Nanoscale Membrane Curvature Research Articles

Nanoscale membrane curvature sorts lipid phases and alters lipid diffusion

The precise spatiotemporal control of nanoscale membrane shape and composition is the result of a complex interplay of individual and collective molecular behaviors. Here, we employed single-molecule localization microscopy and computational simulations to observe single-lipid diffusion and sorting in model membranes with varying compositions, phases, temperatures, and curvatures. Supported lipid bilayers were created over 50-nm-radius nanoparticles to mimic the size of naturally occurring membrane buds, such as endocytic pits and the formation of viral envelopes. The curved membranes recruited liquid-disordered lipid phases while altering the diffusion and sorting of tracer lipids. Disorder-preferring fluorescent lipids sorted to and experienced faster diffusion on the nanoscale curvature only when embedded in a membrane capable of sustaining lipid phase separation at low temperatures. The curvature-induced sorting and faster diffusion even occurred when the sample temperature was above the miscibility temperature of the planar membrane, implying that the nanoscale curvature could induce phase separation in otherwise homogeneous membranes. Further confirmation and understanding of these results are provided by continuum and coarse-grained molecular dynamics simulations with explicit and spontaneous curvature-phase coupling, respectively. The curvature-induced membrane compositional heterogeneity and altered dynamics were achieved only with a coupling of the curvature with a lipid phase separation. These cross-validating results demonstrate the complex interplay of lipid phases, molecular diffusion, and nanoscale membrane curvature that are critical for membrane functionality.

Membrane curvature-facilitated oncogenic RAS clustering in cells

As a dominant oncogenic protein, Ras GTPases are found segregated into nanoclusters on plasma membrane enabling downstream effector binding and signaling activation. Recent studies shed light on the stimulation effect of membrane curvature as a mechanical trigger for Ras accumulation. However, it remains an unclear picture of why Ras can respond to membrane curvature, especially in live cell environment. It is largely due to the lack of efficient tool to manipulate membrane curvature in cells. Here, we utilized the vertically-aligned nanobar arrays to study the regulation effect of nanoscale membrane curvature on oncogenic Ras clustering in live cells.

Coarse-Grained Simulations Suggest the Epsin N-Terminal Homology Domain Can Sense Membrane Curvature without Its Terminal Amphipathic Helix.

Nanoscale membrane curvature is a common feature in cell biology required for functions such as endocytosis, exocytosis and cell migration. These processes require the cytoskeleton to exert forces on the membrane to deform it. Cytosolic proteins contain specific motifs which bind to the membrane, connecting it to the internal cytoskeletal machinery. These motifs often bind charged phosphatidylinositol phosphate lipids present in the cell membrane which play significant roles in signaling. These lipids are important for membrane deforming processes, such as endocytosis, but much remains unknown about their role in the sensing of membrane nanocurvature by protein domains. Using coarse-grained molecular dynamics simulations, we investigated the interaction of a model curvature active protein domain, the epsin N-terminal homology domain (ENTH), with curved lipid membranes. The combination of anionic lipids (phosphatidylinositol 4,5-bisphosphate and phosphatidylserine) within the membrane, protein backbone flexibility, and structural changes within the domain were found to affect the domain’s ability to sense, bind, and localize with nanoscale precision at curved membrane regions. The findings suggest that the ENTH domain can sense membrane curvature without the presence of its terminal amphipathic α helix via another structural region we have denoted as H3, re-emphasizing the critical relationship between nanoscale membrane curvature and protein function.

Light-Inducible Generation of Membrane Curvature in Live Cells with Engineered BAR Domain Proteins.

