Control of synaptic vesicle release probability via VAMP4 targeting to endolysosomes
VAMP4 is a molecular rheostat that adjusts neurotransmitter release to both the activity and degradative status of neurons.
Ivanova, Daniela↗
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The effect of low temperature on poly(3-methyl- N -vinylcaprolactam)- b -poly( N -vinylpyrrolidone) diblock copolymer nanovesicles assembled from all-aqueous media
Nanosized polymeric vesicles (polymersomes) self-assembled from double hydrophilic copolymers of poly(3-methyl-N-vinylcaprolactam) n -b-poly(N-vinylpyrrolidone) m (PMVC n -b-PVPON m ) using all aqueous media are a promising platform for biomedical applications, because of their superior stability over liposomes in vivo and high loading capacity. Herein, we explored the temperature-sensitive behavior of PMVC 58 -b-PVPON 65 vesicles using transmission electron microscopy (TEM), dynamic light scattering (DLS), atomic force microscopy (AFM), and small-angle neutron scattering (SANS) in response to lowering the solution temperature from 37 to 25, 20, 14 and 4 °C. The copolymer vesicles with an average size of 350 nm at 37 °C were assembled from the diblock copolymer dissolved in aqueous solution at 4 °C. We show that while the polymersome's size gradually decreases upon the temperature decrease from 37 to 4 °C, the average shell thickness increases from 17 nm to 25 nm, respectively. SANS study revealed that the PMVC 58 -b-PVPON 65 vesicle undergoes a gradual structure evolution from a dense-shell vesicle at 37–25 °C to a highly-hydrated shell vesicle at 20–14 °C to molecular chain aggregates at 4 °C. From SANS contrast matching study, this vesicle behavior is found to be driven by the gradual rehydration of PMVC block at 37–14 °C. The shell hydration at 20–14 °C also correlated with the 4.4-fold decrease in the relative fluorescence intensity from vesicle-encapsulated fluorescent dye, indicating ~80% of the dye release within 12 hours after the vesicle exposure to 14 °C. No significant (<5%) dye release was observed for the vesicle solutions at 37–20 °C, indicating excellent cargo retention inside the vesicles. Our study provides new fundamental insights on temperature-sensitive polymer vesicles and demonstrates that the copolymer assembly into polymersomes can be achieved by decreasing a copolymer aqueous solution temperature below 14 °C followed by solution exposure to ≥20 °C. This type of all-aqueous assembly, instead of nanoprecipitation from organic solvents or solvent exchange, can be highly desirable for encapsulating a wide range of biological molecules, including proteins, peptides, and nucleic acids, into stable polymer vesicles without a need for organic solvents for dissolution of the copolymers that are amphiphilic at physiologically relevant temperatures of 20–37 °C.
