Pore-forming (poly)peptides originating from invading pathogens cause plasma membrane damage in target cells, with consequences as diverse as proliferation or cell death. However, the factors that define the outcome remain unknown. We show that in cells maintaining an intracellular Ca 2 þ concentration [Ca 2 þ ] i below a critical threshold of 10 lM, repair mechanisms seal off 'hot spots' of Ca 2 þ entry and shed them in the form of microparticles, leading to [ Plasma membrane pores formed by cytotoxic proteins and peptides disrupt the permeability barrier in a target cell. Pathogens gain access and kill host cells by secreting pore-forming toxins, whereas the blood complement system utilises the pore-forming proteins of membrane attack complexes to eliminate both pathogens and the pathogen-invaded cells. 1,2 As in particular, cells of the blood and the vascular systems are permanently exposed to potential deadly attacks by a variety of pore-forming (poly)peptides, it is not surprising that mechanisms repairing the damaged plasma membrane have evolved. [3][4][5][6] Biological effects occurring in the wake of membrane permeabilisation and its subsequent repair are multifaceted. Apart from the two obvious end points, complete recovery or death, recovering cells can newly acquire numerous (patho)physiological functions. 3,7,8 A rise in intracellular Ca 2 þ concentration [Ca 2 þ ] i is critical for successful plasma membrane repair and cell recovery, 9,10 whereas an intracellular Ca 2 þ overload is held responsible for the death of pore-bearing cells. 11 In addition, Ca 2 þ influx, followed by transcriptional activation, is thought to induce a variety of biological responses associated with sublytic effects of pore-forming toxins. 3,4,7,8 Thus, it appears that the extent of [Ca 2 þ ] i elevation following pore formation determines the fate of a targeted cell. Consequently, Morgan et al. 11 suggested that in nucleated cells, an initial increase in [Ca 2 þ ] i stimulates the recovery processes, allowing the cell to withstand a limited complement attack. The recovery might be associated with cellular activation and the production of inflammatory modulators, which, in turn, amplify an ongoing inflammatory response. 3,11 The authors further hypothesised that a more severe membrane damage causes a sharp rise in [Ca 2 þ ] i , which overwhelms all recovery processes. 11 However, how [Ca 2 þ ] i determines cell fate, how the acquisition of novel functions is initiated and how the 'point of no return' is defined remain unknown.In this study, we have undertaken a simultaneous, real-time characterisation of [Ca 2 þ ] i and plasma membrane dynamics in living cells permeabilised with the bacterial pore-forming toxin streptolysin O (SLO). Our data show that the fate of SLOperforated cells is dependent on their ability to control the extent of a pore-induced elevation in [Ca 2 þ ] i . We detail Ca 2 þ -dependent mechanisms that elicit either repair or irreversible structural changes in the plasma membrane and show how intracellula...

The plasma membrane constitutes a barrier that maintains the essential differences between the cytosol and the extracellular environment. Plasmalemmal injury is a common event during the life of many cells that often leads to their premature, necrotic death. Blebbing -a display of plasmalemmal protrusions -is a characteristic feature of injured cells. In this study, we disclose a previously unknown role for blebbing in furnishing resistance to plasmalemmal injury. Blebs serve as precursors for injuryinduced intracellular compartments that trap damaged segments of the plasma membrane. Hence, loss of cytosol and the detrimental influx of extracellular constituents are confined to blebs that are sealed off from the cell body by plugs of annexin A1 -a Ca 2 þ -and membrane-binding protein. Our findings shed light on a fundamental process that contributes to the survival of injured cells. By targeting annexin A1/blebbing, new therapeutic approaches could be developed to avert the necrotic loss of cells in a variety of human pathologies.

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...

Understanding the impact of active dendritic properties on network activity in vivo has so far been restricted to studies in anesthetized animals. However, to date no study has been made to determine the direct effect of the anesthetics themselves on dendritic properties. Here, we investigated the effects of three types of anesthetics commonly used for animal experiments (urethane, pentobarbital and ketamine/xylazine). We investigated the generation of calcium spikes, the propagation of action potentials (APs) along the apical dendrite and the somatic firing properties in the presence of anesthetics in vitro using dual somatodendritic whole cell recordings. Calcium spikes were evoked with dendritic current injection and high-frequency trains of APs at the soma. Surprisingly, we found that the direct actions of anesthetics on calcium spikes were very different. Two anesthetics (urethane and pentobarbital) suppressed dendritic calcium spikes in vitro, whereas a mixture of ketamine and xylazine enhanced them. Propagation of spikes along the dendrite was not significantly affected by any of the anesthetics but there were various changes in somatic firing properties that were highly dependent on the anesthetic. Last, we examined the effects of anesthetics on calcium spike initiation and duration in vivo using high-frequency trains of APs generated at the cell body. We found the same anesthetic-dependent direct effects in addition to an overall reduction in dendritic excitability in anesthetized rats with all three anesthetics compared with the slice preparation.