Coronaviruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble ...on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding.
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Endosomal sorting complexes required for transport (ESCRT)-III family proteins catalyze membrane remodeling processes that stabilize and constrict membrane structures. It has been proposed that ...stable ESCRT-III complexes containing CHMP2B could establish diffusion barriers at the post-synaptic spine neck. In order to better understand this process, we developed a novel method based on fusion of giant unilamellar vesicles to reconstitute ESCRT-III proteins inside GUVs, from which membrane nanotubes are pulled. The new assay ensures that ESCRT-III proteins polymerize only when they become exposed to physiologically relevant membrane topology mimicking the complex geometry of post-synaptic spines. We establish that CHMP2B, both full-length and with a C-terminal deletion (ΔC), preferentially binds to membranes containing phosphatidylinositol 4,5-bisphosphate PI(4,5)P2. Moreover, we show that CHMP2B preferentially accumulates at the neck of membrane nanotubes, and provide evidence that CHMP2B-ΔC prevents the diffusion of PI(4,5)P2 lipids and membrane-bound proteins across the tube neck. This indicates that CHMP2B polymers formed at a membrane neck may function as a diffusion barrier, highlighting a potential important function of CHMP2B in maintaining synaptic spine structures.
A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging ...(FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of
pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with
accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
An important goal for bottom-up synthetic biology is to construct tissue-like structures from artificial cells. The key is the ability to control the assembly of the individual artificial cells. ...Unlike most methods resorting to external fields or sophisticated devices, inspired by the hanging drop method used for culturing spheroids of biological cells, we employ a capillary-driven approach to assemble giant unilamellar vesicles (GUVs)-based protocells into colonized prototissue arrays by means of a coverslip with patterned wettability. By spatially confining and controllably merging a mixed population of lipid-coated double-emulsion droplets that hang on a water/oil interface, an array of synthetic tissue-like constructs can be obtained. Each prototissue module in the array comprises multiple tightly packed droplet compartments where interfacial lipid bilayers are self-assembled at the interfaces both between two neighboring droplets and between the droplet and the external aqueous environment. The number, shape, and composition of the interconnected droplet compartments can be precisely controlled. Each prototissue module functions as a processer, in which fast signal transports of molecules via cell–cell and cell-environment communications have been demonstrated by molecular diffusions and cascade enzyme reactions, exhibiting the ability to be used as biochemical sensing and microreactor arrays. Our work provides a simple yet scalable and programmable method to form arrays of prototissues for synthetic biology, tissue engineering, and high-throughput assays.
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•Electoformation protocol used to form GUVs is extensively varied.•Assessment of GUV population is done by characterizing vesicles’ diameters, structure and amount of lipid.•Prolonged ...duration of electoformation did not influence the quality of GUVs populations.•Frequency and voltage of applied field did influence the quality of GUVs populations.•State and defects in electrodes did influence the quality of GUVs populations.
Giant unilamellar vesicles (GUVs) have become one of extensively studied biological bilayer models especially when investigating topological and mechanical properties of cell membranes. They are also used to visualize membrane-related phenomena. However, the method of preparation and the effects of parameters of preparation on the vesicular structure are extensively varied. Therefore, it is important to understand how the process of formation of GUVs influences the outcome population, as it can influence the outcome of the experiment that is planned. Therefore, in this study, we investigated the effects of protocol parameters of electroformation on properties of homogeneous population of POPC GUVs. The parameters investigated in this study are duration of electroformation, usage of electrodes and frequency of applied AC field and its voltage. The properties investigated, which can be used to describe GUV populations are average diameter of vesicle, the amount of lipid molecules in population, and structure of vesicles. According to our results, prolonged time (greater than 4 h) does not influence outcome; however, parameters of applied electrical field (voltage and frequency) did significantly influence the properties of obtained POPC GUV populations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Membranes are the key structures to separate and spatially organize cellular systems. Their rich dynamics and transformations during the cell cycle are orchestrated by specific membrane‐targeted ...molecular machineries, many of which operate through energy dissipation. Likewise, man‐made light‐activated molecular rotary motors have previously shown drastic effects on cellular systems, but their physical roles on and within lipid membranes remain largely unexplored. Here, the impact of rotary motors on well‐defined biological membranes is systematically investigated. Notably, dramatic mechanical transformations are observed in these systems upon motor irradiation, indicative of motor‐induced membrane expansion. The influence of several factors on this phenomenon is systematically explored, such as motor concentration and membrane composition., Membrane fluidity is found to play a crucial role in motor‐induced deformations, while only minor contributions from local heating and singlet oxygen generation are observed. Most remarkably, the membrane area expansion under the influence of the motors continues as long as irradiation is maintained, and the system stays out‐of‐equilibrium. Overall, this research contributes to a comprehensive understanding of molecular motors interacting with biological membranes, elucidating the multifaceted factors that govern membrane responses and shape transitions in the presence of these remarkable molecular machines, thereby supporting their future applications in chemical biology.
