Perforation Of Plasma Membrane Research Articles

Chemical Plasma Membrane Perforation Generated by a Microfluidic Probe for Single-Cell Intracellular Protein Delivery.

Efficient and convenient delivery of exogenous molecules into cells is important for cell biology research. However, many intracellular delivery methods require carrier-mediated or physical field assistance, complicating the delivery process. Here, a general, simple, and effective method for in situ single-cell intracellular delivery is reported. A solution containing digitonin and cargo is precisely applied to single cells using a microfluidic probe. Digitonin binds to cholesterol in the plasma membrane to induce perforation, and the cargo enters the cell through the pore. By optimizing parameters, propidium iodide (0.67 kDa) and FITC-dextran (10, 40, and 150 kDa) can be successfully introduced into single cells within 3 min while maintaining cell viability. To prove the potential of this method for cell research, we delivered cytochrome C (13 kDa) and cyclophilin A (18 kDa) into cells by this method. The delivered cytochrome C successfully induces cell apoptosis by activating the caspase pathway, and cyclophilin A performs an antioxidant effect in the cells, which may enhance the drug resistance of glioma cells. It is believed that this method will be an attractive tool for single-cell intracellular delivery.

The palmitoylation of gasdermin D directs its membrane translocation and pore formation during pyroptosis.

Plasma membrane perforation elicited by caspase cleavage of the gasdermin D (GSDMD) N-terminal domain (GSDMD-NT) triggers pyroptosis. The mechanisms underlying GSDMD membrane translocation and pore formation are not fully understood. Here, using a proteomic approach, we identified fatty acid synthase (FASN) as a GSDMD-binding partner. S-palmitoylation of GSDMD at Cys191/Cys192 (human/mouse), catalyzed by palmitoyl acyltransferases ZDHHC5 and ZDHHC9 and facilitated by reactive oxygen species (ROS), directly mediated membrane translocation of GSDMD-NT but not full-length GSDMD (GSDMD-FL). Palmitoylation of GSDMD-FL could be induced before inflammasome activation by stimuli such as lipopolysaccharide (LPS), consequently serving as an essential molecular event in macrophage priming. Inhibition of GSDMD palmitoylation suppressed macrophage pyroptosis and IL-1β release, mitigated organ damage, and enhanced the survival of septic mice. Thus, GSDMD-NT palmitoylation is a key regulatory mechanism controlling GSDMD membrane localization and activation, which may offer an additional target for modulating immune activity in infectious and inflammatory diseases.

Gasdermins gone wild: new roles for GSDMs in regulating cellular homeostasis.

Since their discovery, members of the gasdermin (GSDM) family of proteins have been firmly established as executors of pyroptosis, with the N-terminal fragment of most GSDMs capable of forming pores in the plasma membrane. More recent findings suggest that some GSDMs can drive additional cell death pathways, such as apoptosis and necroptosis, through mechanisms independent of plasma membrane perforation. There is also emerging evidence that by associating with cellular compartments such as mitochondria, peroxisomes, endosomes, and the nucleus, GSDMs regulate cell death-independent aspects of cellular homeostasis. Here, we review the diversity of GSDM function across several cell types and explore how various cellular stresses can promote relocalization - and thus refunctionalization - of GSDMs.

Development of a direct point electron beam exposure system to investigate the biological functions of subcellular domains in a living biological cell

Electron microscopy studies have demonstrated that the diameter of a focused electron beam is small enough to probe or manipulate subcellular domains of a single biological cell. Here, we report the development of a direct point electron beam irradiation system to investigate the biological functions of subcellular domains in a living cell. Subcellular structures of a single living cell cultured on a thin film can be selectively irradiated by the point electron beam generated by our system. We have demonstrated controlled beam positioning capability to selectively irradiate 500 nm size structure with a point electron beam. We determined beam irradiation parameters that did not cause irreversible plasma membrane perforation after beam exposure and the irradiation caused intracellular Ca2+ elevation in an irradiated neuronal cell. Since the neuronal cell express fine subcellular structures such as neurites, we tried to position a beam on the structure and observed a Ca2+ wave originated from the intended point, which showed that our system had enough selectivity to target a subcellular structure. Point electron beam exposure is expected to be employed for various cellular stimulation protocols, and this enables the investigation of the biological functions of subcellular domains.

Ultrasound-Mediated Drug Delivery: Sonoporation Mechanisms, Biophysics, and Critical Factors.