36 MATERIALS SCIENCE↗
Mafic explosive volcanism at Llaima Volcano: 3D x-ray microtomography reconstruction of pyroclasts to constrain shallow conduit processes
Abstract Mafic volcanic activity is dominated by effusive to mildly explosive eruptions. Plinian and ignimbrite-forming mafic eruptions, while rare, are also possible; however, the conditions that promote such explosivity are still being explored. Eruption style is determined by the ability of gas to escape as magma ascends, which tends to be easier in low-viscosity, mafic magmas. If magma permeability is sufficiently high to reduce bubble overpressure during ascent, volatiles may escape from the magma, inhibiting violent explosive activity. In contrast, if the permeability is sufficiently low to retain the gas phase within the magma during ascent, bubble overpressure may drive magma fragmentation. Rapid ascent may induce disequilibrium crystallization, increasing viscosity and affecting the bubble network with consequences for permeability, and hence, explosivity. To explore the conditions that promote strongly explosive mafic volcanism, we combine microlite textural analyses with synchrotron x-ray computed microtomography of 10 pyroclasts from the 12.6 ka mafic Curacautín Ignimbrite (Llaima Volcano, Chile). We quantify microlite crystal size distributions (CSD), microlite number densities, porosity, bubble interconnectivity, bubble number density, and geometrical properties of the porous media to investigate the role of magma degassing processes at mafic explosive eruptions. We use an analytical technique to estimate permeability and tortuosity by combing the Kozeny-Carman relationship, tortuosity factor, and pyroclast vesicle textures. The groundmass of our samples is composed of up to 44% plagioclase microlites, > 85% of which are < 10 µm in length. In addition, we identify two populations of vesicles in our samples: (1) a convoluted interconnected vesicle network produced by extensive coalescence of smaller vesicles (> 99% of pore volume), and (2) a population of very small and completely isolated vesicles (< 1% of porosity). Computed permeability ranges from 3.0 × 10 −13 to 6.3 × 10 −12 m 2 , which are lower than the similarly explosive mafic eruptions of Tarawera (1886; New Zealand) and Etna (112 BC; Italy). The combination of our CSDs, microlite number densities, and 3D vesicle textures evidence rapid ascent that induced high disequilibrium conditions, promoting rapid syn-eruptive crystallization of microlites within the shallow conduit. We interpret that microlite crystallization increased viscosity while simultaneously forcing bubbles to deform as they grew together, resulting in the permeable by highly tortuous network of vesicles. Using the bubble number densities for the isolated vesicles (0.1-3 −3 × 10 4 bubbles per mm 3 ), we obtain a minimum average decompression rate of 1.4 MPa/s. Despite the textural evidence that the Curacautín magma reached the percolation threshold, we propose that rapid ascent suppressed outgassing and increased bubble overpressures, leading to explosive fragmentation. Further, using the porosity and permeability of our samples, we estimated that a bubble overpressure > 5 MPa could have been sufficient to fragment the Curacautín magma. Other mafic explosive eruptions report similar disequilibrium conditions induced by rapid ascent rate, implying that syn-eruptive disequilibrium conditions may control the explosivity of mafic eruptions more generally.
Valdivia, Pedro↗
Poly( N -vinylpyrrolidone)- block -Poly(dimethylsiloxane)- block -Poly( N -vinylpyrrolidone) Triblock Copolymer Polymersomes for Delivery of PARP1 siRNA to Breast Cancers
Nearly 20% of HER2-positive breast cancers develop resistance to HER2-targeted therapies requiring the use of advanced therapies. Silencing RNA therapy may be a powerful modality for treating resistant HER2 cancers due to its high specificity and low toxicity. However, the systemic administration of siRNAs requires a safe and efficient delivery platform because of siRNA’s low stability in physiological fluids, inefficient cellular uptake, immunoreactivity, and rapid clearance. We have developed theranostic polymeric vesicles to overcome these hurdles for encapsulation and delivery of small functional molecules and PARP1 siRNA for in vivo delivery to breast cancer tumors. The 100 nm polymer vesicles were assembled from biodegradable and non-ionic poly(N-vinylpyrrolidone) 14 -block-poly(dimethylsiloxane) 47 -block-poly(N-vinylpyrrolidone) 14 triblock copolymer PVPON 14 -PDMS 47 -PVPON 14 using nanoprecipitation and thin-film hydration. We demonstrated that the vesicles assembled from the copolymer covalently tagged with the Cy5.5 fluorescent dye for in vivo imaging could also encapsulate the model drug with high loading efficiency (40%). The dye-loaded vesicles were accumulated in tumors after 18 h circulation in 4TR breast tumor-bearing mice via passive targeting. We found that PARP1 siRNA encapsulated into the vesicles was released intact (13%) into solution by the therapeutic ultrasound treatment as quantified by gel electrophoresis. Additionally, the PARP1 siRNA-loaded polymersomes inhibited the proliferation of MDA-MB-361TR cells by 34% after 6 days of treatment by suppressing the NF-kB signaling pathway, unlike their scrambled siRNA-loaded counterparts. Finally, the treatment by PARP1 siRNA-loaded vesicles prolonged the survival of the mice bearing 4T1 breast cancer xenografts, with the 4-fold survival increase, unlike the untreated mice after 3 weeks following the treatment. These biodegradable, non-ionic PVPON 14 -PDMS 47 -PVPON 14 polymeric nanovesicles capable of the efficient encapsulation and delivery of PARP1 siRNA to successfully knock down PARP1 in vivo can provide an advanced platform for the development of precision-targeted therapeutic carriers, which could help develop highly effective drug delivery nanovehicles for breast cancer gene therapy.
