Reversible Membrane Permeabilization Research Articles

Influence of Microwave-Assisted Drying on Structural Integrity and Viability of Testicular Tissues from Adult and Prepubertal Domestic Cats.

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above -20°C, which is one step closer to future storage at supra-zero temperatures.

Comb-like Pseudopeptides Enable Very Rapid and Efficient Intracellular Trehalose Delivery for Enhanced Cryopreservation of Erythrocytes.

Cell cryopreservation plays a key role in the development of reproducible and cost-effective cell-based therapies. Trehalose accumulated in freezing- and desiccation-tolerant organisms in nature has been sought as an attractive nontoxic cryoprotectant. Herein, we report a coincubation method for very rapid and efficient delivery of membrane-impermeable trehalose into ovine erythrocytes through reversible membrane permeabilization using pH-responsive, comb-like pseudopeptides. The pseudopeptidic polymers containing relatively long alkyl side chains were synthesized to mimic membrane-anchoring fusogenic proteins. The intracellular trehalose delivery efficiency was optimized by manipulating the side chain length, degree of substitution, and concentration of the pseudopeptides with different hydrophobic alkyl side chains, the pH, temperature, and time of incubation, as well as the polymer-to-cell ratio and the concentration of extracellular trehalose. Treatment of erythrocytes with the comb-like pseudopeptides for only 15 min yielded an intracellular trehalose concentration of 177.9 ± 8.6 mM, which resulted in 90.3 ± 0.7% survival after freeze-thaw. The very rapid and efficient delivery was found to be attributed to the reversible, pronounced membrane curvature change as a result of strong membrane insertion of the comb-like pseudopeptides. The pseudopeptides can enable efficient intracellular delivery of not only trehalose for improved cell cryopreservation but also other membrane-impermeable cargos.

Minireview: Biophysical Mechanisms of Cell Membrane Sonopermeabilization. Knowns and Unknowns.

Microbubble-assisted ultrasound has emerged as a promising method for the delivery of low-molecular-weight chemotherapeutic molecules, nucleic acids, therapeutic peptides, and antibodies in vitro and in vivo. Its clinical applications are under investigation for local delivery drug in oncology and neurology. However, the biophysical mechanisms supporting the acoustically mediated membrane permeabilization are not fully established. This review describes the present state of the investigations concerning the acoustically mediated stimuli (i.e., mechanical, chemical, and thermal stimuli) as well as the molecular and cellular actors (i.e., membrane pores and endocytosis) involved in the reversible membrane permeabilization process. The different hypotheses, which were proposed to give a biophysical description of the membrane permeabilization, are critically discussed.

Spatial-Temporal Cellular Bioeffects from Acoustic Droplet Vaporization.

One of the major challenges in developing acoustic droplet vaporization (ADV)-associated therapy as an effective and safe strategy is the precise determination of the spatial cellular bioeffects after ADV (cell death or cell membrane permeabilization). In this study, we combined high-speed camera imaging and live-cell microscopic imaging to observe the transient dynamics of droplets during ADV and to evaluate the mechanical force on cells.Methods: C6 glioma cells were co-incubated with DiI-labeled droplets (radius: 1.5, 2.25, and 3.0 μm). We used an acousto-optical system for high-speed bright-field (500 kfps) and fluorescence (40 kfps) microscopic imaging in order to visualize the dynamics of droplets under ultrasound excitation (frequency = 5 MHz, pressure = 5-8 MPa, cycle number = 3, pulse number = 1). Live-cell microscopic imaging was used to monitor the cell morphology, cell membrane permeabilization, and cell viability by membrane-anchored Lyn-yellow fluorescence protein, propidium Iodide staining, and calcein blue AM staining, respectively.Results: We discovered that the spatial distribution of ADV-induced bioeffects could be mapped to the physical dynamics of droplet vaporization. For droplets with a 1.5 μm radius, the distance threshold for ADV-induced cell death (5.5±1.9 μm) and reversible membrane permeabilization (11.3±3.5 μm) was well correlated with the distance of ADV-bubble pressing downward to the floor (5.7±1.3 μm) and maximum distance of droplet expansion (11.5±2.6 μm), respectively. These distances were enlarged by increasing the droplet sizes and insonation acoustic pressures. The live-cell imaging results show that ADV-bubbles can directly disrupt the cell membrane layer and induce intensive intracellular substance leakage. Further, the droplets shed the payload onto nearby cells during ADV, suggesting ADV could directly induce adjacent cell death by physical force and enhancement of chemotherapy to distant cells.Conclusion: This study provide new insights into the ADV-mediated physicochemical synergic effect for medical applications.

