No 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...

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.

Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50–150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies.

Protein kinase Cα (PKCα) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCα in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCα to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCα knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCα membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCα membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCα with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCα. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCα even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCα scaffold to promote PKCα-mediated EGFR inactivation through increased membrane targeting of PKCα and EGFR/PKCα complex formation.