ER calcium depletion as a key driver for impaired ER-to-mitochondria calcium transfer and mitochondrial dysfunction in Wolfram syndrome.

Wolfram syndrome is a rare genetic disease caused by mutations in the WFS1 or CISD2 gene. A primary defect in Wolfram syndrome involves poor ER Ca2+ handling, but how this disturbance leads to the disease is not known. The current study, performed in primary neurons, the most affected and disease-relevant cells, involving both Wolfram syndrome genes, explains how the disturbed ER Ca2+ handling compromises mitochondrial function and affects neuronal health. Loss of ER Ca2+ content and impaired ER-mitochondrial contact sites in the WFS1- or CISD2-deficient neurons is associated with lower IP3R-mediated Ca2+ transfer from ER to mitochondria and decreased mitochondrial Ca2+ uptake. In turn, reduced mitochondrial Ca2+ content inhibits mitochondrial ATP production leading to an increased NADH/NAD+ ratio. The resulting bioenergetic deficit and reductive stress compromise the health of the neurons. Our work also identifies pharmacological targets and compounds that restore Ca2+ homeostasis, enhance mitochondrial function and improve neuronal health.

Tetrameric, active PKM2 indirectly inhibits IP3 receptors, potentially requiring GRP75 as a tertiary partner.

Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme interacting with the inositol 1,4,5-trisphosphate receptor (IP3R). This interaction suppresses IP3R-mediated cytosolic [Ca2+] rises. As PKM2 exists in monomeric, dimeric and tetrameric forms displaying different properties including catalytic activity, we investigated the molecular determinants of PKM2 enabling its interaction with IP3Rs. Treatment of HeLa cells with TEPP-46, a compound stabilizing the tetrameric form of PKM2, increased both its catalytic activity and the suppression of IP3R-mediated Ca2+ signals. Consistently, in PKM2 knock-out HeLa cells, PKM2C424L, a tetrameric, highly active PKM2 mutant, but not inactive PKM2K270M or the less active PKM2K305Q, suppressed IP3R-mediated Ca2+ release. Surprisingly, however, in vitro assays did not reveal a direct interaction between purified PKM2 and either the purified Fragment 5 of IP3R1 (a.a. 1932-2216) or the therein located D5SD peptide (a.a. 2078-2098 of IP3R1), the presumed interaction sites of PKM2 on the IP3R. Moreover, on-nucleus patch clamp of heterologously expressed IP3R1 in DT40 cells devoid of endogenous IP3Rs did not reveal any functional effect of purified wild-type PKM2, mutant PKM2 or PKM1 proteins. These results indicate that an additional factor mediates the regulation of the IP3R by PKM2 in cellulo. Immunoprecipitation of GRP75 using HeLa cell lysates co-precipitated IP3R1, IP3R3 and PKM2. Moreover, the D5SD peptide not only disrupted PKM2:IP3R, but also PKM2:GRP75 and GRP75:IP3R interactions. Our data therefore support a model in which catalytically active, tetrameric PKM2 suppresses Ca2+ signaling via the IP3R through a multiprotein complex involving GRP75.

Sustainability in a broad sense: An essential aspect of scientific conferences.

This article reflects on sustainability in the context of scientific conferences with emphasis on environmental, diversity, inclusivity, and intellectual aspects. We argue that it is imperative to embrace sustainability as a broad concept during conference organization. In-person conferences have an obvious environmental impact but mitigating strategies can be implemented, such as incentivizing low-emission travel, offering fellowships to support sustainable traveling, and promoting use of public transport or car-pooling. Utilizing eco-conscious venues, catering, and accommodations, along with minimizing resource wastage, further reduces environmental impact. Additional considerations include facilitating hybrid format conferences that allow both in-person and online attendance. Hybrid conferences enhance global participation whilst reducing resource consumption and environmental impact. Often-overlooked benefits can arise from the simple recording of talks to enable asynchronous viewing for people unable to attend in person, in addition to providing a legacy of knowledge that, for example, could support the training of early career researchers (ECRs) or newcomers in the field. The longevity of a research field, intellectual sustainability, requires an inclusive conference atmosphere, offering optimal opportunities for ECRs, minority groups, and researchers from emerging countries. Diversity and inclusivity not only enrich conference experiences but also enhances creativity and innovation.

10th European Calcium Society symposium: The Ca2+-signaling toolkit in cell function, health and disease.

