Spontaneous unbinding transition of nanoparticles adsorbing onto biomembranes: interplay of electrostatics and crowding.

Cellular membranes are constantly bombarded with biomolecules and nanoscale particles, and cell functionality depends on the fraction of the bound/internalized entities. Understanding the biophysical parameters underlying this complex process is very difficult in live cells. Model membranes provide an ideal platform to obtain insight into the minimal and essential parameters involved in determining cell membrane-nanoparticle (NP) interaction. Here we report spontaneous binding and unbinding of semiconductor NPs, carrying different net charges and interacting with model biomembranes, using in situ neutron reflectivity (NR) and fluorescence microscopy studies. We observe a critical concentration of NPs above which they spontaneously unbind along with lipids from lipid monolayer membranes, leaving behind fewer bound NPs. This critical concentration varies depending on whether the NPs carry a net charge or are neutral, and is also governed by the extent of NP crowding for a fixed NP charge. The observations suggest a subtle interplay between electrostatics, membrane fluidity, and NP crowding effects, which eventually determines the adsorbed concentration for unbinding transition. Our study provides valuable microscopic insight into the parameters that could determine the biophysical process underlying NP uptake and ejection by cells which, in turn, can be utilized for their potential applications in bioimaging and drug delivery.

Spontaneous formation and growth kinetics of lipid nanotubules induced by passive nanoparticles.

Lipid nanotubules (LNTs) are conduits that form on the membranes of cells and organelles, and they are ubiquitous in all forms of life from archaea and bacteria to plants and mammals. The formation, shape and dynamics of these LNTs are critical for cellular functions, supporting the transport of myriad cellular cargoes as well as communication within and between cells, and they are also widely believed to be responsible for exploitation of host cells by pathogens for the spread of infection and diseases. In vitro kinetic control of LNT formation can considerably enhance the scope of utilization of these structures for disease control and therapy. Here we report a new paradigm for spontaneous lipid nanotubulation, capturing the dynamical regimes of growth, stabilization and retraction of the tubes through the binding of synthetic nanoparticles on supported lipid bilayers (SLBs). The tubulation is determined by the spontaneous binding-unbinding of nanoparticles on the LNTs. The presented methodology could be used to rectify malfunctioning cellular tubules or to prevent the pathogenic spread of diseases through inhibition of cell-to-cell nanotubule formation.

Modification of bacterial cell membrane dynamics and morphology upon exposure to sub inhibitory concentrations of ciprofloxacin.

Ciprofloxacin (CPX), a second generation fluoroquinolone antibiotic, is used as a primary antibiotic for treatment against gastroenteritis, drug-resistant tuberculosis, and malignant otitis externa. CPX is a broad spectrum antibiotic that targets the DNA gyrase of both Gram-positive and Gram-negative bacteria. Irrational and improper usage of CPX results in emergence of CPX resistant organisms emphasizing the importance of using lethal doses of CPX. Here, we have systematically analysed the effect of CPX at sub lethal concentrations on live E. coli membrane and growth dynamics. As a result of CPX interaction at sub-lethal concentrations, we detected filamentation of the bacterial cells during cell division. Although CPX is a DNA targeting antibiotic and did not result in considerable increase of live E. coli cell surface roughness, we observed significant enhancement in the lipid diffusion coefficients possibly due to disrupted lipid packing or altered lipid composition. Interestingly, we seem to observe slightly higher extent of lipid diffusion alteration when bacterial inner membrane specific label FM4-64 was used in comparison to the non-specific membrane dye. Both these results are contrary to that observed in bacterial cells for colistin, a membrane targeting antibiotics. Our work highlights the need for using multiple, complementary surface and depth sensitive techniques to obtain information on the realistic nature of bacterial cell membrane remodelling due to non-membrane targeting antibiotics. Our work could have implications for identification of potential biomembrane markers at sub-lethal concentrations even for antibiotics which are non-membrane targeting that could help in unravelling pathways for emergence of antimicrobial resistance.

Escherichia coli response to subinhibitory concentrations of colistin: insights from a study of membrane dynamics and morphology.

