prevent lysis. Here we provide evidence that both mechanisms operate in tandem but fulfill diverse cellular needs. The prevalence of the repair strategy varies between cell types and is guided by the severity and the localization of the initial toxin-induced damage, by the morphology of a cell and, most important, by the incidence of the secondary mechanical damage. The surgically precise action of microvesicle shedding is best suited for the instant elimination of individual toxin pores, whereas lysosomal repair is indispensable for mending of self-inflicted mechanical injuries following initial plasmalemmal permeabilization by bacterial toxins. Our study provides new insights into the functioning of non-immune cellular defenses against bacterial pathogens.

The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described ␤ 2 -adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-␤-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the downstream events such as recruitment of protein kinases.

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