Mechanisms of inactivation of Candida humilis and Saccharomyces cerevisiae by pulsed electric fields

AimsThis study aimed to determine how electric field strength, pulse width and shape, and specific energy input relate to the effect of pulsed electric fields (PEF) on viability and membrane permeabilization in Candida humilis and Saccharomyces cerevisiae suspended in potassium phosphate buffer. Methods and resultsCells were treated with a micro-scale system with parallel plate electrodes. Propidium iodide was added before or after treatments to differentiate between reversible and irreversible membrane permeabilization. Treatments of C. humilis with 71kV/cm and 48kJ/kg reduced cell counts by 3.9±0.6 log (cfu/mL). Pulse shape or width had only a small influence on the treatment lethality. Variation of electric field strength (17–71kV/cm), pulse width (0.086–4μs), and specific energy input (8–46kJ/kg) demonstrated that specific energy input correlated to the membrane permeabilization (r2=0.84), while other parameters were uncorrelated. A minimum energy input of 3 and 12kJ/kg was required to achieve reversible membrane permeabilization and a reduction of cell counts, respectively, of C. humilis. ConclusionsEnergy input was the parameter that best described the inactivation efficiency of PEF. Significance and impact of studyThis study is an important step to identify key process parameters and to facilitate process design for improved cost-effectiveness of commercial PEF treatment.

The Perforin Pore Facilitates the Delivery of Cationic Cargos

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.

Time-resolved luminescence resonance energy transfer imaging of protein–protein interactions in living cells

Förster resonance energy transfer (FRET) with fluorescent proteins permits high spatial resolution imaging of protein-protein interactions in living cells. However, substantial non-FRET fluorescence background can obscure small FRET signals, making many potential interactions unobservable by conventional FRET techniques. Here we demonstrate time-resolved microscopy of luminescence resonance energy transfer (LRET) for live-cell imaging of protein-protein interactions. A luminescent terbium complex, TMP-Lumi4, was introduced into cultured cells using two methods: (i) osmotic lysis of pinocytic vesicles; and (ii) reversible membrane permeabilization with streptolysin O. Upon intracellular delivery, the complex was observed to bind specifically and stably to transgenically expressed Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins. LRET between the eDHFR-bound terbium complex and green fluorescent protein (GFP) was detected as long-lifetime, sensitized GFP emission. Background signals from cellular autofluorescence and directly excited GFP fluorescence were effectively eliminated by imposing a time delay (10 micros) between excitation and detection. Background elimination made it possible to detect interactions between the first PDZ domain of ZO-1 (fused to eDHFR) and the C-terminal YV motif of claudin-1 (fused to GFP) in single microscope images at subsecond time scales. We observed a highly significant (P<10(-6)), six-fold difference between the mean, donor-normalized LRET signal from cells expressing interacting fusion proteins and from control cells expressing noninteracting mutants. The results show that time-resolved LRET microscopy with a selectively targeted, luminescent terbium protein label affords improved speed and sensitivity over conventional FRET methods for a variety of live-cell imaging and screening applications.

Single Molecule Sensitive Multivalent Polyethylene Glycol Probes for RNA Imaging

The imaging of RNA in live cells presents a methodological challenge both in the mechanism by which the probes are delivered to the cell and in the sensitivity of the probe. Probes must be delivered to the correct cellular compartment and, once inside the cell, should bind to the target RNA rapidly and with enough sensitivity to detect small numbers of RNA molecules. Here, we report the characterization of a single-molecule sensitive, multivalent RNA imaging probe that utilizes an eight-armed poly(ethylene glycol) core. This probe allowed for the accurate imaging of the human respiratory syncytial virus (hRSV) genomic RNA when delivered into live cells using either reversible membrane permeabilization or TAT-peptide mediated membrane transduction. Neither reversible membrane permeabilization nor TAT mediated delivery proceeded through the endosomal pathway, and both delivery methods demonstrated fast binding kinetics of less than 10 min. Subcellular distributions of RNA were visualized in live cells, as well as RNA-protein colocalization, after fixation and immunostaining of the hRSV RNA binding protein N.

Observation of NMR Signals from Proteins Introduced into Living Mammalian Cells by Reversible Membrane Permeabilization Using a Pore-Forming Toxin, Streptolysin O

We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.

Pore Forming Properties Of Antimicrobial Peptides In Different Natural Lipid Environment

The biophysical characteristics and the pore formation dynamics of synthetic, naturally occurring peptides forming membrane-spanning channels, were investigated by using cells having high membrane resistance recorded in whole-cell configuration. The peptides were applied to (and removed from) the recorded cells in ≤50 ms that were: isolated photoreceptor rod outer segments (OS) of low vertebrates, their red blood cells (RBC), and human RBC. The OS membrane resistance was ≥5 GΩ once the OS main endogenous conductance was blocked with light; the RBC had lower resistances but high enough to allow the detection of the exogenous current produced by the peptides up to single channel resolution. Surprisingly, the synthetic, naturally occurring alamethicin F50/5 and selected analogs produced current amplitudes once inserted in the OS at least twice the ones in RBC: therefore, the lack of hemolysis does not ensure the lack of side-effects of an antimicrobial peptide. Once in the RBC membrane, the alameticines produced pores whose voltage-dependency and single channel properties were similar to the ones recorded in OS, showing that the pore forms only as a barrel-stave, independently by the lipid environment. Other modes of pore formation were investigated with cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH2), that produced reversible membrane permeabilization at concentrations up to 10 μM. This reversibility at such high concentrations is not expected in the case of a carpet model of pore formation, where micellation should be severe. At difference with alamethicines, membrane permeabilization was voltage-independent, repetitive peptide application caused the progressive increase of the steady-state current amplitude, and no discernible single-channel events were detected at low peptide concentrations. Collectively, these results indicate that cecropin-melittin hybrid peptide permeabilize the membrane according to a toroidal model of pore formation (instead of barrel-stave or carpet one).