60 APPLIED LIFE SCIENCES↗
Aqueous Self‐Assembly of Cylindrical and Tapered Bottlebrush Block Copolymers
The self‐assembly of amphiphilic bottlebrush block copolymers (BCPs), featuring backbones densely grafted with two types of side chains, is less well understood compared to linear BCPs. In particular, the solution self‐assembly of tapered bottlebrush BCPs—cone‐shaped BCPs with hydrophilic or hydrophobic tips—remains unexplored. This study investigates eight tapered and four cylindrical bottlebrush BCPs with varied ratios of hydrophobic polystyrene (PS) and hydrophilic poly(acrylic acid) (PAA) side chains, synthesized via sequential addition of macromonomers using ring‐opening metathesis polymerization (SAM‐ROMP). Self‐assembled nanostructures formed in water were analyzed using cryogenic transmission electron microscopy, small‐angle neutron scattering, and dynamic light scattering. Most BCPs generated multiple nanostructures with surface protrusions, including spherical micelles, cylindrical micelles, and vesicles, alongside transitional forms like ellipsoids and semi‐vesicles. Coarse‐grained molecular dynamics simulations supported the experimental findings, which revealed two distinct self‐assembly pathways. The first involved micelle fusion, producing elliptical and cylindrical aggregates, sometimes forming Y‐junctions. The second pathway featured micelle maturation into semivesicles, which developed into vesicles or large compound vesicles. This work provides the first experimental evidence of vesicle formation via semivesicles in bottlebrush BCPs and demonstrates the significant influence of cone directionality on self‐assembly behavior in these cone‐shaped polymeric amphiphiles.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Sculpting 2D Crystals via Membrane Contractions before and during Solidification
When phospholipids crystallize within the otherwise fluid membranes of giant unilamellar vesicles, the resulting molecularly thin “2D” solids exhibit great variety in their morphology evolution. For example, within membranes containing moderate amounts of the crystallizing component, crystals grow with a fixed morphology depending on vesicle size. Conversely for membranes containing large amounts of the crystallizing species, we find small compact crystals on vesicles of all sizes. However, on large vesicles, growing crystals sprout flower petals that lengthen progressively. These behaviors result from two combined mechanisms: first, like other 2D solids, the shear rigidity of phospholipid crystals renders them intolerant to morphologies with nonzero Gaussian curvature. As a result and especially at elevated membrane tension, the cost of bending elasticity is reduced at the expense of line energy by the formation of flowers as opposed to compact crystals. Second, the composition-dependent tension rise during cooling relaxes via water permeation of the membrane with a time constant scaling as R2. The amount of crystal formed for a small decrease in temperature determines this composition-dependent increase in stress from thermal contractions versus solidification. Surface Evolver computations were motivated using the predicted tension evolution to develop a processing space that maps to experimental observations for initial and growing crystal morphology. Important variable groups are identified, including a scaled ratio of bending to line energy, a vesicle-size-independent group for membrane contractions, and a time constant for stress relaxation. Though processing stresses ultimately relax, the crystal morphology persists well beyond the processing window.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Stable nanovesicles formed by intrinsically planar bilayers
