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A cell-free system has been developed to image vesicle budding and fission events. This method reveals an important role for the F-BAR protein FBP17 in regulating tubulation and clathrin-dependent budding. Cell-free reconstitution of membrane traffic reactions and the morphological characterization of membrane intermediates that accumulate under these conditions have helped to elucidate the physical and molecular mechanisms involved in membrane transport 1 , 2 , 3 . To gain a better understanding of endocytosis, we have reconstituted vesicle budding and fission from isolated plasma membrane sheets and imaged these events. Electron and fluorescence microscopy, including subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) 4 , 5 , 6 , revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked by GTPγS. Triggering fission by replacing GTPγS with GTP led not only to separation of clathrin-coated buds, but also to vesicle formation by fragmentation of the tubules. These results suggest a functional link between FBP17-dependent membrane tubulation and clathrin-dependent budding. They also show that clathrin spatially directs plasma membrane invaginations that lead to the generation of endocytic vesicles larger than those enclosed by the coat.

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

BAR domain proteins contribute to membrane deformation in diverse cellular processes. The inverted-BAR (I-BAR) protein IRSp53, for instance, is found on the inner leaflet of the tubular membrane of filopodia; however its role in the formation of these structures is incompletely understood. Here we develop an original assay in which proteins are encapsulated in giant unilamellar vesicles connected to membrane nanotubes. Our results demonstrate that I-BAR dimers sense negative membrane curvature. Experiment and theory reveal that the I-BAR displays a non-monotonic sorting with curvature, and expands the tube at high imposed tension while constricting it at low tension. Strikingly, at low protein density and tension, protein-rich domains appear along the tube. This peculiar behaviour is due to the shallow intrinsic curvature of I-BAR dimers. It allows constriction of weakly curved membranes coupled to local protein enrichment at biologically relevant conditions. This might explain how IRSp53 contributes in vivo to the initiation of filopodia. The inverted-BAR domain protein IRSp53 associates with the inner leaflet of tubular membranes such as filopodia. Here, Prévost et al . demonstrate that the I-BAR domain of IRSp53 senses negative membrane curvature, and undergoes phase separation which may aid its clustering upon filopodia generation.

Recent studies have described the role of shedding vesicles as physiological conveyers of intracellular components between neighboring cells. Here we report that melanosomes are one example of shedding vesicle cargo, but are processed by a previously unreported mechanism. Pigment globules were observed to be connected to the filopodia of melanocyte dendrites, which have previously been shown to be conduits for melanosomes. Pigment globules containing multiple melanosomes were released from various areas of the dendrites of normal human melanocytes derived from darkly pigmented skin. The globules were then captured by the microvilli of normal human keratinocytes, also derived from darkly pigmented skin, which incorporated them in a protease-activated receptor-2 (PAR-2)-dependent manner. After the pigment globules were ingested by the keratinocytes, the membrane that surrounded each melanosome cluster was gradually degraded, and the individual melanosomes then spread into the cytosol and were distributed primarily in the perinuclear area of each keratinocyte. These results suggest a melanosome transfer pathway wherein melanosomes are transferred from melanocytes to keratinocytes via the shedding vesicle system. This packaging system generates pigment globules containing multiple melanosomes in a unique manner.

Using Caenorhabditis elegans genetic screens, we identified receptor‐mediated endocytosis (RME)‐4 and RME‐5/RAB‐35 as important regulators of yolk endocytosis in vivo . In rme‐4 and rab‐35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME‐4 and RAB‐35 function downstream of clathrin, upstream of RAB‐7, and act synergistically with recycling regulators RAB‐11 and RME‐1. We find that RME‐4 is a conserved DENN domain protein that binds to RAB‐35 in its GDP‐loaded conformation. GFP–RME‐4 also physically interacts with AP‐2, is enriched on clathrin‐coated pits, and requires clathrin but not RAB‐5 for cortical association. GFP–RAB‐35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme‐4 mutants. We propose that RME‐4 functions on coated pits and/or vesicles to recruit RAB‐35, which in turn functions in the endosome to promote receptor recycling.

This paper presents unique approaches to enable control and quantification of ultrasound-mediated cell membrane disruption, or sonoporation, at the single-cell level. Ultrasound excitation of microbubbles that were targeted to the plasma membrane of HEK-293 cells generated spatially and temporally controlled membrane disruption with high repeatability. Using whole-cell patch clamp recording combined with fluorescence microscopy, we obtained time-resolved measurements of single-cell sonoporation and quantified the size and resealing rate of pores. We measured the intracellular diffusion coefficient of cytoplasmic RNA/DNA from sonoporation-induced transport of an intercalating fluorescent dye into and within single cells. We achieved spatiotemporally controlled delivery with subcellular precision and calcium signaling in targeted cells by selective excitation of microbubbles. Finally, we utilized sonoporation to deliver calcein, a membrane-impermeant substrate of multidrug resistance protein-1 (MRP1), into HEK-MRP1 cells, which overexpress MRP1, and monitored the calcein efflux by MRP1. This approach made it possible to measure the efflux rate in individual cells and to compare it directly to the efflux rate in parental control cells that do not express MRP1.