Reversible Membrane Permeabilization of Mammalian Cells Treated with Digitonin and Its Use for Inducing Nuclear Reprogramming byXenopusEgg Extracts

Plasma membranes can be reversibly permeabilized by Streptolysin O. The permeabilized cells can be reprogrammed and partially dedifferentiated in the cell-free system from egg extracts. However, the permeabilizing activity of Streptolysin O is not stable, and therefore it is difficult to control its activity. An alternative method for reversible permeabilization is useful for establishing a cell-free system. Here, we used a nonionic detergent, digitonin, for permeabilization. A low concentration of digitonin induced reversible permeabilization of the plasma membrane in bovine, mouse, and porcine somatic cells. The permeabilized cells were treated with Xenopus laevis egg extracts. The treated cells showed exchange of nuclear proteins from extracts such as incorporation of Xenopus-specific histone B4 and Lamin LIII into nuclei. After resealing of the membrane, the cells showed upregulation of OCT4, SOX2, and NANOG expression. Our results suggest that reversible permeabilization with digitonin can be used to induce nuclear reprogramming and to activate pluripotent genes by a cell-free system.

Enhancement of biomolecule transport by electroporation: A review of theory and practical application to transformation of Corynebacterium glutamicum

Selective and reversible permeabilization of the cell wall permeability barrier is the focus for many biotechnological applications. In this article, the basic principles for reversible membrane permeabilization, based on biological, chemical, and physical methods are reviewed. Emphasis is given to electroporation (electropermeabilization) which tends to be the most popular method for membrane permeabilization and for introduction of foreign molecules into the cells. The applications of this method in industrial processes as well as the critical factors and parameters which affect the success of this approach are discussed. The different strategies developed throughout the years for increased transformation efficiencies of the industrially important amino acid-overproducing bacterium Corynebacterium glutamicum, are also summarized.

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 10(5)-10(6) molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca(2+)-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.

Control of ultrasound-mediated reversible membrane permeabilization for targeted drug delivery

Ultrasound-mediated permeabilization of cell membranes is a potentially useful method to noninvasively enhance delivery of therapeutic compounds into targeted cells. Unlike previous studies concerned only with cell death, in this study the effects of ultrasound exposure parameters on reversible membrane permeabilization of prostate cancer cells were determined. Cells were suspended in a solution containing a membrane-impermeant marker (calcein) and exposed to 24-kHz ultrasound. Viability and molecular uptake were quantified using flow cytometry. Average uptake increased with incident ultrasound pressure above a threshold and reached a plateau (20%–25% of external calcein concentration); increased with total exposure time; was weakly dependent on pulse length (3.3 ms to 1.0 s) with a small maximum at 10–33 ms and a sharp decline at 1.0 ms; and was independent of duty cycle (0.5%–50%). The fraction of viable cells decreased continuously with increasing pressure, decreased as uptake increased for most other examined condition was approximately 25% for maximum uptake under optimal conditions. Uptake decreased approximately exponentially with the time at which calcein was added to the cell suspension after exposure, with a time constant of about 1 min. The membrane disruptions allows uptake of albumin (MW=66 000) but not R-phycoerythrin (MW=240 000).

P2X7 purinoceptor expression in Xenopus oocytes is not sufficient to produce a pore-forming P2Z-like phenotype

The purinergic rP2X7 receptor expressed in a number of heterologous systems not only functions as a cation channel but also gives rise to a P2Z-like response, i.e. a reversible membrane permeabilization that allows the passage of molecules with molecular masses of ≥300 Da. We investigated the properties of rP2X7 receptors expressed in Xenopus oocytes. In two-electrode voltage-clamp experiments, ATP or BzATP caused inward currents that were abolished or greatly diminished when NMDG + or choline + replaced Na + as the principal external cation. In fluorescent dye experiments, BzATP application did not result in entry of the fluorophore YO-PRO-1 2+. Thus, rP2X7 expression in Xenopus oocytes does not by itself give rise to the pore-forming P2Z phenotype, suggesting that ancillary factors are involved.

Detection of Ribonuclease H-Generated mRNA Fragments in Human Leukemia Cells Following Reversible Membrane Permeabilization in the Presence of Antisense Oligodeoxynucleotides

The involvement of ribonuclease H (RNase H) in antisense phenomena in intact cells has, to date, only been adequately demonstrated for microinjected Xenopus systems. The significance of RNase H for the antisense effects of oligodeoxynucleotides observed in human and other mammalian cell cultures has remained obscure, in part because of inadequate analytic methods. In this report we show that the "reverse ligation-mediated PCR" (RL-PCR) procedure permits amplification of RNA fragments produced by oligodeoxynucleotide-directed RNase H activity. We have used this procedure to demonstrate RNase H-dependent antisense effects in irreversibly permeabilized (dead) cells and reversibly permeabilized (live) cells.