Quatsome nanovesicles, formed through the self-assembly of cholesterol (CHOL) and cetyltrimethylammonium bromide (CTAB) in water, have shown long-term stability in terms of size and morphology, while at the same time exhibiting high CHOL-CTAB intermolecular binding energies. We hypothesize that CHOL/CTAB quatsomes are indeed thermodynamically stable nanovesicles, and investigate the mechanism underlying their formation. A systematic study was performed to determine whether CHOL/CTAB quatsomes satisfy the experimental requisites of thermodynamically stable vesicles. Coarse-grain molecular dynamics simulations were used to investigate the molecular organization in the vesicle membrane, and the characteristics of the simulated vesicle were corroborated with experimental data obtained by cryo–electron microscopy, small- and wide-angle X-ray scattering, and multi-angle static light scattering. CHOL/CTAB quatsomes fulfill the requisites of thermodynamically stable nanovesicles, but they do not exhibit the classical membrane curvature induced by a composition asymmetry between the bilayer leaflets, like catanionic nanovesicles. Instead, CHOL/CTAB quatsomes are formed through the association of intrinsically planar bilayers in a faceted vesicle with defects, indicating that distortions in the organization and orientation of molecules can play a major role in the formation of thermodynamically stable nanovesicles.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Standalone Block Copolymer Nanoballoons: Decoupling Self-Assembly from Implementation in Nanomanufacturing
Here we report a facile method to produce isolatable hollow-core elastomeric vesicles, “nanoballoons”, prepared via block copolymer self-assembly in a polymer blend. Poly(isoprene-block-dimethylsiloxane) (PI-PDMS) diblock copolymers are blended with PDMS homopolymers (h-PDMS) as a “solvent” phase to template the self-assembly of PDMS-tethered vesicles with PI walls. The walls are subsequently crosslinked to yield mechanically stabilized elastomeric vesicles. The h-PDMS inner-core and matrix are separated from the vesicles by dialysis to yield the matrix-free nanoballoons. These objects, 0.3 – 1 μm in diameter, can be further reincorporated into a crosslinkable PDMS. Throughout the self-assembly, recovery, and reincorporation processes we apply several techniques including solvent dispersion/dynamic light scattering (DLS) measurements, transmission electron microscopy (TEM)/energy-dispersive X-ray spectroscopy (EDS), and small-angle X-ray scattering (SAXS) to provide a consilient body of evidence that the nanoballoon morphology is retained. Furthermore, this work presents advanced nanomanufacturing schema that illustrate the decoupling of the thermodynamic and dynamic factors that govern macromolecular self-assembly from the environment in which the self-assembled objects are deployed.
36 MATERIALS SCIENCE↗
Effect of hydration on morphology of thin phosphonate block copolymer electrolyte membranes studied by electron tomography
The morphological changes of phosphonate polypeptoid electrolyte membranes, poly-N-(2-ethyl)hexylglycine-block-poly-N-phosphonomethylglycine (pNeh m -b-pNpm n ), in hydrated and dry states were characterized by cryogenic transmission electron microscopy (cryo-TEM) and cryogenic electron tomography (cryo-ET). The analysis of 3D tomograms revealed that the pNeh 9 -b-pNpm 9 thin films absorbed a large amount of water, resulting in the formation of membranes that were nearly flat and giant multicompartment vesicles dispersed in the water phase. A simple lamellar phase appeared when the films were dried. In contrast, pNeh 18 -b-pNpm 18 thin films absorbed little water and formed small highly curved unilamellar and multilamellar vesicles. Water was located mainly outside the closely-packed vesicles. When water was removed by drying, the walls of adjacent vesicles collapsed to form honeycomb-like capsules. Here, the changes in domain size reflected changes in chain conformations. The pNpm9 blocks were saturated by water and fully extended, while pNpm 18 blocks were neither saturated by water nor fully extended. In addition, the thicknesses of hydrophobic blocks in the hydrated films of both pNeh 9 -b-pNpm 9 and pNeh 18 -b-pNpm 18 were smaller than those in the dry films, reflecting an increase of the average distance between the neighboring junctions of polypeptoid molecules.
