Zheng Shi

Membrane tension and peripheral protein density mediate membrane shape transitions

Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation, and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature coupling proteins and therefore a wide range of endocytic proteins.

Exploiting imperfections in the bulk to direct assembly of surface colloids

We exploit the long-ranged elastic fields inherent to confined nematic liquid crystals to assemble colloidal particles trapped at the liquid crystal interface into reconfigurable structures with complex symmetries and packings. Spherical colloids with homeotropic anchoring trapped at the interface between air and the nematic liquid crystal 5CB create quadrupolar distortions in the director field causing particles to repel and consequently form close-packed assemblies with a triangular habit. Here we report on complex, open structures organized via interactions with defects in the bulk. Specifically, by confining the nematic liquid crystal in an array of microposts with homeotropic anchoring conditions, we cause defect rings to form at well-defined locations in the bulk of the sample. These defects source elastic deformations that direct the assembly of the interfacially-trapped colloids into ring-like assemblies, which recapitulate the defect geometry even when the microposts are completely immersed in the nematic. When the surface density of the colloids is high, they form a ring near the defect and a hexagonal lattice far from it. Since topographically complex substrates are easily fabricated and liquid crystal defects are readily reconfigured, this work lays the foundation for a new, robust mechanism to dynamically direct assembly over large areas by controlling surface anchoring and associated bulk defect structure.Significance In this research, we develop new means of directing colloids at an interface to assemble into complex configurations by exploiting defects in a liquid crystal (LC). Through confinement of a nematic LC over a topographically patterned surface, we demonstrate the formation of defects at precise locations in the LC bulk. These defects source elastic distortion fields that guide the assembly of colloids constrained to the LC–air interface. This work significantly extends prior work in which LCs confined in film or droplet geometries guide colloidal assembly beyond simple triangular lattices and chains. Here, we demonstrate colloidal assembly at precise locations, with particle-rich and -poor regions, determined remotely by defects deliberately seeded in the LC bulk. Experimental results are supported by numerical and analytical investigation. We exploit the long-ranged elastic fields inherent to confined nematic liquid crystals (LCs) to assemble colloidal particles trapped at the LC interface into reconfigurable structures with complex symmetries and packings. Spherical colloids with homeotropic anchoring trapped at the interface between air and the nematic LC 4-cyano-4′-pentylbiphenyl create quadrupolar distortions in the director field causing particles to repel and consequently form close-packed assemblies with a triangular habit. Here, we report on complex open structures organized via interactions with defects in the bulk. Specifically, by confining the nematic LC in an array of microposts with homeotropic anchoring conditions, we cause defect rings to form at well-defined locations in the bulk of the sample. These defects source elastic deformations that direct the assembly of the interfacially trapped colloids into ring-like assemblies, which recapitulate the defect geometry even when the microposts are completely immersed in the nematic. When the surface density of the colloids is high, they form a ring near the defect and a hexagonal lattice far from it. Because topographically complex substrates are easily fabricated and LC defects are readily reconfigured, this work lays the foundation for a versatile, robust mechanism to direct assembly dynamically over large areas by controlling surface anchoring and associated bulk defect structure.

Kinetics of Endophilin N-BAR Domain Dimerization and Membrane Interactions

Background: Endocytosis can involve dimerization and membrane association of the protein endophilin. Results: We found subnanomolar affinity for endophilin N-BAR dimerization. Membrane dissociation is substantially slower than association, and membrane-bound protein density-dependent. Conclusion: Endophilin binds membranes as dimers that subsequently oligomerize. Significance: Our findings illuminate the membrane binding mechanism of endophilin, which is important in understanding the regulation of membrane trafficking events. The recruitment to plasma membrane invaginations of the protein endophilin is a temporally regulated step in clathrin-mediated endocytosis. Endophilin is believed to sense or stabilize membrane curvature, which in turn likely depends on the dimeric structure of the protein. The dynamic nature of the membrane association and dimerization of endophilin is thus functionally important and is illuminated herein. Using subunit exchange Förster resonance energy transfer (FRET), we determine dimer dissociation kinetics and find a dimerization equilibrium constant orders of magnitude lower than previously published values. We characterize N-BAR domain membrane association kinetics under conditions where the dimeric species predominates, by stopped flow, observing prominent electrostatic sensitivity of membrane interaction kinetics. Relative to membrane binding, we find that protein monomer/dimer species equilibrate with far slower kinetics. Complementary optical microscopy studies reveal strikingly slow membrane dissociation and an increase of dissociation rate constant for a construct lacking the amphipathic segment helix 0 (H0). We attribute the slow dissociation kinetics to higher-order protein oligomerization on the membrane. We incorporate our findings into a kinetic scheme for endophilin N-BAR membrane binding and find a significant separation of time scales for endophilin membrane binding and subsequent oligomerization. This separation may facilitate the regulation of membrane trafficking phenomena.

Mutations in BIN1 associated with centronuclear myopathy disrupt membrane remodeling by affecting protein density and oligomerization.

The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease.

Biophysics of α-synuclein induced membrane remodelling.

