Peptide-Induced Fusion of Dynamic Membrane Nanodomains: Implications in a Viral Entry.

Enveloped viruses infect host cells via protein-mediated membrane fusion. However, insights into the microscopic rearrangement induced by the viral proteins and peptides have not yet emerged. Here, we report a new methodology to extract viral fusion peptide (FP)-mediated biomembrane dynamical nanodomain fusion parameter, λ, based on stimulated emission depletion microscopy coupled with fluorescence correlation spectroscopy. We also define another dynamical parameter membrane gradient, defined in terms of the ratio of average lipid diffusion coefficients across dynamic crossover length scales, ξ. Significantly, we observe that λ as well as these mobility gradients are larger in the stiffer liquid-ordered (Lo) phase compared to the liquid-disordered phase and are more effective at the smaller nanodomain interfaces, which are only present in the Lo phase. The results could possibly help to resolve a long-standing puzzle about the enhanced fusogenicity of FP in the Lo phase. Results obtained from the diffusion results have been correlated with the human immunodeficiency virus gp41 FP-induced membrane fusion.

Interactions of Surfactants with the Bacterial Cell Wall and Inner Membrane: Revealing the Link between Aggregation and Antimicrobial Activity.

Surfactants with their intrinsic ability to solubilize lipid membranes are widely used as antibacterial agents, and their interactions with the bacterial cell envelope are complicated by their differential aggregation tendencies. We present a combined experimental and molecular dynamics investigation to unravel the molecular basis for the superior antimicrobial activity and faster kill kinetics of shorter-chain fatty acid surfactant, laurate, when compared with the longer-chain surfactants studied in contact time assays with live Escherichia coli (E. coli). From all-atom molecular dynamics simulations, translocation events across peptidoglycan were the highest for laurate followed by sodium dodecyl sulfate, myristate, palmitate, oleate, and stearate. The translocation kinetics were positively correlated with the critical micellar concentration, which determined the free monomer surfactant concentration available for translocation across peptidoglycan. Interestingly, aggregates showed a lower propensity to translocate across the peptidoglycan layer and longer translocation times were observed for oleate, thereby revealing an intrinsic sieving property of the bacterial cell wall. Molecular dynamics simulations with surfactant-incorporated bacterial inner membranes revealed the greatest hydrophobic mismatch and membrane thinning in the presence of laurate when compared with the other surfactants. The enhanced antimicrobial efficacy of laurate over oleate was further verified by experiments with giant unilamellar vesicles, and electroporation molecular dynamics simulations revealed greater inner membrane poration tendency in the presence of laurate when compared with the longer-chain surfactants. Our study provides molecular insights into surfactant translocation across peptidoglycan and chain length-induced structural disruption of the inner membrane, which correlate with contact time kill efficacies observed as a function of chain length with E. coli. The insights gained from our study uncover unexplored barrier properties of the bacterial cell envelope to rationalize the development of antimicrobial formulations and therapeutics.

Compressibility of Multicomponent, Charged Model Biomembranes Tunes Permeation of Cationic Nanoparticles.

Cells respond to external stress by altering their membrane lipid composition to maintain fluidity, integrity and net charge. However, in interactions with charged nanoparticles (NPs), altering membrane charge could adversely affect its ability to transport ions across the cell membrane. Hence, it is important to understand possible pathways by which cells could alter zwitterionic lipid composition to respond to NPs without compromising membrane integrity and charge. Here, we report in situ synchrotron X-ray reflectivity (XR) measurements to monitor the interaction of cationic NPs in the form of quantum dots, with phase-separated supported lipid bilayers of different compositions containing an anionic lipid and zwitterionic lipids having variable degrees of stiffness. We observe that the extent of NP penetration into the respective membranes, as estimated from XR data analysis, is inversely related to membrane compression moduli, which was tuned by altering the stiffness of the zwitterionic lipid component. For a particular membrane composition with a discernible height difference between ordered and disordered phases, we were able to observe subtle correlations between the extent of charge on the NPs and the specificity to bind to the charged and ordered phase, contrary to that observed earlier for phase-separated model biomembranes containing no charged lipids. Our results provide microscopic insight into the role of membrane rigidity and electrostatics in determining membrane permeation. This can lead to great potential benefits in rational designing of NPs for bioimaging and drug delivery applications as well as in assessing and alleviating cytotoxicity of NPs.

Penetration and preferential binding of charged nanoparticles to mixed lipid monolayers: interplay of lipid packing and charge density.