Sonoporation, or the use of ultrasound in the presence of cavitation nuclei to induce plasma membrane perforation, is well considered as an emerging physical approach to facilitate the delivery of drugs and genes to living cells. Nevertheless, this emerging drug delivery paradigm has not yet reached widespread clinical use, because the efficiency of sonoporation is often deemed to be mediocre due to the lack of detailed understanding of the pertinent scientific mechanisms. Here, we summarize the current observational evidence available on the notion of sonoporation, and we discuss the prevailing understanding of the physical and biological processes related to sonoporation. To facilitate systematic understanding, we also present how the extent of sonoporation is dependent on a multitude of factors related to acoustic excitation parameters (ultrasound frequency, pressure, cavitation dose, exposure time), microbubble parameters (size, concentration, bubble-to-cell distance, shell composition), and cellular properties (cell type, cell cycle, biochemical contents). By adopting a science-backed approach to the realization of sonoporation, ultrasound-mediated drug delivery can be more controllably achieved to viably enhance drug uptake into living cells with high sonoporation efficiency. This drug delivery approach, when coupled with concurrent advances in ultrasound imaging, has potential to become an effective therapeutic paradigm.

Plasma membrane perforation by GSDME during apoptosis-driven secondary necrosis

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.

Plasma Membrane Blebbing Dynamics Involved in the Reversibly Perforated Cell by Ultrasound-Driven Microbubbles

The perforation of plasma membrane by ultrasound-driven microbubbles (i.e., sonoporation) provides a temporary window for transporting macromolecules into the cytoplasm that is promising with respect to drug delivery and gene therapy. To improve the efficacy of delivery while ensuring biosafety, membrane resealing and cell recovery are required to help sonoporated cells defy membrane injury and regain their normal function. Blebs are found to accompany the recovery of sonoporated cells. However, the spatiotemporal characteristics of blebs and the underlying mechanisms remain unclear. With a customized platform for ultrasound exposure and 2-D/3-D live single-cell imaging, localized membrane perforation was induced with ultrasound-driven microbubbles, and the cellular responses were monitored using multiple fluorescent probes. The results indicated that localized blebs undergoing four phases (nucleation, expansion, pausing and retraction) on a time scale of tens of seconds to minutes were specifically involved in the reversibly sonoporated cells. The blebs spatially correlated with the membrane perforation site and temporally lagged (about tens of seconds to minutes) the resealing of perforated membrane. Their diameter (about several microns) and lifetime (about tens of seconds to minutes) positively correlated with the degree of sonoporation. Further studies revealed that intracellular calcium transients might be an upstream signal for triggering blebbing nucleation; exocytotic lysosomes not only contributed to resealing of the perforated membrane, but also to the increasing bleb volume during expansion; and actin components accumulation facilitated bleb retraction. These results provide new insight into the short-term strategies that the sonoporated cell employs to recover on membrane perforation and to remodel membrane structure and a biophysical foundation for sonoporation-based therapy.

Mortalin peptides exert antitumor activities and act as adjuvants to antibody-mediated complement-dependent cytotoxicity.

Cancer cells have developed numerous strategies to maintain their proliferative capacity and to withstand different kinds of stress. The mitochondrial stress‑70protein named glucose regulated protein 75 (GRP75), also known as mortalin, is an intriguing cancer pro‑survival factor. It is constitutively expressed in normal tissues but is upregulated in many tumors, and was shown to be a cancer prognostic biomarker. Mortalin is an inhibitor of complement‑dependent cytotoxicity(CDC) and may therefore protect cells from antibody‑based immunotherapy. To target mortalin for cancer therapy, our laboratory designed several mortalin mimetic peptides with sequences predicted to be involved in mortalin binding to its client proteins. The peptides were synthesized with a C‑terminal transactivator of transcription sequence. By using cell death methodologies, the mechanism of action of the mortalin mimetic peptides on cancer cells was studied. Two peptides in particular, Mot‑P2 and Mot‑P7, were found to be highly toxic to lymphoma and ovarian, breast and prostate carcinoma cells. The analysis of their mode of action revealed that they may induce, within minutes, plasma membrane perturbations and mitochondrial stress. Furthermore, Mot‑P2 and Mot‑P7 activated necrotic cell death, leading to plasma membrane perforation, mitochondrial inner membrane depolarization and decrease in ATP level. In addition, Mot‑P7, but not Mot‑P2, required extracellular calcium ions to fully mediate cell death and was partially inhibited by plasma membrane cholesterol. At sub‑toxic concentrations, the two peptides moderately inhibited cancer cell proliferation and blocked cell cycle at G2/M. Both peptides may bind intracellularly to mortalin and/or a mortalin‑binding protein, hence knocking down mortalin expression reduced cell death. Combining treatment with Mot‑P2 or Mot‑P7 and CDC resulted in increased cell death. This study identified highly cytotoxic mortalin mimetic peptides that may be used as monotherapy or combined with complement‑activating antibody therapy to target mortalin for precision cancer therapy.