Explore the nuanced relationship between light‐driven molecular motors and lipid membranes in this research. Delve into the continuous interplay, observing how these motors, akin to dynamic springs fueled by light, orchestrate subtle yet fascinating shape transitions in membranes. Observe how these out‐of‐equilibrium systems can modulate area expansion through their action in lipid membranes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The results of the analysis of changes in electrotelluric potentials (ETP) during the observation of intense solar flare events and intense magnetic storms on Sakhalin are presented. The data were ...studied in the period from July 20 to October 12, 2023. The absence of characteristic changes in the ETP (integral amplification or attenuation of noise in the low-frequency region) depending on the presence or absence of a solar flare event is shown. At the same time, in some cases, the strongest flashes were found to coincide with the appearance of signals of the GUV type (Geyser type ULF Variation). For almost three months of observations, five cases of quasi-periodic GUV series have been identified, four of which coincide completely or partially with the times of solar flares and magnetic storms. It should be noted that earlier in the literature, the appearance of these signals was not correlated with any physical process. At the same time, the identification of such patterns is an integral part of extensive work on identifying predictive signs of earthquake preparation in the ETP.
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•The effect of saponins on model lecithin and lecithin/cholesterol (4:1mol/mol) bilayers is described.•The bilayers were in the form of Giant Unilamellar Vesicles (GUV), Large ...Unilamellar Vesicles (LUV) and Supported Lipid Bilayers (SLB).•Four triterpene (α-hederin, hederacoside C, ammonium glycyrrhizate, Quillaja bark saponins (QBS)) and one steroid (digitonin) saponins were studied.•Fluorescence microscopy, dynamic light scattering and neutron reflectivity provided consistent results.•Cholesterol significantly enhances (for α-hederin) or triggers the attack of QBS and digitonin on model bilayers.
We describe the effect of four triterpene (α-hederin, hederacoside C, ammonium glycyrrhizate, Quillaja bark saponins (QBS)) and one steroid (digitonin) saponins on lecithin and lecithin-cholesterol (4:1mol/mol) model bilayers. For this purpose the Giant Unilamellar Vesicles (GUV), Large Unilamellar Vesicles (LUV) and Supported Lipid Bilayers (SLB) were probed with fluorescence microscopy, dynamic light scattering and neutron reflectivity, respectively. In most cases the results provided from the three techniques were consistent. In cholesterol-free systems, only α-hederin was able to penetrate SLB and induce macroscopic changes in LUV and GUV. Digitonin can adsorb to a small extent on the surface of cholesterol-free SLB, but does not affect macroscopically the LUV and GUV. In the presence of cholesterol, not only α-hederin but also QBS and digitonin affect the liposomes both microscopically and macroscopically. The present data clearly confirm that cholesterol significantly enhances the effect of membrane-active saponins on bilayers and that the effect on model bilayers correlates well with the membrane-activity of the saponins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The low membrane permeability of candidate drug molecules is a major challenge in drug development, and insufficient permeability is one reason for the failure of antibiotic treatment against ...bacteria. Quantifying drug transport across specific pathways in living systems is challenging because one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological pH and salt concentrations, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin and ciprofloxacin across biomimetic liposomes.
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