α-Synuclein is an intrinsically disordered protein whose aggregation is a hallmark of Parkinsons disease. In neurons, α-synuclein is thought to play important roles in mediating both endo- and exocytosis of synaptic vesicles through interactions with either the lipid bilayer or other proteins. Upon membrane binding, the N-terminus of α-synuclein forms a helical structure and inserts into the hydrophobic region of the outer membrane leaflet. However, membrane structural changes induced by α-synuclein are still largely unclear. Here we report a substantial membrane area expansion induced by the binding of α-synuclein monomers. This measurement is accomplished by observing the increase of membrane area during the binding of α-synuclein to pipette-aspirated giant vesicles. The extent of membrane area expansion correlates linearly with the density of α-synuclein on the membrane, revealing a constant area increase induced by the binding per α-synuclein molecule. The area expansion per synuclein is found to exhibit a strong dependence on lipid composition, but is independent of membrane tension and vesicle size. Fragmentation or tubulation of the membrane follows the membrane expansion process. However, contrary to BAR domain proteins, no distinct tubulation-transition density can apparently be identified for α-synuclein, suggesting a more complex membrane curvature generation mechanism. Consideration of α-synucleins membrane binding free energy and biophysical properties of the lipid bilayer leads us to conclude that membrane expansion by α-synuclein results in thinning of the bilayer. These membrane thinning and tubulation effects may underlie α-synucleins role in mediating cell trafficking processes such as endo- and exocytosis.

Ring around the colloid

In this work, we show that Janus washers, genus-one colloids with hybrid anchoring conditions, form topologically required defects in nematic liquid crystals. Experiments under crossed polarizers reveal the defect structure to be a rigid disclination loop confined within the colloid, with an accompanying defect in the liquid crystal. When confined to a homeotropic cell, the resulting colloid-defect ring pair tilts relative to the far field director, in contrast to the behavior of toroidal colloids with purely homeotropic anchoring. We show that this tilting behavior can be reversibly suppressed by the introduction of a spherical colloid into the center of the toroid, creating a new kind of multi-shape colloidal assemblage.

DNA Island Formation on Binary Block Copolymer Vesicles

Here, we report DNA-induced polymer segregation and DNA island formation in binary block copolymer assemblies. A DNA diblock copolymer of polymethyl acrylate-block-DNA (PMA-b-DNA) and a triblock copolymer of poly(butadiene)-block-poly(ethylene oxide)-block-DNA (PBD-b-PEO-b-DNA) were synthesized, and each was coassembled with a prototypical amphiphilic polymer of poly(butadiene)-block-poly(ethylene oxide) (PBD-b-PEO). The binary self-assembly of PMA-b-DNA and PBD-b-PEO resulted in giant polymersomes with DNA uniformly distributed in the hydrophilic PEO shell. When giant polymersomes were connected through specific DNA interactions, DNA block copolymers migrated to the junction area, forming DNA islands within polymersomes. These results indicate that DNA hybridization can induce effective lateral polymer segregation in mixed polymer assemblies. The polymer segregation and local DNA enrichment have important implications in DNA melting properties, as mixed block copolymer assemblies with low DNA block copolymer contents can still exhibit useful DNA melting properties that are characteristic of DNA nanostructures with high DNA density.

Bar Domain Proteins can Differ Substantially in their Capacity to Generate Membrane Curvature

BAR (Bin/amphiphysin/Rvs) domains bind to and reshape cell membranes. Numerous BAR proteins are known to participate in endocytosis pathways. Whether their function is redundant or specifically different has remained unclear. Here we first compared the membrane curvature generation abilities of three N-BAR domains involved in endocytosis: endophilin, amphiphysin and SNX9. For this purpose, we used a membrane shape stability assay recently developed in our lab that allows determination of shape stability diagrams obtained via pipette aspirated giant unilamellar vesicles (GUVs). Significantly different membrane stability phase diagrams were observed for these three proteins, suggesting their functional non-redundant. We further compared the membrane binding and curvature generating of endophilin with an F-BAR protein: FCHo2, which arrives at clathrin coated pits (CCPs) earlier than endophilin. We observed interesting competitive membrane binding between FCHo2 and endophilin, as well as stronger curvature induction abilities of FCHo2 when compared with endophilin. These observations suggest a membrane binding regulation mechanism by the BAR domains, and promising to explain why FCHo2 act as the nucleator of the CCPs.

Membrane Shape Transition Mediated by Curvature-Inducing Proteins, Membrane Tension, and Macrocrowders

Classical BAR (Bin/amphiphysin/Rvs) domains and their distant relatives, I-BAR domains, can bind to and reshape membranes in vitro and in vivo through their intrinsic “crescent-shaped” and “zeppelin-shaped” homodimeric structure. However, the mechanisms by which plasma membrane intrusions induced by classical BAR domains, or extrusions induced by I-BAR domains, are barely studied. Here we used a GUV membrane shape stability assay to quantitatively investigate the key factors regulating BAR- and I-BAR-induced membrane shape transition processes. We found that the IMD-induced membrane curvature transition depends on protein density and membrane tension, as we found earlier for the endophilin N-BAR and full-length protein. Furthermore, we demonstrated that macromolecular crowding on the membrane surface significantly influences the BAR proteins’ ability to induce membrane curvature changes.