Designing of nanoparticles (NPs) for biomedical applications or mitigating their cytotoxic effects requires microscopic understanding of their interactions with cell membranes. Such insight is best obtained by studying model biomembranes which, however, need to replicate actual cell membranes, especially their compositional heterogeneity and charge. In this work we have investigated the role of lipid charge density and packing of phase separated Langmuir monolayers in the penetration and phase specificity of charged quantum dot (QD) binding. Using an ordered and anionic charged lipid in combination with uncharged but variable stiffness lipids we demonstrate how the subtle interplay of zwitterionic lipid packing and anionic lipid charge density can affect cationic nanoparticle penetration and phase specific binding. Under identical subphase pH, the membrane with higher anionic charge density displays higher NP penetration. We also observe coalescence of charged lipid rafts floating amidst a more fluidic zwitterionic lipid matrix due to the phase specificity of QD binding. Our results suggest effective strategies which can be used to design NPs for diverse biomedical applications as well as to devise remedial actions against their harmful cytotoxic effects especially against respiratory diseases.

Assessing Barriers for Antimicrobial Penetration in Complex Asymmetric Bacterial Membranes: A Case Study with Thymol.

The bacterial cell envelope is a complex multilayered structure evolved to protect bacteria in hostile environments. An understanding of the molecular basis for the interaction and transport of antibacterial therapeutics with the bacterial cell envelope will enable the development of drug molecules to combat bacterial infections and suppress the emergence of drug-resistant strains. Here we report the successful creation of an in vitro supported lipid bilayer (SLB) platform of the outer membrane (OM) of E. coli, an archetypical Gram-negative bacterium, containing the full smooth lipopolysaccharide (S-LPS) architecture of the membrane. Using this platform, we performed fluorescence correlation spectroscopy (FCS) in combination with molecular dynamics (MD) simulations to measure lipid diffusivities and provide molecular insights into the transport of natural antimicrobial agent thymol. Lipid diffusivities measured on symmetric supported lipid bilayers made up of inner membrane lipids show a distinct increase in the presence of thymol as also corroborated by MD simulations. However, lipid diffusivities in the asymmetric OM consisting of only S-LPS are invariant upon exposure to thymol. Increasing the phospholipid content in the LPS-containing outer leaflet improved the penetration toward thymol as reflected in slightly higher relative diffusivity changes in the inner leaflet when compared with the outer leaflet. Free-energy computations reveal the presence of a barrier (∼6 kT) only in the core-saccharide region of the OM for the translocation of thymol while the external O-antigen part is easily traversed. In contrast, thymol spontaneously inserts into the inner membrane. In addition to providing leaflet-resolved penetration barriers in bacterial membranes, we also assess the ability of small molecules to penetrate various membrane components. With rising bacterial resistance, our study opens up the possibility of screening potential antimicrobial drug candidates using these realistic model platforms for Gram-negative bacteria.

Unraveling complex nanoscale lipid dynamics in simple model biomembranes: Insights from fluorescence correlation spectroscopy in super-resolution stimulated emission depletion mode.

Dynamic heterogeneity (DH) at nanoscale due to lipid-lipid and/or lipid-protein interactions in cell membranes plays a crucial role in determining a broad range of important cell functions. In cell membranes, the dimensions of these nanodomains have been postulated to be in the order of 10's of nm and transient in nature. While the structural features of membranes have been studied in detail, little is known about their dynamical characteristics due to paucity of techniques which can probe nanoscale phenomena with simultaneous high temporal resolution. A combination of super-resolution stimulated emission depletion (STED) and fluorescence correlation spectroscopy (FCS) technique can overcome this limitation and provide information about the nanoscale dynamic heterogeneity in cell membranes. Using STED-FCS and FCS diffusion law, we provide an understanding of how nanoscale dynamically organizing lipid platforms can emerge in minimal system of model biomembranes. To illustrate the utility of the technique we have chosen cholesterol containing supported lipid bilayers and demonstrated the role of cholesterol concentration and/or added pore-forming protein, Listeriolysin O (LLO) in determining onset of lipid DH. In addition we have also looked at multi-component lipid bilayers with and without cholesterol to infer about the role of phospholipid and cholesterol composition on lipid dynamics. These results on simple biomimetic systems provide insights into fundamental pathways for the emergence of complex nanodomain substructures with implications for a wide variety of membrane mediated cellular events and depict the significant contribution that STED-FCS can make in resolving several outstanding issues in membrane biology.

Nanoscale Heterogeneities Drive Enhanced Binding and Anomalous Diffusion of Nanoparticles in Model Biomembranes.

