Membrane Resealing Research Articles

Plasma membrane wound repair is characterized by extensive membrane lipid and protein rearrangements in vascular endothelial cells

Vascular endothelial cells are subject to mechanical stress resulting from blood flow and interactions with leukocytes. Stress occurs at the apical, vessel-facing cell surface and leads to membrane ruptures that have to be resealed to ensure cell survival. To mimic this process, we developed a laser ablation protocol selectively inducing wounds in the apical plasma membrane of endothelial cells. We show that Ca2+-dependent membrane resealing is initiated following this wounding protocol and that the process is accompanied by substantial membrane lipid dynamics at the wound site. Specifically, phosphatidylinositol (4,5)-bisphosphate, phosphatidylserine and phosphatidic acid rapidly accumulate at membrane wounds forming potential interaction platforms for Ca2+/phospholipid binding proteins of the annexin (Anx) family that are also recruited within seconds after wounding. Depletion of one annexin, AnxA2, and its putative binding partner S100A11 interferes with membrane resealing suggesting that Ca2+-dependent annexin-phospholipid interactions are required for efficient membrane wound repair in endothelial cells.

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.

Poly(Ethylene Glycol) Interacts with Hyaluronan in Aqueous Media.

We report here the first evidence for the interaction of poly(ethylene glycol) (PEG) with hyaluronan (HA) in aqueous solutions. PEG-HA complexes (Kapp = 45,000 ± 8000 M-1) contained about 3.3 ± 0.1 of ethylene glycol units per disaccharide of HA. The carboxyl of the D-glucuronic acid and the amide of the N-acetyl-D-glucosamine did not participate in PEG binding. Similar experiments performed with dextran and monosaccharides showed that multiple free primary hydroxyls regularly distributed along the polysaccharide chain are necessary for PEG binding. Another novelty of our study is contraction of HA upon PEG binding. The effect was observed with HA in solution or adsorbed on positively charged liposomes. The thickness of the HA layer on the liposomes decreased 2-fold upon PEG addition. HA compaction induced by PEG may underlie the changes in the plasma membrane properties and resealing of mechanical injuries induced by Pluronics.

Eradication of Saccharomyces cerevisiae by Pulsed Electric Field Treatments.

One of the promising technologies that can inactivate microorganisms without heat is pulsed electric field (PEF) treatment. The aim of this study was to examine the influence of PEF treatment (2.9 kV cm−1, 100 Hz, 5000 pulses in trains mode of 500 pulses with a pulse duration of 10 µs) on Saccharomyces cerevisiae eradication and resealing in different conditions, such as current density (which is influenced by the medium conductivity), the sort of medium (phosphate buffered saline (PBS) vs. yeast malt broth (YMB) and a combined treatment of PEF with the addition of preservatives. When the S. cerevisiae were suspended in PBS, increasing the current density from 0.02 to 3.3 A cm−2 (corresponding to a total specific energy of 22.04 to 614.59 kJ kg−1) led to an increase of S. cerevisiae eradication. At 3.3 A cm−2, a total S. cerevisiae eradication was observed. However, when the S. cerevisiae in PBS was treated with the highest current density of 3.3 A cm−2, followed by dilution in a rich YMB medium, a phenomenon of cell membrane resealing was observed by flow cytometry (FCM) and CFU analysis. The viability of S. cerevisiae was also examined when the culture was exposed to repeating PEF treatments (up to four cycles) with and without the addition of preservatives. This experiment was performed when the S. cerevisiae were suspended in YMB containing tartaric acid (pH 3.4) and ethanol to a final concentration of 10% (v/v), which mimics wine. It was shown that one PEF treatment cycle led to a reduction of 1.35 log10, compared to 2.24 log10 when four cycles were applied. However, no synergic effect was observed when the preservatives, free SO2, and sorbic acid were added. This study shows the important and necessary knowledge about yeast eradication and membrane recovery processes after PEF treatment, in particular for application in the liquid food industry.

Nanofountain Probe Electroporation Enables Versatile Single-Cell Intracellular Delivery and Investigation of Postpulse Electropore Dynamics.