K092A and K092B, Two Peptides Isolated from the Dogfish (Scyliorhinus canicula L.), with Potential Antineoplastic Activity Against Human Prostate and Breast Cancer Cells.

Cancer therapy is currently a major challenge within the research community, especially in reducing the side effects of treatments and to develop new specific strategies against cancers that still have a poor prognosis. In this context, alternative strategies using biotechnologies, such as marine peptides, have been developed based on their promise of effectivity associated with a low toxicity for healthy cells. The purpose of the present paper is to investigate the active mechanism of two peptides that were isolated from the epigonal tissue of the lesser spotted dogfish Scyliorhinus canicula L., identified NFDTDEQALEDVFSKYG (K092A) and EAPPEAAEEDEW (K092B) on the in vitro growth inhibition of ZR-75-1 mammary carcinoma cells and MDA-Pca-2b prostate cancer cells. The effects of the peptides on cell proliferation and cell death mechanisms were studied by the flow cytometry and immunofluorescence microscopy approaches. The results have shown the onset of both K092A- and K092B-induced early cytoskeleton changes, and then cell cycle perturbations followed by non-apoptotic cell death. Moreover, impedance perturbation and plasma membrane perforation in ZR-75-1 K092A-treated cell cultures and autophagy inhibition in MDA-Pca-2b K092B-treated cells have been observed. In conclusion, these two bioactive peptides from dogfish exhibit antineoplastic activity on the human prostate and breast cancer cells in vitro.

Multiple Parameters Beyond Lipid Binding Affinity Drive Cytotoxicity of Cholesterol-Dependent Cytolysins.

The largest superfamily of bacterial virulence factors is pore-forming toxins (PFTs). PFTs are secreted by both pathogenic and non-pathogenic bacteria. PFTs sometimes kill or induce pro-pathogen signaling in mammalian cells, all primarily through plasma membrane perforation, though the parameters that determine these outcomes are unclear. Membrane binding, calcium influx, pore size, and membrane repair are factors that influence PFT cytotoxicity. To test the contribution of membrane binding to cytotoxicity and repair, we compared the closely related, similarly-sized PFTs Perfringolysin O (PFO) from Clostridium perfringens and Streptolysin O (SLO) from Streptococcus pyogenes. Cell death kinetics for PFO and SLO were different because PFO increased in cytotoxicity over time. We introduced known L3 loop mutations that swap binding affinity between toxins and measured hemolytic activity, nucleated cell death kinetics and membrane repair using viability assays, and live cell imaging. Altered hemolytic activity was directly proportional to toxin binding affinity. In contrast, L3 loop alterations reduced nucleated cell death, and they had limited effects on cytotoxicity kinetics and membrane repair. This suggests other toxin structural features, like oligomerization, drives these parameters. Overall, these findings suggest that repair mechanisms and toxin oligomerization add constraints beyond membrane binding on toxin evolution and activity against nucleated cells.

Relative Roles of Listeriolysin O, InlA, and InlB in Listeria monocytogenes Uptake by Host Cells.

Listeria monocytogenes is a facultative intracellular pathogen that infects a wide variety of cells, causing the life-threatening disease listeriosis. L. monocytogenes virulence factors include two surface invasins, InlA and InlB, known to promote bacterial uptake by host cells, and the secreted pore-forming toxin listeriolysin O (LLO), which disrupts the phagosome to allow bacterial proliferation in the cytosol. In addition, plasma membrane perforation by LLO has been shown to facilitate L. monocytogenes internalization into epithelial cells. In this work, we tested the host cell range and importance of LLO-mediated L. monocytogenes internalization relative to the canonical invasins, InlA and InlB. We measured the efficiencies of L. monocytogenes association with and internalization into several human cell types (hepatocytes, cytotrophoblasts, and endothelial cells) using wild-type bacteria and isogenic single, double, and triple deletion mutants for the genes encoding InlA, InlB and LLO. No role for InlB was detected in any tested cells unless the InlB expression level was substantially enhanced, which was achieved by introducing a mutation (prfA*) in the gene encoding the transcription factor PrfA. In contrast, InlA and LLO were the most critical invasion factors, although they act in a different manner and in a cell-type-dependent fashion. As expected, InlA facilitates both bacterial attachment and internalization in cells that express its receptor, E-cadherin. LLO promotes L. monocytogenes internalization into hepatocytes, but not into cytotrophoblasts and endothelial cells. Finally, LLO and InlA cooperate to increase the efficiency of host cell invasion by L. monocytogenes.