Electroporation uses electric pulses to promote delivery of DNA and drugs into cells. This study presents a model of electroporation in a spherical cell exposed to an electric field. The model determines transmembrane potential, number of pores, and distribution of pore radii as functions of time and position on the cell surface. For a 1-ms, 40 kV/m pulse, electroporation consists of three stages: charging of the cell membrane (0–0.51 μs), creation of pores (0.51–1.43 μs), and evolution of pore radii (1.43 μs to 1 ms). This pulse creates ∼341,000 pores, of which 97.8% are small (≈1 nm radius) and 2.2% are large. The average radius of large pores is 22.8 ± 18.7 nm, although some pores grow to 419 nm. The highest pore density occurs on the depolarized and hyperpolarized poles but the largest pores are on the border of the electroporated regions of the cell. Despite their much smaller number, large pores comprise 95.3% of the total pore area and contribute 66% to the increased cell conductance. For stronger pulses, pore area and cell conductance increase, but these increases are due to the creation of small pores; the number and size of large pores do not increase.

The initiation of action potentials (APs) by membrane depolarization occurs after a brief vulnerability period, during which excitation can be abolished by the reversal of the stimulus polarity. This vulnerability period is determined by the time needed for gating of voltage-gated sodium channels (VGSC). We compared nerve excitation by ultra-short uni- and bipolar stimuli to define the time frame of bipolar cancellation and of AP initiation. Propagating APs in isolated frog sciatic nerve were elicited by cathodic pulses (200 ns–300 µs), followed by an anodic (canceling) pulse of the same duration after a 0–200-µs delay. We found that the earliest and the latest boundaries for opening the critical number of VGSC needed to initiate AP are, respectively, between 11 and 20 µs and between 100 and 200 µs after the onset of depolarization. Stronger depolarization accelerated AP initiation, apparently due to faster VGSC opening, but not beyond the 11-µs limit. Bipolar cancellation was augmented by reducing pulse duration, shortening the delay between pulses, decreasing the amplitude of the cathodic pulse, and increasing the amplitude of the anodic one. Some of these characteristics contrasted the bipolar cancellation of cell membrane electroporation (Pakhomov et al. in Bioelectrochemistry 122:123–133, 2018; Gianulis et al. in Bioelectrochemistry 119:10–19, 2017), suggesting different mechanisms. The ratio of nerve excitation thresholds for a unipolar cathodic pulse and a symmetrical bipolar pulse increased as a power function as the pulse duration decreased, in remarkable agreement with the predictions of SENN model of nerve excitation (Reilly and Diamant in Health Phys 83(3):356–365, 2002).

The effect of pulsed electric field (PEF) treatments of different intensities on the electroporation of the cytoplasmatic membrane of Chlorella vulgaris, and on the extraction of carotenoids and chlorophylls were investigated. Staining the cells with propidium iodide before and after the PEF treatment revealed the existence of reversible and irreversible electroporation. Application of PEF treatments in the range of 20–25 kV cm −1 caused most of the population of C. vulgaris to be irreversibly electroporated even at short treatment times (5 pulses of 3 µs). However, at lower electric field strengths (10 kV cm −1 ), cells that were reversibly electroporated were observed even after 50 pulses of 3 µs. The electroporation of C. vulgaris cells by PEF higher than 15 kV cm −1 and duration is higher than 15 µs increased significantly the extraction yield of intracellular components of C. vulgaris. The application of a 20 kV cm −1 for 75 μs increased the extraction yield just after the PEF treatment of the carotenoids, and chlorophylls a and b 0.5, 0.7, and 0.8 times, respectively. However, further increments in electric field strength and treatment time did not cause significant increments in the extraction yield. The extraction of carotenoids from PEF-treated C. vulgaris cells after 1 h of the application of the treatment significantly increased the extraction yield in comparison to the yield obtained from the cells extracted just after the PEF treatment. After PEF treatment at 20 kV cm −1 for 75 µs, extraction yield for carotenoids, and chlorophylls a and b increased 1.2, 1.6, and 2.1 times, respectively. A high correlation was observed between irreversible electroporation and percentage of yield increase when the extraction was conducted after 1 h of the application of PEF treatment ( R : 0.93), but not when the extraction was conducted just after PEF treatment ( R : 0.67).

The cell membrane is a complex and highly regulated system that is composed of lipid bilayer and proteins. One of the main functions of the cell membrane is the regulation of cell entry. Cell-penetrating peptides (CPPs) are defined as peptides that can cross the plasma membrane and deliver their cargo inside the cell. The uptake of a peptide is determined by its sequence and biophysicochemical properties. At the same time, the uptake mechanism and efficiency are shown to be dependent on local peptide concentration, cell membrane lipid composition, characteristics of the cargo, and experimental methodology, suggesting that a highly efficient CPP in one system might not be as productive in another. To better understand the dependence of CPPs on the experimental system, we present a review of the in vitro assays that have been employed in the literature to evaluate CPPs and CPP-cargos. Our comprehensive review suggests that utilization of orthogonal assays will be more effective for deciphering the true ability of CPPs to translocate through the membrane and enter the cell cytoplasm.