Introducing exogenous molecules into cells with high efficiency and dosage control is a crucial step in basic research as well as clinical applications. Here, the capability of the nanofountain probe electroporation (NFP-E) system to deliver proteins and plasmids in a variety of continuous and primary cell types with appropriate dosage control is reported. It is shown that the NFP-E can achieve fine control over the relative expression of two cotransfected plasmids. Finally, the dynamics of electropore closure after the pulsing ends with the NFP-E is investigated. Localized electroporation has recently been utilized to demonstrate the converse process of delivery (sampling), in which a small volume of the cytosol is retrieved during electroporation without causing cell lysis. Single-cell temporal sampling confers the benefit of monitoring the same cell over time and can provide valuable insights into the mechanisms underlying processes such as stem cell differentiation and disease progression. NFP-E parameters that maximize the membrane resealing time, which is essential for increasing the sampled volume and in meeting the challenge of monitoring low copy number biomarkers, are identified. Its application in CRISPR/Cas9 gene editing, stem cell reprogramming, and single-cell sampling studies is envisioned.

Ischemia reperfusion injury provokes adverse left ventricular remodeling in dysferlin-deficient hearts through a pathway that involves TIRAP dependent signaling

Cardiac myocytes have multiple cell autonomous mechanisms that facilitate stabilization and repair of damaged sarcolemmal membranes following myocardial injury. Dysferlin is a protein which facilitates membrane repair by promoting membrane resealing. Although prior studies have shown that dysferlin-deficient (Dysf−/−) mouse hearts have an impaired recovery from acute ischemia/reperfusion (I/R) injury ex vivo, the role of dysferlin in mediating the recovery from myocardial injury in vivo is unknown. Here we show that Dysf−/− mice develop adverse LV remodeling following I/R injury secondary to the collateral damage from sustained myocardial inflammation within the infarct zone. Backcrossing Dysf−/− mice with mice lacking signaling through the Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein (Tirap−/−), attenuated inflammation and abrogated adverse LV remodeling following I/R injury. Subsequent studies using Poloxamer 188 (P188), a membrane resealing reagent, demonstrated that P188 did not attenuate inflammation nor prevent adverse LV remodeling in Dysf−/− mice following I/R injury. Viewed together these studies reveal a previously unappreciated role for the importance of membrane sealing and the resolution of inflammation following myocardial injury.

Premature rupture of the membranes at 16weeks: report of asuccessful outcome of pregnancy and review of the literature.

Prelabor rupture of the fetal membranes (premature rupture of membranes, PROM) before or at the limit of fetal viability is condition associated with significant and serious pediatric morbidity and mortality. It is arare problem, with an estimated incidence between 0.1 and 0.7%. Management of this condition is one of the most challenging clinical situations in obstetrics. We report the case of apregnant woman presenting at 16weeks gestation with ruptured membranes. The course of pregnancy was further complicated by complete placenta previa. Expectant management was undertaken, with term delivery and successful outcome of pregnancy. Expectant management is areasonable approach in properly selected patients. Better understanding of the mechanisms of spontaneous membrane resealing is needed in order to improve poor outcomes. More published data and evidence are necessary to standardize treatment options for this rare condition.

Annexin-A6 in Membrane Repair of Human Skeletal Muscle Cell: A Role in the Cap Subdomain.

Defects in membrane repair contribute to the development of some muscular dystrophies, highlighting the importance to decipher the membrane repair mechanisms in human skeletal muscle. In murine myofibers, the formation of a cap subdomain composed notably by annexins (Anx) is critical for membrane repair. We applied membrane damage by laser ablation to human skeletal muscle cells and assessed the behavior of annexin-A6 (AnxA6) tagged with GFP by correlative light and electron microscopy (CLEM). We show that AnxA6 was recruited to the site of membrane injury within a few seconds after membrane injury. In addition, we show that the deficiency in AnxA6 compromises human sarcolemma repair, demonstrating the crucial role played by AnxA6 in this process. An AnxA6-containing cap-subdomain was formed in damaged human myotubes in about one minute. Through transmission electron microscopy (TEM), we observed that extension of the sarcolemma occurred during membrane resealing, which participated in forming a dense lipid structure in order to plug the hole. By properties of membrane folding and curvature, AnxA6 helped in the formation of this tight structure. The compaction of intracellular membranes—which are used for membrane resealing and engulfed in extensions of the sarcolemma—may also facilitate elimination of the excess of lipid and protein material once cell membrane has been repaired. These data reinforce the role played by AnxA6 and the cap subdomain in membrane repair of skeletal muscle cells.

Naturally occurring and tumor-associated variants of RNF167 promote lysosomal exocytosis and plasma membrane resealing.