Interaction of functional nanoparticles with cells and model biomembranes has been widely studied to evaluate the effectiveness of the particles as potential drug delivery vehicles and bioimaging labels as well as in understanding nanoparticle cytotoxicity effects. Charged nanoparticles, in particular, with tunable surface charge have been found to be effective in targeting cellular membranes as well as the subcellular matrix. However, a microscopic understanding of the underlying physical principles that govern nanoparticle binding, uptake, or diffusion on cells is lacking. Here, we report the first experimental studies of nanoparticle diffusion on model biomembranes and correlate this to the existence of nanoscale dynamics and structural heterogeneities using super-resolution stimulated emission depletion (STED) microscopy. Using confocal and STED microscopy coupled with fluorescence correlation spectroscopy (FCS), we provide novel insight on why these nanoparticles show enhanced binding on two-component lipid bilayers as compared to single-component membranes and how binding and diffusion is correlated to subdiffraction nanoscale dynamics and structure. The enhanced binding is also dictated, in part, by the presence of structural and dynamic heterogeneity, as revealed by STED-FCS studies, which could potentially be used to understand enhanced nanoparticle binding in raft-like domains in cell membranes. In addition, we also observe a clear correlation between the enhanced nanoparticle diffusion on membranes and the extent of membrane penetration by the nanoparticles. Our results not only have a significant impact on our understanding of nanoparticle binding and uptake as well as diffusion in cell and biomembranes, but have very strong implications for uptake mechanisms and diffusion of other biomolecules, like proteins on cell membranes and their connections to functional membrane nanoscale platform.

Pathways for creation and annihilation of nanoscale biomembrane domains reveal alpha and beta-toxin nanopore formation processes.

Raft-like functional domains with putative sizes of 20-200 nm and which are evolving dynamically are believed to be the most crucial regions in cellular membranes which determine cell signaling and various functions of cells. While the actual sizes of these domains are believed to vary from cell to cell no direct determination of their sizes and their evolution when cells interact with external agents like toxins and relevant biomolecules exists. Here, we report the first direct determination of the size of these nanoscale regions in model raft-forming biomembranes using the method of super-resolution stimulated emission depletion nanoscopy coupled with fluorescence correlation spectroscopy (STED-FCS). We also show that the various pathways for creation and destruction of such nanoscale membrane regions due to interaction with prototypical α and ß nanopore-forming toxins, can reveal the nature of the respective pore formation processes. The methodology, in turn, establishes a new nano-biotechnological protocol which could be very useful in preventing their cytotoxic effects in particular but also enable microscopic understanding of biomolecule-cell membrane interactions in general.

Super-resolution Stimulated Emission Depletion-Fluorescence Correlation Spectroscopy Reveals Nanoscale Membrane Reorganization Induced by Pore-Forming Proteins.

Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that the affinity for cholesterol in the membrane binding motifs of the LLO subdomains induce cholesterol and lipid reorganization to a greater extent in the distal (upper) leaflet when compared with the proximal (lower) leaflet. The observed length scale-dependent membrane reorganization that occurs due to invasion by LLO could be generalized to other cholesterol-dependent cytolysins and emphasizes the significant advantage of using super-resolution STED nanoscopy to unravel complex lipid-protein interactions in membrane and cellular biophysics.

Nanoscale dynamics of phospholipids reveals an optimal assembly mechanism of pore-forming proteins in bilayer membranes.

Cell membranes are believed to be highly complex dynamical systems having compositional heterogeneity involving several types of lipids and proteins as the major constituents. This dynamical and compositional heterogeneity is suggested to be critical to the maintenance of active functionality and response to chemical, mechanical, electrical and thermal stresses. However, delineating the various factors responsible for the spatio-temporal response of actual cell membranes to stresses can be quite challenging. In this work we show how biomimetic phospholipid bilayer membranes with variable lipid fluidity determine the optimal assembly mechanism of the pore-forming protein, listeriolysin O (LLO), belonging to the class of cholesterol dependent cytolysins (CDCs). By combining atomic force microscopy (AFM) and super-resolution stimulated emission depletion (STED) microscopy imaging on model membranes, we show that pores formed by LLO in supported lipid bilayers can have variable conformation and morphology depending on the fluidity of the bilayer. At a fixed cholesterol concentration, pores formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes were larger, flexible and more prone to coalescence when compared with the smaller and more compact pores formed in the lower fluidity 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. In contrast, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes did not show any evidence of pore formation. Fluorescence correlation spectroscopy (FCS) in STED mode revealed the appearance of a length scale, ξ, below which lipid dynamics, under the influence of LLO protein binding and assembly, becomes anomalous. Interestingly, the magnitude of ξ is found to correlate with both lipid fluidity and pore dimensions (and flexibility) in DOPC and POPC bilayers. However this length scale dependent crossover, signalling the onset of anomalous diffusion, was not observed in DMPC bilayers. Our study highlights the subtle interplay of lipid membrane mediated protein assembly and lipid fluidity in determining proteo-lipidic complexes formed in biomembranes and the significant insight that STED microscopy provides in unraveling critical aspects of nanoscale membrane biophysics.