The 10th European Calcium Society symposium, organized in Leuven, Belgium on November 15-17, 2023, focused on the role of Ca2+ signaling in cell function, health and disease. The symposium featured six scientific sessions, 16 invited speakers - of whom two were postdoctoral researchers - and 14 short talks. The talks covered various aspects of intracellular Ca2+ signaling and its implications in pathology. Each session was opened by one or more invited speakers, followed by a series of presentations from speakers selected from submitted abstracts. Through short talks, poster presentations, awards, and sustainable travel fellowships, the symposium also fostered opportunities for the active participation of early-career researchers. At least half of the short talks were allocated to early-career researchers, thereby offering a platform for the presentation of ongoing work and unpublished results. Presentations were also broadcast in real-time for online attendees. In this Meeting Review, we aim to capture the spirit of the meeting and discuss the main take-home messages that emerged during the symposium.

Recent advances in canonical versus non-canonical Ca2+-signaling-related anti-apoptotic Bcl-2 functions and prospects for cancer treatment.

Cell fate is tightly controlled by a continuous balance between cell survival and cell death inducing mechanisms. B-cell lymphoma 2 (Bcl-2)-family members, composed of effectors and regulators, not only control apoptosis at the level of the mitochondria but also by impacting the intracellular Ca2+ homeostasis and dynamics. On the one hand, anti-apoptotic protein Bcl-2, prevents mitochondrial outer membrane permeabilization (MOMP) by scaffolding and neutralizing proapoptotic Bcl-2-family members via its hydrophobic cleft (region composed of BH-domain 1-3). On the other hand, Bcl-2 suppress pro-apoptotic Ca2+ signals by binding and inhibiting IP3 receptors via its BH4 domain, which is structurally exiled from the hydrophobic cleft by a flexible loop region (FLR). As such, Bcl-2 prevents excessive Ca2+ transfer from ER to mitochondria. Whereas regulation of both pathways requires different functional regions of Bcl-2, both seem to be connected in cancers that overexpress Bcl-2 in a life-promoting dependent manner. Here we discuss the anti-apoptotic canonical and non-canonical role, via calcium signaling, of Bcl-2 in health and cancer and evolving from this the proposed anti-cancer therapies with their shortcomings. We also argue how some cancers, with the major focus on diffuse large B-cell lymphoma (DLBCL) are difficult to treat, although theoretically prime marked for Bcl-2-targeting therapeutics. Further work is needed to understand the non-canonical functions of Bcl-2 also at organelles beyond the mitochondria, the interaction partners outside the Bcl-2 family as well as their ability to target or exploit these functions as therapeutic strategies in diseases.

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS.

IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.

AIM: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation. METHODS: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays. RESULTS: IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 µM) < IP3 R3 (~4.3 µM) < IP3 R1 (~9.0 µM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide. CONCLUSION: IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.

Cellular effects of BAPTA: Are they only about Ca2+ chelation?

Intracellular Ca2+ signals play a vital role in a broad range of cell biological and physiological processes in all eukaryotic cell types. Dysregulation of Ca2+ signaling has been implicated in numerous human diseases. Over the past four decades, the understanding of how cells use Ca2+ as a messenger has flourished, largely because of the development of reporters that enable visualization of Ca2+ signals in different cellular compartments, and tools that can modulate cellular Ca2+ signaling. One such tool that is frequently used is BAPTA; a fast, high-affinity Ca2+-chelating molecule. By making use of a cell-permeable acetoxymethyl ester (AM) variant, BAPTA can be readily loaded into the cytosol of cells (referred to as BAPTAi), where it is trapped and able to buffer changes in cytosolic Ca2+. Due to the ease of loading of the AM version of BAPTA, this reagent has been used in hundreds of studies to probe the role of Ca2+ signaling in specific processes. As such, for decades, researchers have almost universally attributed changes in biological processes caused by BAPTAi to the involvement of Ca2+ signaling. However, BAPTAi has often been used without any form of control, and in many cases has neither been shown to be retained in cells for the duration of experiments nor to buffer any Ca2+ signals. Moreover, increasing evidence points to off-target cellular effects of BAPTA that are clearly not related to Ca2+ chelation. Here, we briefly introduce Ca2+ signaling and the history of Ca2+ chelators and fluorescent Ca2+ indicators. We highlight Ca2+-independent effects of BAPTAi on a broad range of molecular targets and describe some of BAPTAi's impacts on cell functions that occur independently of its Ca2+-chelating properties. Finally, we propose strategies for determining whether Ca2+ chelation, the binding of other metal ions, or off-target interactions with cell components are responsible for BAPTAi's effect on a particular process and suggest some future research directions.