Lysosomal exocytosis and resealing of damaged plasma membrane are essential for cellular homeostasis and tumor invasion. However, very little is known of the molecular machinery that regulates these physiological processes. Moreover, no mutations in any of the known regulators of lysosomal exocytosis in primary tumors of patients have been characterized. Here we demonstrate that RNF167-a, a lysosomal-associated ubiquitin ligase, negatively regulates lysosomal exocytosis by inducing perinuclear clustering of lysosomes. Importantly, we also characterized a set of novel natural mutations in RNF167-a, which are commonly found in diverse tumor types. We found that RNF167-a-K97N mutant, unlike the wild type, localizes in the cytoplasm and does not promote perinuclear lysosomal clustering. Furthermore, cells expressing RNF167-a-K97N exhibit dispersed lysosomes, increased exocytosis and enhanced plasma membrane repair. Interestingly, these functional features of RNF167-a-K97N were shared with a naturally occurring short version of RNF167 (isoform RNF167-b). In brief, the results presented here reveal a novel role of RNF167-a, as well as its natural variants RNF167-a-K97N and RNF167-b, as an upstream regulator of lysosomal exocytosis and plasma membrane resealing.

Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin.

Pore-forming toxins are alluring tools for delivering biologically-active, impermeable cargoes to intracellular environments by introducing large conductance pathways into cell membranes. However, the lack of regulation often leads to the dissipation of electrical and chemical gradients, which might significantly affect the viability of cells under scrutiny. To mitigate these problems, we explored the use of lysenin channels to reversibly control the barrier function of natural and artificial lipid membrane systems by controlling the lysenin’s transport properties. We employed artificial membranes and electrophysiology measurements in order to identify the influence of labels and media on the lysenin channel’s conductance. Two cell culture models: Jurkat cells in suspension and adherent ATDC5 cells were utilized to demonstrate that lysenin channels may provide temporary cytosol access to membrane non-permeant propidium iodide and phalloidin. Permeability and cell viability were assessed by fluorescence spectroscopy and microscopy. Membrane resealing by chitosan or specific media addition proved to be an effective way of maintaining cellular viability. In addition, we loaded non-permeant dyes into liposomes via lysenin channels by controlling their conducting state with multivalent metal cations. The improved control over membrane permeability might prove fruitful for a large variety of biological or biomedical applications that require only temporary, non-destructive access to the inner environment enclosed by natural and artificial membranes.

Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions.

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca2+, Cd2+, Zn2+, and Ba2+ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca2+ and Ba2+ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within <1 ms. The threshold for one 300-ns pulse was at 1.5–2 kV/cm, much lower than >7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba2+ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca2+ entry could elicit calcium-induced calcium release (CICR) followed by Ca2+ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca2+ and Ba2+ proved as sensitive nanoporation markers, with Ba2+ being more reliable for monitoring membrane damage and resealing.

Extracellular-Ca2+-Induced Decrease in Small Molecule Electrotransfer Efficiency: Comparison between Microsecond and Nanosecond Electric Pulses.

Electroporation—a transient electric-field-induced increase in cell membrane permeability—can be used to facilitate the delivery of anticancer drugs for antitumour electrochemotherapy. In recent years, Ca2+ electroporation has emerged as an alternative modality to electrochemotherapy. The antitumor effect of calcium electroporation is achieved as a result of the introduction of supraphysiological calcium doses. However, calcium is also known to play a key role in membrane resealing, potentially altering the pore dynamics and molecular delivery during electroporation. To elucidate the role of calcium for the electrotransfer of small charged molecule into cell we have performed experiments using nano- and micro-second electric pulses. The results demonstrate that extracellular calcium ions inhibit the electrotransfer of small charged molecules. Experiments revealed that this effect is related to an increased rate of membrane resealing. We also employed mathematical modelling methods in order to explain the differences between the CaCl2 effects after the application of nano- and micro-second duration electric pulses. Simulation showed that these differences occur due to the changes in transmembrane voltage generation in response to the increase in specific conductivity when CaCl2 concentration is increased.

Myofibers deficient in connexins 43 and 45 expression protect mice from skeletal muscle and systemic dysfunction promoted by a dysferlin mutation