Enhancing Carrier Diffusion Length and Quantum Efficiency through Photoinduced Charge Transfer in Layered Graphene-Semiconducting Quantum Dot Devices.

Hybrid devices consisting of graphene or transition metal dichalcogenides (TMDs) and semiconductor quantum dots (QDs) were widely studied for potential photodetector and photovoltaic applications, while for photodetector applications, high internal quantum efficiency (IQE) is required for photovoltaic applications and enhanced carrier diffusion length is also desirable. Here, we reported the electrical measurements on hybrid field-effect optoelectronic devices consisting of compact QD monolayer at controlled separations from single-layer graphene, and the structure is characterized by high IQE and large enhancement of minority carrier diffusion length. While the IQE ranges from 10.2% to 18.2% depending on QD-graphene separation, ds, the carrier diffusion length, LD, estimated from scanning photocurrent microscopy (SPCM) measurements, could be enhanced by a factor of 5-8 as compared to that of pristine graphene. IQE and LD could be tuned by varying back gate voltage and controlling the extent of charge separation from the proximal QD layer due to photoexcitation. The obtained IQE values were remarkably high, considering that only a single QD layer was used, and the parameters could be further enhanced in such devices significantly by stacking multiple layers of QDs. Our results could have significant implications for utilizing these hybrid devices as photodetectors and active photovoltaic materials with high efficiency.

Observation of photonic spin-momentum locking due to coupling of achiral metamaterials and quantum dots.

Chiral interfaces provide a new platform to execute quantum control of light-matter interactions. One phenomenon which has emerged from engineering such nanophotonic interfaces is spin-momentum locking akin to similar reports in electronic topological materials and phases. While there are reports of spin-momentum locking with combination of chiral emitters and/or chiral metamaterials with directional far field excitation it is not readily observable with both achiral emitters and metamaterials. Here, we report the observation of photonic spin-momentum locking in the form of directional and chiral emission from achiral quantum dots (QDs) evanescently coupled to achiral hyperbolic metamaterials (HMM). Efficient coupling between QDs and the metamaterial leads to emergence of these photonic topological modes which can be detected in the far field. We provide theoretical explanation for the emergence of spin-momentum locking through rigorous modeling based on photon Green's function where pseudo spin of light arises from coupling of QDs to evanescent modes of HMM.

Room Temperature Weak-to-Strong Coupling and the Emergence of Collective Emission from Quantum Dots Coupled to Plasmonic Arrays.

Colloidal quantum dot (CQD) assemblies exhibit interesting optoelectronic properties when coupled to optical resonators ranging from Purcell-enhanced emission to the emergence of hybrid electronic and photonic polariton states in the weak and strong coupling limits, respectively. Here, experiments exploring the weak-to-strong coupling transition in CQD-plasmonic lattice hybrid devices at room temperature are presented for varying CQD concentrations. To interpret these results, generalized retarded Fano-Anderson and effective medium models are developed. Individual CQDs are found to interact locally with the lattice yielding Purcell-enhanced emission. At high CQD densities, polariton states emerge as two-peak structures in the photoluminescence, with a third polariton peak, due to collective CQD emission, appearing at still higher CQD concentrations. Our results demonstrate that CQD-lattice plasmon devices represent a highly flexible platform for the manipulation of collective spontaneous emission using lattice plasmons, which could find applications in optoelectronics, ultrafast optical switches, and quantum information science.

Complex dynamics at the nanoscale in simple biomembranes.

Nature is known to engineer complex compositional and dynamical platforms in biological membranes. Understanding this complex landscape requires techniques to simultaneously detect membrane re-organization and dynamics at the nanoscale. Using super-resolution stimulated emission depletion (STED) microscopy coupled with fluorescence correlation spectroscopy (FCS), we reveal direct experimental evidence of dynamic heterogeneity at the nanoscale in binary phospholipid-cholesterol bilayers. Domain formation on the length scale of ~200-600 nm due to local cholesterol compositional heterogeneity is found to be more prominent at high cholesterol content giving rise to distinct intra-domain lipid dynamics. STED-FCS reveals unique dynamical crossover phenomena at length scales of ~100-150 nm within each of these macroscopic regions. The extent of dynamic heterogeneity due to intra-domain hindered lipid diffusion as reflected from the crossover length scale, is driven by cholesterol packing and organization, uniquely influenced by phospholipid type. These results on simple binary model bilayer systems provide novel insights into pathways leading to the emergence of complex nanodomain substructures with implications for a wide variety of membrane mediated cellular events.