Nanoscale membrane curvature is now understood to play an active role in essential cellular processes such as endocytosis, exocytosis, and actin dynamics. Previous studies have shown that membrane curvature can directly affect protein function and intracellular signaling. However, few methods are able to precisely manipulate membrane curvature in live cells. Here, we report the development of a new method of generating nanoscale membrane curvature in live cells that is controllable, reversible, and capable of precise spatial and temporal manipulation. For this purpose, we make use of Bin/Amphiphysin/Rvs (BAR) domain proteins, a family of well-studied membrane-remodeling and membrane-sculpting proteins. Specifically, we engineered two optogenetic systems, opto-FBAR and opto-IBAR, that allow light-inducible formation of positive and negative membrane curvature, respectively. Using opto-FBAR, blue light activation results in the formation of tubular membrane invaginations (positive curvature), controllable down to the subcellular level. Using opto-IBAR, blue light illumination results in the formation of membrane protrusions or filopodia (negative curvature). These systems present a novel approach for light-inducible manipulation of nanoscale membrane curvature in live cells.

Light-Inducible Generation of Membrane Curvature in Live Cells with Engineered Bar Domain Proteins

Nanoscale membrane curvature is now understood to play an active role in many subcellular processes. Studies of nanoscale membrane curvature effects on protein function thus far have primarily consisted of in vitro studies, as few methods are able to induce membrane curvature in live cells. A new method of generating nanoscale membrane curvature that is dynamic, reversible, and capable of stimulating a large population of cells is desired. Here, we report the development of two optogenetic systems: opto-FBAR and opto-IBAR. Derived from the human FBP17 and IRSp53 proteins, respectively, these systems allow us to induce the formation of positive and negative membrane curvature with fine spatiotemporal precision. In opto-FBAR, blue light activation results in the formation of membrane tubules, controllable down to the subcellular level. Using a modified image analysis program, we are able to quantify levels of plasma membrane tubulation in cells. opto-IBAR activation results in the formation or elongation of cellular membrane protrusions and filopodia. These systems present a novel approach for controlling nanoscale membrane curvature in live cells.

QUANTIFICATION OF MITOCHONDRIAL MEMBRANE CURVATURE BY THREE-DIMENSIONAL LOCALIZATION MICROSCOPY.

Mitochondrial membrane organization is important for many biological functions, and is implicated in a number of diseases, but conventional microscopy has insufficient resolution to image biologically relevant structures. We present methods to quantify nanoscale membrane curvature using three-dimensional localization-based super-resolution microscopy. Localizations are analyzed using a cluster algorithm followed by principal component analysis to determine local membrane curvature. Results are shown for mitochondria in C2C12 mouse myotubes labeled with Tom20-Dendra2.

Single-Nanometer Changes in Nanopore Geometry Influence Curvature, Local Properties, and Protein Localization in Membrane Simulations

Nanoporous surfaces are used in many applications in intracellular sensing and drug delivery. However, the effects of such nanostructures on cell membrane properties are still far from understood. Here, we use coarse-grained molecular dynamics simulations to show that nanoporous substrates can stimulate membrane-curvature effects and that this curvature-sensing effect is much more sensitive than previously thought. We define a series of design parameters for inducing a nanoscale membrane curvature and show that nanopore taper plays a key role in membrane deformation, elucidating a previously unexplored fabrication parameter applicable to many nanostructured biomaterials. We report significant changes in the membrane area per lipid and thickness at regions of curvature. Finally, we demonstrate that regions of the nanopore-induced membrane curvature act as local hotspots for an increased bioactivity. We show spontaneous binding and localization of the epsin N-terminal homology (ENTH) domain to the regions of curvature. Understanding this interplay between the membrane curvature and nanoporosity at the biointerface helps both explain recent biological results and suggests a pathway for developing the next generation of cell-active substrates.

Single-lipid tracking on nanoscale membrane buds: The effects of curvature on lipid diffusion and sorting

