Plasma membrane perforation by GSDME during apoptosis-driven secondary necrosis

Cellular and Molecular Life Sciences (2022) 79:19 https://doi.org/10.1007/s00018-021-04078-0 Cellular and Molecular Life Sciences ORIGINAL ARTICLE Plasma membrane perforation by GSDME during apoptosis‑driven secondary necrosis Elke De Schutter1,2,3 · Jana Ramon4 · Benjamin Pfeuty5 · Caroline De Tender6,7 · Stephan Stremersch4 · Koen Raemdonck4 · Ken Op de Beeck3,8 · Wim Declercq1,2 · Franck B. Riquet1,2,9 · Kevin Braeckmans4 · Peter Vandenabeele1,2 Received: 3 June 2021 / Revised: 14 October 2021 / Accepted: 19 October 2021 © The Author(s) 2021 Abstract Secondary necrosis has long been perceived as an uncontrolled process resulting in total lysis of the apoptotic cell. Recently, it was shown that progression of apoptosis to secondary necrosis is regulated by Gasdermin E (GSDME), which requires activation by caspase-3. Although the contribution of GSDME in this context has been attributed to its pore-forming capacity, little is known about the kinetics and size characteristics of this. Here we report on the membrane permeabilizing features of GSDME by monitoring the influx and efflux of dextrans of different sizes into/from anti-Fas-treated L929sAhFas cells undergoing apoptosis-driven secondary necrosis. We found that GSDME accelerates cell lysis measured by SYTOX Blue staining but does not affect the exposure of phosphatidylserine on the plasma membrane. Furthermore, loss of GSDME expression clearly hampered the influx of fluorescently labeled dextrans while the efflux happened independently of the presence or absence of GSDME expression. Importantly, both in- and efflux of dextrans were dependent on their molecular weight. Altogether, our results demonstrate that GSDME regulates the passage of compounds together with other plasma membrane destabilizing subroutines. Keywords Gasdermins · Cell death · Membrane permeabilization · Influx · Efflux · Dextrans Introduction Elke De Schutter and Jana Ramon (shared first authorship) contributed equally to this work. Franck B. Riquet, Kevin Braeckmans and Peter Vandenabeele share senior authorship. * Peter Vandenabeele 1 VIB Center for Inflammation Research, 9052 Ghent, Belgium 2 Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium 3 Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, 2650 Antwerp, Belgium 4 Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium Apoptosis, the best-known form of regulated cell death, is essentially a containment and recycling program that prepares the cell corpse for efficient phagocytosis [1]. However, when phagocytes are absent or the phagocytic capacity is insufficient, apoptotic cells progress to necrotic plasma membrane permeabilization called apoptosis-driven secondary necrosis, which results in a more inflammatory 5 Université de Lille, CNRS, UMR 8523-PhLAM-Physique des Lasers Atomes et Molécules,, 59000 Lille, France 6 Department of Applied Mathematics, Computer Science and Statistics, Ghent University, 9000 Ghent, Belgium 7 Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food, 9820 Merelbeke, Belgium 8 Center for Oncological Research, University of Antwerp and Antwerp University Hospital, 2610 Antwerp, Belgium 9 Université de Lille, 59000 Lille, France 13 Vol.:(0123456789) 19 Page 2 of 17 environment [2–5]. The gasdermin (GSDM) protein family gained a lot of interest as plasma membrane permeabilizers during regulated cell death [6–8]. Gasdermin D (GSDMD) is proteolytically activated by caspase-1 and -4 leading to inflammasome-mediated pyroptosis [9, 10] and the GSDMD-dependent release of pro-inflammatory cytokines such as interleukin-1β [11]. Similarly, apoptosis-driven secondary necrosis is driven by the activation of gasdermin E (GSDME) [12, 13]. To entail its effect, caspase-3-mediated cleavage induces the release of GSDME’s cytotoxic N-terminal p30 fragment from the auto-inhibiting C-terminal domain, which is followed by plasma membrane recruitment and plasma membrane permeabilization [12, 14, 15]. Nevertheless, GSDME may not be the only mechanism responsible for secondary necrosis. GSDME expression is dispensable for secondary necrosis following NLRC4-mediated apoptosis in macrophages [16] or UV irradiation-induced apoptosis in human T cells and monocytes [17]. After cleavage, GSDM proteins were shown to oligomerize and bind to plasma membrane phospholipids [14, 18], suggesting that GSDMs establish plasma membrane permeabilization by perforation. Moreover, the structure of GSDMD and mouse GSDMA3 revealed more mechanistic insights in how the N-terminal domain is able to form pores [19, 20]. Using cryo-electron microscopy, it was discovered that the N-terminal domains of GSDMA3 form a large, 27-fold β-barrel-stave protein pore with an inner diameter of 18 nm [19]. In addition, 26- and 28-fold oligomerization structures were reported with similar dimensions as the dominant 27-subunit GSDMA3 pore [19]. In contrast, GSDMD assemblies were reported to have a 31- to 34-fold symmetry [20], suggesting significant variability in oligomerization among different GSDM proteins. In addition, the N-terminal domain of GSDMD assembles into dynamic arc- and slit-shaped oligomers before they finally transform to stable ring-shaped oligomers with varying diameters ranging from 13.5 till 33.5 nm [21, 22]. Unlike GSDMA3 and GSDMD, the characteristics of GSDME pore formation are currently unknown. Therefore, to gain insight into the membrane permeabilizing behavior of GSDME and its role in apoptosis-driven secondary necrosis, we applied two in vitro approaches. With the assumption that GSDME forms pores in the plasma membrane, influx or efflux of macromolecules, such as fluorescently labeled dextrans, is expected to happen when cells are exposed to apoptotic stimuli. Monitoring the uptake of fluorescently labeled dextrans in apoptotic cells is quite straightforward, only requiring the addition of the dextrans to the culture medium after apoptosis induction. However, monitoring efflux is less obvious as the cells should be pre-loaded with the dextrans in a manner that does not interfere with cellular processes such as proliferation or without inducing apoptosis by itself. Therefore, we selected nanoparticle-sensitized 13 E. De Schutter et al. photoporation, which is an emerging intracellular delivery technique that enables direct cytosolic delivery of membrane-impermeable macromolecules in virtually every cell type with minimal impact on the cellular homeostasis [23–29]. This technique makes use of photothermal nanoparticles, such as gold nanoparticles (AuNPs), which are incubated with cells and bind to the plasma membrane. Upon irradiation by a short, yet intense laser pulse, the AuNPs become heated, resulting in the evaporation of the surrounding water and the formation of quickly expanding water vapor na (...truncated)