36 MATERIALS SCIENCE↗
Complex motion of steerable vesicular robots filled with active colloidal rods
Abstract While the collective motion of active particles has been studied extensively, effective strategies to navigate particle swarms without external guidance remain elusive. We introduce a method to control the trajectories of two-dimensional swarms of active rod-like particles by confining the particles to rigid bounding membranes (vesicles) with non-uniform curvature. We show that the propelling agents spontaneously form clusters at the membrane wall and collectively propel the vesicle, turning it into an active superstructure. To further guide the motion of the superstructure, we add discontinuous features to the rigid membrane boundary in the form of a kinked tip, which acts as a steering component to direct the motion of the vesicle. We report that the system’s geometrical and material properties, such as the aspect ratio and Péclet number of the active rods as well as the kink angle and flexibility of the membrane, determine the stacking of active particles close to the kinked confinement and induce a diverse set of dynamical behaviors of the superstructure, including linear and circular motion both in the direction of, and opposite to, the kink. From a systematic study of these various behaviors, we design vesicles with switchable and reversible locomotions by tuning the confinement parameters. The observed phenomena suggest a promising mechanism for particle transportation and could be used as a basic element to navigate active matter through complex and tortuous environments.
42 ENGINEERING↗
Visualizing subcellular rearrangements in intact β cells using soft x-ray tomography
Characterizing relationships between cell structures and functions requires mesoscale mapping of intact cells showing subcellular rearrangements following stimulation; however, current approaches are limited in this regard. Here, we report a unique application of soft x-ray tomography to generate three-dimensional reconstructions of whole pancreatic β cells at different time points following glucose-stimulated insulin secretion. Reconstructions following stimulation showed distinct insulin vesicle distribution patterns reflective of altered vesicle pool sizes as they travel through the secretory pathway. Our results show that glucose stimulation caused rapid changes in biochemical composition and/or density of insulin packing, increased mitochondrial volume, and closer proximity of insulin vesicles to mitochondria. Costimulation with exendin-4 (a glucagon-like peptide-1 receptor agonist) prolonged these effects and increased insulin packaging efficiency and vesicle maturation. This study provides unique perspectives on the coordinated structural reorganization and interactions of organelles that dictate cell responses.
59 BASIC BIOLOGICAL SCIENCES↗
Small-Angle Neutron Scattering for Studying Lipid Bilayer Membranes
Small-angle neutron scattering (SANS) is a powerful tool for studying biological membranes and model lipid bilayer membranes. The length scales probed by SANS, being from 1 nm to over 100 nm, are well-matched to the relevant length scales of the bilayer, particularly when it is in the form of a vesicle. However, it is the ability of SANS to differentiate between isotopes of hydrogen as well as the availability of deuterium labeled lipids that truly enable SANS to reveal details of membranes that are not accessible with the use of other techniques, such as small-angle X-ray scattering. In this work, an overview of the use of SANS for studying unilamellar lipid bilayer vesicles is presented. The technique is briefly presented, and the power of selective deuteration and contrast variation methods is discussed. Approaches to modeling SANS data from unilamellar lipid bilayer vesicles are presented. Finally, recent examples are discussed. While the emphasis is on studies of unilamellar vesicles, examples of the use of SANS to study intact cells are also presented.
59 BASIC BIOLOGICAL SCIENCES↗
A bacterial membrane sculpting protein with BAR domain-like activity
Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature. Cryo-TEM reveals that a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubules or narrow chains produced by the wild-type strain. Overexpression of BdpA promotes OME formation during planktonic growth of S. oneidensis where they are not typically observed. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli . Based on the ability of BdpA to alter membrane architecture in vivo, we propose that BdpA and its homologs comprise a newly identified class of bacterial BAR domain-like proteins.