Prevalence of widespread bacterial infections brings forth a critical need to understand the molecular mechanisms of the antibiotics as well as the bacterial response to those antibiotics. Improper use of antibiotics, which can be in sub-lethal concentrations is one among the multiple reasons for acquiring antibiotic resistance which makes it vital to understand the bacterial response towards sub-lethal concentrations of antibiotics. In this work, we have used colistin, a well-known membrane active antibiotic used to treat severe bacterial infections and explored the impact of its sub-minimum inhibitory concentration (MIC) on the lipid membrane dynamics and morphological changes of E. coli. Upon investigation of live cell membrane properties such as lipid dynamics using fluorescence correlation spectroscopy, we observed that colistin disrupts the lipid membrane at sub-MIC by altering the lipid diffusivity. Interestingly, filamentation-like cell elongation was observed upon colistin treatment which led to further exploration of surface morphology with the help of atomic force spectroscopy. The changes in the surface roughness upon colistin treatment provides additional insight on the colistin-membrane interaction corroborating with the altered lipid diffusion. Although altered lipid dynamics could be attributed to an outcome of lipid rearrangement due to direct disruption by antibiotic molecules on the membrane or an indirect consequence of disruptions in lipid biosynthetic pathways, we were able to ascertain that altered bacterial membrane dynamics is due to direct disruptions. Our results provide a broad overview on the consequence of the cyclic polypeptide colistin on membrane-specific lipid dynamics and morphology of a live Gram-negative bacterial cell.

Pore Forming Protein Induced Biomembrane Reorganization and Dynamics: A Focused Review.

Pore forming proteins are a broad class of pathogenic proteins secreted by organisms as virulence factors due to their ability to form pores on the target cell membrane. Bacterial pore forming toxins (PFTs) belong to a subclass of pore forming proteins widely implicated in bacterial infections. Although the action of PFTs on target cells have been widely investigated, the underlying membrane response of lipids during membrane binding and pore formation has received less attention. With the advent of superresolution microscopy as well as the ability to carry out molecular dynamics (MD) simulations of the large protein membrane assemblies, novel microscopic insights on the pore forming mechanism have emerged over the last decade. In this review, we focus primarily on results collated in our laboratory which probe dynamic lipid reorganization induced in the plasma membrane during various stages of pore formation by two archetypal bacterial PFTs, cytolysin A (ClyA), an α-toxin and listeriolysin O (LLO), a ß-toxin. The extent of lipid perturbation is dependent on both the secondary structure of the membrane inserted motifs of pore complex as well as the topological variations of the pore complex. Using confocal and superresolution stimulated emission depletion (STED) fluorescence correlation spectroscopy (FCS) and MD simulations, lipid diffusion, cholesterol reorganization and deviations from Brownian diffusion are correlated with the oligomeric state of the membrane bound protein as well as the underlying membrane composition. Deviations from free diffusion are typically observed at length scales below ∼130 nm to reveal the presence of local dynamical heterogeneities that emerge at the nanoscale-driven in part by preferential protein binding to cholesterol and domains present in the lipid membrane. Interrogating the lipid dynamics at the nanoscale allows us further differentiate between binding and pore formation of ß- and α-PFTs to specific domains in the membrane. The molecular insights gained from the intricate coupling that occurs between proteins and membrane lipids and receptors during pore formation are expected to improve our understanding of the virulent action of PFTs.

Bacterial protein listeriolysin O induces nonmonotonic dynamics because of lipid ejection and crowding.

Membrane-bound protein complexes involving pore forming toxins (PFTs) released by virulent bacteria are known to form transmembrane pores leading to host cell lysis. Developing alternative strategies against PFT mediated bacterial virulence factors requires an understanding of the cellular membrane response. However, membrane disruption and related lipid reorganization events during attack by PFTs remain largely unexplored. We report counterintuitive and nonmonotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane-bound oligomers of a prototypical cholesterol-dependent cytolysin, listeriolysin O (LLO). The observed dynamical crossover is correlated with concentration dependent transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area-based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events because of a preponderance of ring-like pore states. At higher protein concentrations in which membrane-inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodeling, and mechanical property variations, providing unique insights into the pore forming mechanisms of cholesterol-dependent cytolysins.

Strongly Coupled Exciton-Surface Lattice Resonances Engineer Long-Range Energy Propagation.