Dysferlinopathy is a genetic human disease caused by mutations in the gene that encodes the dysferlin protein (DYSF). Dysferlin is believed to play a relevant role in cell membrane repair. However, in dysferlin-deficient (blAJ) mice (a model of dysferlinopathies) the recovery of the membrane resealing function by means of the expression of a mini-dysferlin does not arrest progressive muscular damage, suggesting the participation of other unknown pathogenic mechanisms. Here, we show that proteins called connexins 39, 43 and 45 (Cx39, Cx43 and Cx45, respectively) are expressed by blAJ myofibers and form functional hemichannels (Cx HCs) in the sarcolemma. At rest, Cx HCs increased the sarcolemma permeability to small molecules and the intracellular Ca2+ signal. In addition, skeletal muscles of blAJ mice showed lipid accumulation and lack of dysferlin immunoreactivity. As sign of extensive damage and atrophy, muscles of blAJ mice presented elevated numbers of myofibers with internal nuclei, increased number of myofibers with reduced cross-sectional area and elevated creatine kinase activity in serum. In agreement with the extense muscle damage, mice also showed significantly low motor performance. We generated blAJ mice with myofibers deficient in Cx43 and Cx45 expression and found that all above muscle and systemic alterations were absent, indicating that these two Cxs play a critical role in a novel pathogenic mechanism of dysfernolophaties, which is discussed herein. Therefore, Cx HCs could constitute an attractive target for pharmacologic treatment of dyferlinopathies.

Cellular functions and intrinsic attributes of the ATP-binding Eps15 homology domain-containing proteins.

Several cellular processes rely on a cohort of dedicated proteins that manage tubulation, fission, and fusion of membranes. A notably large number of them belong to the dynamin superfamily of proteins. Among them is the evolutionarily conserved group of ATP-binding Eps15-homology domain-containing proteins (EHDs). In the two decades since their discovery, EHDs have been linked to a range of cellular processes that require remodeling or maintenance of specific membrane shapes such as during endocytic recycling, caveolar biogenesis, ciliogenesis, formation of T-tubules in skeletal muscles, and membrane resealing after rupture. Recent work has shed light on their structure and the unique attributes they possess in linking ATP hydrolysis to membrane remodeling. This review summarizes some of these recent developments and reconciles intrinsic protein functions to their cellular roles.

Control of Instant Membrane Resealing by Lysosomal Acid Ceramidase in Endothelial Cells

Previous studies have shown that lysosome exocytosis and ceramide platform formation in the outer leaflet of the lipid bilayer are essential for plasma membrane repair. As a lysosomal enzyme catalyzing ceramide hydrolysis, the role of acid ceramidase (AC) in the control of cell membrane repair remains unknown. In the present study, we tested a hypothesis that lysosomal AC‐mediating signaling plays a crucial role in instant membrane resealing of endothelial cells (ECs), which help maintain the integrity of EC membrane and related functional activity. By Western blot analysis, it was found that carmofur, an AC inhibitor, decreased the AC expression in murine ECs. On the contrary, Genistein, an AC activator, elevated the level of AC in these cells. Using wavelength‐switching fluorescent microscopy with propidium iodide (PI), a fluorescent membrane‐impermeant dye as membrane resealing marker and fura‐2 as intracellular Ca2+ indicator, we monitored instant membrane resealing following saponin‐induced membrane disruption simultaneously with Ca2+ influx recording in murine ECs. The speed of PI fluorescence increase (df/dt) within ECs and time to a peak of PI fluorescence elevation were used to reflect the instant membrane resealing. Under Ca2+‐free condition, we found that membrane resealing in ECs was enhanced by carmofur as shown by df/dt reduction, but inhibited by genistein (increase in df/dt). In Hanks’ buffer containing Ca2+, however, carmofur and genistein failed to alter membrane resealing of murine ECs when Ca2+ influx was increased. In isolated coronary ECs from WT/WT and Asah1fl/fl/Endocre mice, we demonstrated that AC gene knockout specifically in ECs remarkably enhanced membrane resealing following saponin‐induced membrane interruption, which also mainly occurred under Ca2+‐free condition. These results suggest that lysosomal AC expression and activity are critical for the regulation of instant membrane resealing in ECs after acute injury, which represents a Ca2+‐independent membrane repairing mechanism.Support or Funding InformationThis study was supported by NIH grants DK54927 and DK102539.