Nanoscale membrane curvature in cells is critical for endocytosis/exocytosis and membrane trafficking. However, the biophysical ramifications of nanoscale membrane curvature on the behavior of lipids remain poorly understood. Here, we created an experimental model system of membrane curvature at a physiologically-relevant scale and obtained nanoscopic information on single-lipid distributions and dynamics. Supported lipid bilayers were created over 50 and 70 nm radius nanoparticles to create membrane buds. Single-molecule localization microscopy was performed with diverse mixtures of fluorescent and non-fluorescent lipids. Variations in lipid acyl tales length, saturation, head-group, and fluorescent labeling strategy were tested while maintaining a single fluid lipid phase throughout the membrane. Monte Carlo simulations were used to fit our experimental results and quantify the effects of curvature on the lipid diffusion and sorting. Whereas varying the composition of the non-fluorescent lipids yielded minimal changes to the curvature effects, the labeling strategy of the fluorescent lipids yielded highly varying effects of curvature. Most conditions yield single-population Brownian diffusion throughout the membrane; however, curvature-induced lipid sorting, slowing, and aggregation were observed in some conditions. Head-group labeled lipids such as DPPE-Texas Red and POPE-Rhodamine diffused >2.4× slower on the curved vs. the planar membranes; tail-labeled lipids such as NBD-PPC, TopFluor-PPC, and TopFluor-PIP2, as well as DiIC12 and DiIC18 displayed no significant changes in diffusion due to the membrane curvature. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

Nanoscale Membrane Curvature Generated by Cholera Toxin Subunit B: The Effects of Lipid Cross-Linking and Lipid Phase

Nanoscale Membrane Curvature Generated by Cholera Toxin Subunit B: The Effects of Lipid Cross-Linking and Lipid Phase

Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility.

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity.

The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy

The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. The initiation of membrane bending occurs at a length scale that is irresolvable by most superresolution optical microscopy methods. Here, we report the development of polarized localization microscopy (PLM), a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting. PLM combines polarized total internal reflection fluorescence microscopy and single-molecule localization microscopy to reveal membrane orientation with subdiffraction-limited resolution without reducing localization precision by point spread function manipulation. Membrane curvature detection with PLM requires fewer localization events to detect curvature than three-dimensional single-molecule localization microscopy (e.g., photoactivated localization microscopy or stochastic optical reconstruction microscopy), which enables curvature detection 10× faster via PLM. With rotationally confined lipophilic fluorophores and the polarized incident fluorescence excitation, membrane-bending events are revealed with superresolution. Engineered hemispherical membrane curvature with a radius ≥24 nm was detected with PLM, and individual fluorophore localization precision was 13 ± 5 nm. Further, deciphering molecular mobility as a function of membrane topology was enabled. The diffusion coefficient of individual DiI molecules was 25 ± 5× higher in planar supported lipid bilayers than within nanoscale membrane curvature. Through the theoretical foundation and experimental demonstration provided here, PLM is poised to become a powerful technique for revealing the underlying biophysical mechanisms of membrane bending at physiological length scales.

Polarized Localization Microscopy Detects Membrane Curvature and Reveals the Interplay between Membrane Orientation and Protein Dynamics

Nanoscale membrane curvature is a necessary component of countless cellular processes. However, the understanding of these processes is limited by the experimental techniques and its inability to detect and resolve nanoscale membrane curvature. Here we present Polarized Localization Microscopy (PLM), a novel optical microscopy technique that detects and resolves membrane curvature with 15 nm resolution. PLM combines the advantages of polarized total internal reflection fluorescence microscopy (TIRF) and fluorescence localization microscopy to reveal single-fluorophore locations and orientations without sacrificing the localization precision of the point spread function. We demonstrate PLM capability of resolving membrane orientations by draping a supported lipid bilayer over polystyrene nanoparticles of varying sizes on a glass coverslip, thus creating a model membrane with coexisting flat and curved regions and membrane radii of curvature as small as 20 nm. Further, we introduce theoretical analysis that enables mapping out the membrane topology from the density of detected localizations in fluorescence excitation polarization. Using this approach, the interplay between membrane topology and cholera toxin subunit B (CTxB) dynamics was addressed. CTxB partitioned to curved membrane over the nanoparticle and further drove formation of lipid tubules. PLM detected the binding of an exocytosis protein complex to nanoscale membrane curvature. PLM was utilized to perform high throughput single particle tracking to reveal the correlated effects of membrane curvature, dynamics, and protein distribution. By providing super-resolution of membrane topology, PLM is able to reveal the critical components in various cellular processes such as endocytosis/exocytosis, viral infections, and neurotransmission.