Achieving propagation lengths in hybrid plasmonic systems beyond typical values of tens of micrometers is important for quantum plasmonics applications. We report long-range optical energy propagation due to excitons in semiconductor quantum dots (SQDs) being strongly coupled to surface lattice resonance (SLRs) in silver nanoparticle arrays. Photoluminescence (PL) measurements provide evidence of an exciton-SLR (ESLR) mode extending at least 600 µm from the excitation region. We also observe additional energy propagation with range well beyond the ESLR mode and with dependency on the coupling strength, g, between SQDs and SLR. Cavity quantum electrodynamics calculations capture the nature of the PL spectra for consistent g values, while coupled dipole calculations show a SQD number-dependent electric field decay profile consistent with the experimental spatial PL profile. Our results suggest an exciting direction wherein SLRs mediate long-range interactions between SQDs, having possible applications in optoelectronics, sensing, and quantum information science.

Strong suppression of emission quenching in core quantum dots coupled to monolayer MoS2.

Non-radiative processes like energy and charge transfer in 0D-2D semiconductor quantum dot (QD)-transition metal dichalcogenides (TMDs) and other two-dimensional layered materials, like graphene and analogs, leading to strong quenching of the photoluminescence (PL) of the usually highly emissive QDs, have been widely studied. Here we report control of the emission efficiency of core QDs placed in close proximity to the monolayers of MoS2. The QDs are transferred in the form of a high-density compact monolayer with the dot-dot separation, δ as well as the MoS2-QD separation, d, being controlled through chemical methods. While at larger separations we observe some quenching due to non-radiative processes, at smaller separations we observe enhanced emission from QDs on MoS2 as compared to the reference despite the presence of significant non-radiative charge transfer. Interestingly, at small separations δ, we see evidence of strong dot-dot interactions and a significant red shift of QD PL which enhances spectral overlap with the B exciton of MoS2. However, we observe significant reduction of PL quenching of QDs relative to longer δ and d cases, despite increased probability of non-radiative resonant energy transfer to MoS2, due to the enhanced spectral overlap, as well as charge transfer. Significantly we observe that simultaneously the intensity of the B exciton of MoS2 increases significantly suggesting the possibility of coherent and resonant radiative energy exchange between the 0D excitons in QDs and the 2D B exciton in MoS2. Our study reveals interesting nanoscale light-matter interaction effects which can suppress quenching of QDs leading to potential applications of these nanoscale materials in light emitting and photonic devices.

Size-dependent interaction of hydrophilic/hydrophobic ligand functionalized cationic and anionic nanoparticles with lipid bilayers.

We study the nature of nanoparticle (NPs)-membrane interaction as a function of nanoparticle size for different functionalization using molecular dynamics simulation. Zinc sulphide quantum dots of size, 2 nm and 4 nm are used as model NPs, and DLPC and DPPC lipid bilayers are used as model membranes. We use coarse-grained polarizable MARTINI model (MPW) to simulate the NPs and lipid bilayers. Our simulation results show that uncharged bare NPs penetrate the lipid bilayers and embed themselves within the hydrophobic core of the bilayer both in the gel and fluid phases. NPs of size 4 nm are shown to disrupt the bilayer. The bilayer recovers from the damages caused by smaller NPs of size 2 nm. In case of either purely hydrophilic or hybrid (with hydrophilic/hydrophobic ratio of 2:1) ligand-functionalized NPs of smaller size (shell size 2 nm), only cationic NPs bind to the bilayer. However, for larger NPs with a shell size of 4 nm, both anionic and cationic hybrid functionalized NPs bind to the bilayer. The performance of standard Martini (SM) force field for the charged NP/bilayer systems has also been tested and compared with the results obtained using MPW model. Although the overall trend that the cationic NPs interact strongly with the bilayers than their anionic counterparts has been captured correctly using SM, the adsorption behaviour of the functionalized NPs differ significantly in the SM force field. The interaction of anionic NPs with both fluid and gel bilayers has been observed to be least accurately represented in the SM force field.

Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins.

Pore-forming toxins (PFTs) are a class of proteins implicated in a wide range of virulent bacterial infections and diseases. These toxins bind to target membranes and subsequently oligomerize to form functional pores that eventually lead to cell lysis. While the protein undergoes large conformational changes on the bilayer, the connection between intermediate oligomeric states and lipid reorganization during pore formation is largely unexplored. Cholesterol-dependent cytolysins (CDCs) are a subclass of PFTs widely implicated in food poisoning and other related infections. Using a prototypical CDC, listeriolysin O (LLO), we provide a microscopic connection between pore formation, lipid dynamics, and leakage kinetics by using a combination of Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) measurements on single giant unilamellar vesicles (GUVs). Upon exposure to LLO, two distinct populations of GUVs with widely different leakage kinetics emerge. We attribute these differences to the existence of oligomeric intermediates, sampling various membrane-bound conformational states of the protein, and their intimate coupling to lipid rearrangement and dynamics. Molecular dynamics simulations capture the influence of various membrane-bound conformational states on the lipid and cholesterol dynamics, providing molecular interpretations to the FRET and FCS experiments. Our study establishes a microscopic connection between membrane binding and conformational changes and their influence on lipid reorganization during PFT-mediated cell lysis. Additionally, our study provides insights into membrane-mediated protein interactions widely implicated in cell signaling, fusion, folding, and other biomolecular processes.