Muscle Fibers Utilize its Glucose Uptake Machinery to Reseal Sarcolemma Disruptions &amp; Stimulating Affiliated Rab G Proteins Improves Repair Capacity in Muscle Dystrophic Mice

The glucose uptake machinery in cells and muscle fibers is a robust and highly regulated system used to import extracellular glucose for intracellular metabolic processing. The majority of glucose uptake in skeletal muscle is done through stimulated exocytosis and fusion of vesicles to the plasma membrane to enrich the cellular exterior with glucose transporter protein GLUT4. In our investigation, we found evidence that GLUT4 containing vesicles (GSV) and the glucose uptake regulators AS160, rab8A, and rab10 serve an additional role in the plasma membrane repair response. Through the use of mutant plasmids and live cell imaging, we observed that GSVs, as well as affiliated rabs, translocate to injury sites in C2C12 myoblasts and isolated mouse muscle fibers under different modes of damage. We observed that AS160 phosphorylation from the phosphoinositide‐3 kinase (PI3K)/Akt1 signaling axis regulates membrane repair in cultured muscle cells. In addition, we screened GSV associating rab G proteins and saw that knocking down rab8A and rab10 expression reduced the repair capacity of the cells. Importantly, through the electroporation of overexpression or constitutively active rab10 mutant constructs in skeletal muscle of Duchenne muscular dystrophy mdx mice, there was a significant improvement in membrane resealing in vivo. Overall, our findings suggest that the glucose uptake vesicles commonly thought of as important for metabolic homeostasis, serve an additional function in repairing membrane wounds in some cell lines and mouse muscle fibers. Targeting glucose uptake regulators could lead to the development of new therapies for muscle disease and injury.

Neuronal Plasma Membrane Integrity is Transiently Disturbed by Traumatic Loading.

The acute response of neurons subjected to traumatic loading involves plasma membrane disruption, yet the mechanical tolerance for membrane compromise, time course, and mechanisms for resealing are not well understood. We have used an in vitro traumatic neuronal injury model to investigate plasma membrane integrity immediately following a high-rate shear injury. Cell-impermeant fluorescent molecules were added to cortical neuronal cultures prior to insult to assess membrane integrity. The percentage of cells containing the permeability marker was dependent on the molecular size of the marker, as smaller molecules gained access to a higher percentage of cells than larger ones. Permeability increases were positively correlated with insult loading rate. Membrane disruption was transient, evidenced by a membrane resealing within the first minute after the insult. In addition, chelation of either extracellular Ca2+ or intracellular Ca2+ limited membrane resealing. However, injury following chelation of both extracellular and intracellular Ca2+ caused diminished permeability as well as a greater resealing ability compared to chelation of extracellular or intracellular Ca2+ alone. Treatment of neuronal cultures with jasplakinolide, which stabilizes filamentous actin, reduced permeability increases, while latrunculin-B, an actin depolymerizing agent, both reduced the increase in plasma membrane permeability and promoted resealing. This study gives insight into the dynamics of neuronal membrane disruption and subsequent resealing, which was found to be calcium dependent and involve actin in a role that differs from non-neuronal cells. Taken together, these data will lead to a better understanding of the acute neuronal response to traumatic loading.

Multiple poloxamers increase plasma membrane repair capacity in muscle and nonmuscle cells.

Various previous studies established that the amphiphilic tri-block copolymer known as poloxamer 188 (P188) or Pluronic-F68 can stabilize the plasma membrane following a variety of injuries to multiple mammalian cell types. This characteristic led to proposals for the use of P188 as a therapeutic treatment for various disease states, including muscular dystrophy. Previous studies suggest that P188 increases plasma membrane integrity by resealing plasma membrane disruptions through its affinity for the hydrophobic lipid chains on the lipid bilayer. P188 is one of a large family of copolymers that share the same basic tri-block structure consisting of a middle hydrophobic propylene oxide segment flanked by two hydrophilic ethylene oxide moieties [poly(ethylene oxide)80-poly(propylene oxide)27-poly(ethylene oxide)80]. Despite the similarities of P188 to the other poloxamers in this chemical family, there has been little investigation into the membrane-resealing properties of these other poloxamers. In this study we assessed the resealing properties of poloxamers P181, P124, P182, P234, P108, P407, and P338 on human embryonic kidney 293 (HEK293) cells and isolated muscle from the mdx mouse model of Duchenne muscular dystrophy. Cell membrane injuries from glass bead wounding and multiphoton laser injury show that the majority of poloxamers in our panel improved the plasma membrane resealing of both HEK293 cells and dystrophic muscle fibers. These findings indicate that many tri-block copolymers share characteristics that can increase plasma membrane resealing and that identification of these shared characteristics could help guide design of future therapeutic approaches.

The link between yeast cell wall porosity and plasma membrane permeability after PEF treatment

An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure to PEF indicate a strong coupling between these parts of the cell.

Phenotypic Drug Screening for Dysferlinopathy Using Patient-Derived Induced Pluripotent Stem Cells

Dysferlinopathy is a progressive muscle disorder that includes limb‐girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG‐132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient‐derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. stem cells translational medicine 2019;8:1017–1029