The Complex Effects of PKM2 and PKM2:IP3R Disruption on Intracellular Ca2+ Handling and Cellular Functions.

Pyruvate kinase M (PKM) 2 was described to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and suppress its activity. To further investigate the physiological importance of the PKM2:IP3R interaction, we developed and characterized HeLa PKM2 knockout (KO) cells. In the HeLa PKM2 KO cells, the release of Ca2+ to the cytosol appears to be more sensitive to low agonist concentrations than in HeLa wild-type (WT) cells. However, upon an identical IP3-induced Ca2+ release, Ca2+ uptake in the mitochondria is decreased in HeLa PKM2 KO cells, which may be explained by the smaller number of contact sites between the ER and the mitochondria. Furthermore, in HeLa PKM2 KO cells, mitochondria are more numerous, though they are smaller and less branched and have a hyperpolarized membrane potential. TAT-D5SD, a cell-permeable peptide representing a sequence derived from IP3R1 that can disrupt the PKM2:IP3R interaction, induces Ca2+ release into the cytosol and Ca2+ uptake into mitochondria in both HeLa WT and PKM2 KO cells. Moreover, TAT-D5SD induced apoptosis in HeLa WT and PKM2 KO cells but not in HeLa cells completely devoid of IP3Rs. These results indicate that PKM2 separately regulates cytosolic and mitochondrial Ca2+ handling and that the cytotoxic effect of TAT-D5SD depends on IP3R activity but not on PKM2. However, the tyrosine kinase Lck, which also interacts with the D5SD sequence, is expressed neither in HeLa WT nor PKM2 KO cells, and we can also exclude a role for PKM1, which is upregulated in HeLa PKM2 KO cells, indicating that the TAT-D5SD peptide has a more complex mode of action than anticipated.

Loss of INPP5K attenuates IP3-induced Ca2+ responses in the glioblastoma cell line U-251 MG cells.

INPP5K (inositol polyphosphate 5-phosphatase K) is an endoplasmic reticulum (ER)-resident enzyme that acts as a phosphoinositide (PI) 5-phosphatase, capable of dephosphorylating various PIs including PI 4,5-bisphosphate (PI(4,5)P2), a key phosphoinositide found in the plasma membrane. Given its ER localization and substrate specificity, INPP5K may play a role in ER-plasma membrane contact sites. Furthermore, PI(4,5)P2 serves as a substrate for phospholipase C, an enzyme activated downstream of extracellular agonists acting on Gq-coupled receptors or tyrosine-kinase receptors, leading to IP3 production and subsequent release of Ca2+ from the ER, the primary intracellular Ca2+ storage organelle. In this study, we investigated the impact of INPP5K on ER Ca2+ dynamics using a previously established INPP5K-knockdown U-251 MG glioblastoma cell model. We here describe that loss of INPP5K impairs agonist-induced, IP3 receptor (IP3R)-mediated Ca2+ mobilization in intact cells, while the ER Ca2+ content and store-operated Ca2+ influx remain unaffected. To further elucidate the underlying mechanisms, we examined Ca2+ release in permeabilized cells stimulated with exogenous IP3. Interestingly, the absence of INPP5K also disrupted IP3-induced Ca2+ release events. These results suggest that INPP5K may directly influence IP3R activity through mechanisms yet to be resolved. The findings from this study point towards role of INPP5K in modulating ER calcium dynamics, particularly in relation to IP3-mediated signaling pathways. However, further work is needed to establish the general nature of our findings and to unravel the exact molecular mechanisms underlying the interplay between INNP5K function and Ca2+ signaling.

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing MCL-1-addicted cancer cells.

Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we discovered that BAPTAi alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTAi provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTAi diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTAi. Notably, these effects were also induced by a BAPTAi analog with low affinity for Ca2+. Consequently, our findings uncover PFKFB3 inhibition as an Ca2+-independent mechanism through which BAPTAi impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

Human IP3 receptor triple knockout stem cells remain pluripotent despite altered mitochondrial metabolism.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ER Ca2+-release channels that control a broad set of cellular processes. Animal models lacking IP3Rs in different combinations display severe developmental phenotypes. Given the importance of IP3Rs in human diseases, we investigated their role in human induced pluripotent stem cells (hiPSC) by developing single IP3R and triple IP3R knockouts (TKO). Genome edited TKO-hiPSC lacking all three IP3R isoforms, IP3R1, IP3R2, IP3R3, failed to generate Ca2+ signals in response to agonists activating GPCRs, but retained stemness and pluripotency. Steady state metabolite profiling and flux analysis of TKO-hiPSC indicated distinct alterations in tricarboxylic acid cycle metabolites consistent with a deficiency in their pyruvate utilization via pyruvate dehydrogenase, shifting towards pyruvate carboxylase pathway. These results demonstrate that IP3Rs are not essential for hiPSC identity and pluripotency but regulate mitochondrial metabolism. This set of knockout hiPSC is a valuable resource for investigating IP3Rs in human cell types of interest.

LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD), with growing importance also for Crohn's disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology.

The ER-mitochondria interface, where Ca2+ and cell death meet.

Endoplasmic reticulum (ER)-mitochondria contact sites are crucial to allow Ca2+ flux between them and a plethora of proteins participate in tethering both organelles together. Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a pivotal role at such contact sites, participating in both ER-mitochondria tethering and as Ca2+-transport system that delivers Ca2+ from the ER towards mitochondria. At the ER-mitochondria contact sites, the IP3Rs function as a multi-protein complex linked to the voltage-dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane, via the chaperone glucose-regulated protein 75 (GRP75). This IP3R-GRP75-VDAC1 complex supports the efficient transfer of Ca2+ from the ER into the mitochondrial intermembrane space, from which the Ca2+ ions can reach the mitochondrial matrix through the mitochondrial calcium uniporter. Under physiological conditions, basal Ca2+ oscillations deliver Ca2+ to the mitochondrial matrix, thereby stimulating mitochondrial oxidative metabolism. However, when mitochondrial Ca2+ overload occurs, the increase in [Ca2+] will induce the opening of the mitochondrial permeability transition pore, thereby provoking cell death. The IP3R-GRP75-VDAC1 complex forms a hub for several other proteins that stabilize the complex and/or regulate the complex's ability to channel Ca2+ into the mitochondria. These proteins and their mechanisms of action are discussed in the present review with special attention for their role in pathological conditions and potential implication for therapeutic strategies.

Connexin hemichannels as candidate targets for cardioprotective and anti-arrhythmic treatments.

Connexins are crucial cardiac proteins that form hemichannels and gap junctions. Gap junctions are responsible for the propagation of electrical and chemical signals between myocardial cells and cells of the specialized conduction system in order to synchronize the cardiac cycle and steer cardiac pump function. Gap junctions are normally open, while hemichannels are closed, but pathological circumstances may close gap junctions and open hemichannels, thereby perturbing cardiac function and homeostasis. Current evidence demonstrates an emerging role of hemichannels in myocardial ischemia and arrhythmia, and tools are now available to selectively inhibit hemichannels without inhibiting gap junctions as well as to stimulate hemichannel incorporation into gap junctions. We review available experimental evidence for hemichannel contributions to cellular pro-arrhythmic events in ventricular and atrial cardiomyocytes, and link these to insights at the level of molecular control of connexin-43-based hemichannel opening. We conclude that a double-edged approach of both preventing hemichannel opening and preserving gap junctional function will be key for further research and development of new connexin-based experimental approaches for treating heart disease.

Pore-forming proteins as drivers of membrane permeabilization in cell death pathways.

Regulated cell death (RCD) relies on activation and recruitment of pore-forming proteins (PFPs) that function as executioners of specific cell death pathways: apoptosis regulator BAX (BAX), BCL-2 homologous antagonist/killer (BAK) and BCL-2-related ovarian killer protein (BOK) for apoptosis, gasdermins (GSDMs) for pyroptosis and mixed lineage kinase domain-like protein (MLKL) for necroptosis. Inactive precursors of PFPs are converted into pore-forming entities through activation, membrane recruitment, membrane insertion and oligomerization. These mechanisms involve protein-protein and protein-lipid interactions, proteolytic processing and phosphorylation. In this Review, we discuss the structural rearrangements incurred by RCD-related PFPs and describe the mechanisms that manifest conversion from autoinhibited to membrane-embedded molecular states. We further discuss the formation and maturation of membrane pores formed by BAX/BAK/BOK, GSDMs and MLKL, leading to diverse pore architectures. Lastly, we highlight commonalities and differences of PFP mechanisms involving BAX/BAK/BOK, GSDMs and MLKL and conclude with a discussion on how, in a population of challenged cells, the coexistence of cell death modalities may have profound physiological and pathophysiological implications.