Evolution of inter-layer coupling in artificially stacked bilayer MoS2.

In this paper, we show experimentally that for van der Waals heterostructures (vdWh) of atomically-thin materials, the hybridization of bands of adjacent layers is possible only for ultra-clean interfaces. This we achieve through a detailed experimental study of the effect of interfacial separation and adsorbate content on the photoluminescence emission and Raman spectra of ultra-thin vdWh. For vdWh with atomically-clean interfaces, we find the emergence of novel vibrational Raman-active modes whose optical signatures differ significantly from that of the constituent layers. Additionally, we find for such systems a significant modification of the photoluminescence emission spectra with the appearance of peaks whose strength and intensity directly correlate with the inter-layer coupling strength. Our ability to control the intensity of the photoluminescence emission led to the observation of detailed optical features like indirect-band peaks. Our study establishes that it is possible to engineer atomically-thin van der Waals heterostructures with desired optical properties by controlling the inter-layer spacing, and consequently the inter-layer coupling between the constituent layers.

Emergence of compositionally tunable nanoscale dynamical heterogeneity in model binary lipid biomembranes.

While the existence of nanoscale dynamical heterogeneity in biological membranes has been suggested to act as an active functional platform for enabling various cellular processes like signal transduction and viral or bacterial entry, it has been extremely difficult to detect the existence of such domains. Model lipid bilayer membranes have been widely used to detect such dynamical heterogeneity in order to avoid complications arising from the compositional heterogeneity of cellular membranes. However, even in model biological membranes the issue of nanoscale lipid dynamics has remained controversial and unresolved due to the difficulty of detecting the existence of such dynamical heterogeneity on the scale of 10-300 nm. Here we report direct evidence of nanoscale lipid dynamical heterogeneity in model binary lipid bilayer membranes using a combination of super-resolution stimulated emission depletion (STED) microscopy and fluorescence correlation spectroscopy (FCS). We control the phase behavior of the lipid bilayers by varying their composition and discuss how this leads to the emergence of dynamical lipid domains on the scale of 80-150 nm, which is also dependent on the lipid phase in which such dynamics are observed. Notably, our work shows that the presence of cholesterol is not required for the existence of such domains even in fluid like bilayers, as has been widely believed, and specifies the minimal conditions required for the emergence of such dynamical heterogeneity in cellular membranes. Our work will thus not only be of great significance towards understanding the nanoscale dynamic organizing principles of cellular membranes but could also be useful in understanding the dynamics of related soft matter systems and nanoparticle-cell membrane interactions.

Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes.

Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes.

Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-ß Conformation.

Identifying the structures of membrane bound proteins is critical to understanding their function in healthy and diseased states. We introduce a surface enhanced Raman spectroscopy technique which can determine the conformation of membrane-bound proteins, at low micromolar concentrations, and also in the presence of a substantial membrane-free fraction. Unlike conventional surface enhanced Raman spectroscopy, our approach does not require immobilization of molecules, as it uses spontaneous binding of proteins to lipid bilayer-encapsulated Ag nanoparticles. We apply this technique to probe membrane-attached oligomers of Amyloid-ß40 (Aß40), whose conformation is keenly sought in the context of Alzheimer's disease. Isotope-shifts in the Raman spectra help us obtain secondary structure information at the level of individual residues. Our results show the presence of a ß-turn, flanked by two ß-sheet regions. We use solid-state NMR data to confirm the presence of the ß-sheets in these regions. In the membrane-attached oligomer, we find a strongly contrasting and near-orthogonal orientation of the backbone H-bonds compared to what is found in the mature, less-toxic Aß fibrils. Significantly, this allows a "porin" like ß-barrel structure, providing a structural basis for proposed mechanisms of Aß oligomer toxicity.