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

Abstract

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...

Blebbing confers resistance against cell lysis

“…Contraction of the cortical actomyosin was a driving force in bleb formation ( Figure 1b) 9 that causes generalized damage to the phospholipid bilayer and cytoskeleton and leads to a depletion of intracellular ATP levels. 2 Blebbing was a universal cellular response to plasmalemmal injury: all cell types we have tested so far blebbed in response to injury irrespective of the nature of the damaging agent ( Figure 2; Supplementary Movies 1 and 2).…”

“…To evaluate the role of blebbing during plasmalemmal injury, we simultaneously monitored bleb dynamics and the outcome of the damage in individual living cells. The fate of the perforated cells was visualized by monitoring the intracellular dynamics of annexin A1 (Supplementary Figure S1; see also Babiychuk et al, 2009). The number of cells manifesting an irreversible translocation of annexin A1 (lysed cells) was scored in blebbing cells as well as in those that failed to bleb after exposure to SLO (Figure 3a).…”

“…The intrableb Ca 2 þ elevation was indirectly assessed by monitoring the Ca 2 þ -dependent translocation of annexin A1. 9 To demonstrate the localized intra-bleb elevation in Ca 2 þ directly, we used Fluo 5F -a Ca 2 þ -sensitive fluorescent dyewhich permits a monitoring of Ca 2 þ fluxes in the high micromolar range. The elevation, observed in non-transfected HEK 293 cells, is documented in Figure 5 and in Supplementary Movie 8.…”

“…[3][4][5] Nucleated cells survive the disruption of their plasma membrane by a process of resealing. 1,2,[6][7][8][9] If resealing fails, the intracellular contents are lost. But irreversible damage can occur even before this event if the influx of extracellular Ca 2 þ is not restrained.…”

“…But irreversible damage can occur even before this event if the influx of extracellular Ca 2 þ is not restrained. [9][10][11] An abrupt elevation in the intracellular concentration of Ca 2 þ ([Ca 2 þ ] i ) activates intracellular proteinases and phospholipases, thereby causing generalized damage to the phospholipid bilayer and the cytoskeleton. 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.…”