60 APPLIED LIFE SCIENCES↗
Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection
Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA. We show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. Early phage genes are expressed from the EPI vesicle, demonstrating its functionality as a prokaryotic, transcriptionally active, membrane-bound organelle. We also show that the phage nucleus is essential, with genome replication beginning after the injected DNA is transferred from the EPI vesicle to the phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.
59 BASIC BIOLOGICAL SCIENCES↗
Programmable Aggregation of Artificial Cells with DNA Signals
Cell aggregation is a complex behavior, which is closely related to the viability, differentiation, and migration of cells. An effort to create synthetic analogs could lead to considerable advances in cell physiology and biophysics. Rendering and modulating such a dynamic artificial cell system require mechanisms for receiving, transducing, and transmitting intercellular signals, yet effective tools are limited at present. Here we construct synthetic cells from engineered lipids and show their programmable aggregation behaviors using DNA oligonucleotides as a signaling molecule. The artificial cells have transmembrane channels made of DNA origami that are used to recognize and process intercellular signals. We demonstrate that multiple small vesicles aggregate onto a giant vesicle after a transduction of external DNA signals by an intracellular enzyme, and that the small vesicles dissociate when receiving ‘release’ signals. Furthermore, this work provides new possibilities for building synthetic protocells capable of chemical communication and coordination.
59 BASIC BIOLOGICAL SCIENCES↗
Influence of NaCl on shape deformation of polymersomes
Polymersomes frequently appear in the literature as promising candidates for a wide range of applications from targeted drug delivery to nanoreactors. From a cell mimetic point of view, it is important to understand the size and shape changes of the vesicles in the physiological environment since that can influence the drug delivery mechanism. In this work we studied the structural features of polymersomes consisting of poly(ethylene glycol)–poly(dimethylsiloxane)–poly(ethylene glycol) at the nanoscopic length scale in the presence of NaCl, which is a very common molecule in the biotic aqueous environment. Here, we used dynamic light scattering (DLS), cryo-TEM, small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS). We observed transformation of polymersomes from spherical to elongated vesicles at low salt concentration and into multivesicular structures at high salt concentration. Model fitting analysis of SANS data indicated a reduction of vesicle radius up to 47% and from the SAXS data we observed an increase in membrane thickness up to 8% and an increase of the PDMS hydrophobic segment up to 11% indicating stretching of the membrane due to osmotic imbalance. Also, from the increase in the interlamellar repeat distance up to 98% under high salt concentrations, we concluded that the shape and structural changes observed in the polymersomes are a combined result of osmotic pressure change and ion–membrane interactions.
36 MATERIALS SCIENCE↗
An intensity-based post-processing tool for 3D instance segmentation of organelles in soft X-ray tomograms
Investigating the 3D structures and rearrangements of organelles within a single cell is critical for better characterizing cellular function. Imaging approaches such as soft X-ray tomography have been widely applied to reveal a complex subcellular organization involving multiple inter-organelle interactions. However, 3D segmentation of organelle instances has been challenging despite its importance in organelle characterization. Here we propose an intensity-based post-processing tool to identify and separate organelle instances. Our tool separates sphere-like (insulin vesicle) and columnar-shaped organelle instances (mitochondrion) based on the intensity of raw tomograms, semantic segmentation masks, and organelle morphology. We validate our tool using synthetic tomograms of organelles and experimental tomograms of pancreatic β -cells to separate insulin vesicle and mitochondria instances. As compared to the commonly used connected regions labeling, watershed, and watershed + Gaussian filter methods, our tool results in improved accuracy in identifying organelles in the synthetic tomograms and an improved description of organelle structures in β -cell tomograms. In addition, under different experimental treatment conditions, significant changes in volumes and intensities of both insulin vesicle and mitochondrion are observed in our instance results, revealing their potential roles in maintaining normal β -cell function. Our tool is expected to be applicable for improving the instance segmentation of other images obtained from different cell types using multiple imaging modalities.
59 BASIC BIOLOGICAL SCIENCES↗