Effect of non-acoustic parameters on heterogeneous sonoporation mediated by single-pulse ultrasound and microbubbles

Sonoporation—transient plasma membrane perforation elicited by the interaction of ultrasound waves with microbubbles—has shown great potential for drug delivery and gene therapy. However, the heterogeneity of sonoporation introduces complexities and challenges in the realization of controllable and predictable drug delivery. The aim of this investigation was to understand how non-acoustic parameters (bubble related and bubble-cell interaction parameters) affect sonoporation. Using a customized ultrasound-exposure and fluorescence-imaging platform, we observed sonoporation dynamics at the single-cell level and quantified exogenous molecular uptake levels to characterize the degree of sonoporation. Sonovue microbubbles were introduced to passively regulate microbubble-to-cell distance and number, and bubble size. 1MHz ultrasound with 10-cycle pulse duration and 0.6MPa peak negative pressure were applied to trigger the inertial collapse of microbubbles. Our data revealed the impact of non-acoustic parameters on the heterogeneity of sonoporation. (i) The localized collapse of relatively small bubbles (diameter, D<5.5μm) led to predictable sonoporation, the degree of which depended on the bubble-to-cell distance (d). No sonoporation was observed when d/D>1, whereas reversible sonoporation occurred when d/D<1. (ii) Large bubbles (D>5.5μm) exhibited translational movement over large distances, resulting in unpredictable sonoporation. Translation towards the cell surface led to variable reversible sonoporation or irreversible sonoporation, and translation away from the cell caused either no or reversible sonoporation. (iii) The number of bubbles correlated positively with the degree of sonoporation when D<5.5μm and d/D<1. Localized collapse of two to three bubbles mainly resulted in reversible sonoporation, whereas irreversible sonoporation was more likely following the collapse of four or more bubbles. These findings offer useful insight into the relationship between non-acoustic parameters and the degree of sonoporation.

Vital dyes and virtual deaths

Dear Editor, In 2009 and 2012, Cell Death & Differentiation has published recommendations of the Nomenclature Committee on Cell Death (NCCD) for the Classification of cell death and Molecular definitions of cell death subroutines, respectively. Under the subtitle When is a cell ‘dead'?, and in an accompanying table entitled Molecular or morphological criteria to define dead cells, the 2009 paper featured a set of three criteria; the fulfillment of any single one is considered to indicate cell death.1 The first criterion in this list is Loss of plasma membrane integrity, and parameters commonly used to assess integrity of the plasma membrane were mentioned, namely exclusion from cells of vital dyes like trypan blue or propidium iodide. It should be noted that injuries of the plasma membrane may be transient. This is now widely accepted, and here some key publications related to this issue are reviewed. For instance, mechanical stress can cause lesions of the plasma membrane spanning several micrometers, but they can be rapidly repaired by calcium influx-dependent mechanisms.2 Similarly, plasma membrane perforation by various pore forming proteins may be reversible.3 Some pore-forming proteins, for example, streptolysin O, render cells permeable for vital dyes and even for macromolecules, whereas small pore-forming proteins like α-toxin of S. aureus form lesions too small to permit flux of these molecules. However, all kinds of membrane lesions cause a drop of potassium and cellular ATP concentrations, which however, may be reversible if reconstitution of membrane integrity is achieved in time. Transient plasma membrane damage may not only trigger transcriptional responses, and release of mediators,4 but also cell cycle progression.5 Therefore, loss of membrane integrity is not even a reliable predictor of imminent death. Cellular tools for mending perforated membranes are diverse and comprise calcium influx-dependent or calcium influx-independent mechanisms. However, vesicle traffic emerges as a common denominator of resealing processes,6, 7, 8 which appear to be evolutionarily conserved and operate in vivo.8 As all definitions of cell death are necessarily ‘package deals' with the tacit assumption that the observed changes are irreversible, investigators are facing the question for how long they have to observe an indicator of a presumed, irreversible process before they may actually regard it as such. As membrane repair depends on the energy consuming task of vesicle traffic, irreversibility of loss of membrane integrity may be ascertained when cellular ATP is depleted. Although more work is required to fully understand and appreciate the mechanisms and relevance of membrane repair, the available evidence warrants a reassessment of Loss of plasma membrane integrity in itself as a bona fide criterion to define cell death.