Novel Experimental Methods to Resolve Nanoscale Membrane Organizaion and Curvature

The structures and functions of cell membranes are regulated by their diverse compositions, distinctive intermolecular interactions, and dynamic lateral organization. In particular, the combination of nanoscale membrane curvature and lateral organization of membrane components play a driving force in the initiation and regulation of intricate cellular processes such as endocytosis and exocytosis. However, many hypotheses concerning the nanoscale lipid redistribution in response to curvature are unanswered due to the diffraction limit of current optical imaging techniques. We aim to resolve nanoscale membrane curvature and the redistribution of proteins and lipids phase separated into liquid domains by inducing nanoscale curvature on giant unilamellar vesicles and novel microcopy methods. We achieve this through polarized total internal reflection fluorescence localization microscopy to yield sub-diffraction-limited resolution of membrane curvature and molecular organization. Polystyrene nanoparticles melted into agarose hydrogel substrates create a nanoengineered surface with small curved regions with controlled radii of curvature from 20 nm to 2 μm. These substrates work well with the evanescent field for total internal reflection microscopy because they are thin (<40 nm thick) and have a low index of refraction (<1.4). Micropipette aspiration techniques coupled with nanoengineered substrates enable dynamic control of membrane curvature and noticeable differences in lipid domain appearances. Preliminary data shows that when the vesicle is pressed against a surface, vesicles with coexisting liquid-­order and liquid-disorder phases demonstrate an increased propensity for the liquid-disordered phase to localize at the curved regions. Having the capability to resolve these previously irresolvable details allows us to better understand membrane dynamics and molecular sorting, in addition to exploring vital biological and disease related processes that are sensitive to membrane curvature.

Nucleation in mesoscopic systems under transient conditions: Peptide-induced pore formation in vesicles

Attachment of lytic peptides to the lipid membrane of virions or bacteria is often accompanied by their aggregation and pore formation, resulting eventually in membrane rupture and pathogen neutralization. The membrane rupture may occur gradually via formation of many pores or abruptly after the formation of the first pore. In academic studies, this process is observed during interaction of peptides with lipid vesicles. We present an analytical model and the corresponding Monte Carlo simulations focused on the pore formation in such situations. Specifically, we calculate the time of the first nucleation-limited pore-formation event and show the distribution of this time in the regime when the fluctuations of the number of peptides attached to a vesicle are appreciable. The results obtained are used to clarify the mechanism of the pore formation and membrane destabilization observed recently during interaction of highly active α-helical peptide with sub-100-nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature. The model proposed and the analysis presented are generic and may be applicable to other meso- and nanosystems.

Single Vesicle Analysis Reveals Nanoscale Membrane Curvature Selective Pore Formation in Lipid Membranes by an Antiviral α-Helical Peptide

Using tethered sub-100 nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature, we have in this work designed a total internal reflection fluorescence microscopy-based single vesicle assay to investigate how an antiviral amphipathic α-helical (AH) peptide interacts with lipid membranes to induce membrane curvature-dependent pore formation and membrane destabilization. Based on a combination of statistics from single vesicle imaging, binding kinetics data, and theoretical analysis, we propose a mechanistic model that is consistent with the experimentally observed peptide association and pore formation kinetics at medically relevant peptide concentrations (10 nM to 1 μM) and unusually low peptide-to-lipid (P/L) ratio (~1/1000). Importantly, the preference of the AH peptide to selectively rupture virions with sub-100 nm diameters appears to be related to membrane strain-dependent pore formation rather than to previously observed nanoscale membrane curvature facilitated binding of AH peptides. Compared to other known proteins and peptides, the combination of low effective P/L ratio and high specificity for nm-sized membrane curvature lends this particular AH peptide great potential to serve as a framework for developing a highly specific and potent antiviral agent for prophylactic and therapeutic applications while avoiding toxic side effects against host cell membranes.