Study Of Membrane Dynamics Research Articles

Probing macromolecular crowding at the lipid membrane interface with genetically-encoded sensors.

Biochemical processes within the living cell occur in a highly crowded environment, where macromolecules, first of all proteins and nucleic acids, occupy up to 30 % of the volume. The phenomenon of macromolecular crowding is not an exclusive feature of the cytoplasm and can be observed in the densely protein-packed, nonhomogeneous cellular membranes and at the membrane interfaces. Crowding affects diffusional and conformational dynamics of proteins within the lipid bilayer, alters kinetic and thermodynamic properties of biochemical reactions, and modulates the membrane organization. Despite its importance, the non-invasive quantification of the membrane crowding is not trivial. Here, we developed the genetically-encoded fluorescence-based sensor for probing the macromolecular crowding at the membrane interfaces. Two sensor variants, both composed of fluorescent proteins and a membrane anchor, but differing by the flexible linker domains were characterized in vitro, and the procedures for the membrane reconstitution were established. Steric pressure induced by membrane-tethered synthetic and protein crowders altered the sensors' conformation, causing increase in the intramolecular Förster's resonance energy transfer. Notably, the effect of protein crowders only weakly correlated with their molecular weight, suggesting that other factors, such as shape and charge contribute to the crowding via the quinary interactions. Finally, measurements performed in inner membrane vesicles of E. coli validated the crowding-dependent dynamics of the sensors in the physiologically relevant environment. The sensors offer broad opportunities to study interfacial crowding in a complex environment of native membranes, and thus add to the toolbox of methods for studying membrane dynamics and proteostasis. This article is protected by copyright. All rights reserved.

Implications of different membrane compartmentalization models in particle-based in silico studies

Studying membrane dynamics is important to understand the cellular response to environmental stimuli. A decisive spatial characteristic of the plasma membrane is its compartmental structure created by the actin-based membrane-skeleton (fences) and anchored transmembrane proteins (pickets). Particle-based reaction–diffusion simulation of the membrane offers a suitable temporal and spatial resolution to analyse its spatially heterogeneous and stochastic dynamics. Fences have been modelled via hop probabilities, potentials or explicit picket fences. Our study analyses the different approaches’ constraints and their impact on simulation results and performance. Each of the methods comes with its own constraints; the picket fences require small timesteps, potential fences might induce a bias in diffusion in crowded systems, and probabilistic fences, in addition to carefully scaling the probability with the timesteps, induce higher computational costs for each propagation step.

Visualization of Exo- and Endocytosis Membrane Dynamics with Super-Resolution STED Microscopy.

Recent advances in stimulated emission depletion (STED) microscopy offer an unparalleled avenue to study membrane dynamics of exo- and endocytosis, such as fusion pore opening, pore expansion, constriction, and closure, as well as the membrane transformation from flat-shaped to round-shaped vesicles in real time. Here we depict a method of using the state-of-the-art STED microscopy to image these membrane dynamics in bovine chromaffin cells. This method can potentially be applied to study other membrane structure dynamics in other cell model system.

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.

A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3

Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast (Saccharomyces cerevisiae) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (328SRFS331). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3-Env7 kinase cascade.

Cell scientist to watch – Prisca Liberali

ABSTRACT Prisca Liberali studied chemistry at Università degli Studi di Roma ‘La Sapienza’ in Rome, Italy. She then pursued her PhD in physical chemistry, studying membrane dynamics and trafficking with Daniela Corda and Alberto Luini at Fondazioni Mario Negri Sud. Afterwards, Prisca joined Lucas Pelkmans' research group at ETH in Zurich and at the University of Zurich, Switzerland in 2008 for her postdoctoral work on genetic interactions and regulatory networks in membrane trafficking, making use of quantitative biology approaches. In 2015, Prisca became an Assistant Professor at the University of Basel, Switzerland while also starting her own research group at the Friedrich Miescher Institute for Biomedical Research (FMI) in Basel. She was awarded an ERC Starting Grant in 2016 and was selected as an EMBO Young Investigator in 2019. Her lab focuses on the collective characteristics of multicellular systems and how patterning can arise from single-cell properties and behaviour. Her research group uses experimental and theoretical analysis to address the cellular heterogeneity and other relevant questions on stem cell biology and organoid development.

Fluorescence Correlation Spectroscopy to Examine Protein-Lipid Interactions in Membranes.

Fluorescence correlation spectroscopy (FCS) is a versatile technique to study membrane dynamics and protein-lipid interactions. It can provide information about diffusion coefficients, concentrations, and molecular interactions of proteins and lipids in the membrane. These parameters allow for the determination of protein partitioning into different lipid environments, the identification of lipid domains, and the detection of lipid-protein complexes on the membrane. During the last decades, FCS studies were successfully performed on model membrane systems as also on living cells, to characterize protein-lipid interactions. Recent developments of the method described here improved quantitative measurements on membranes and decreased the number of potential artifacts. The aim of this chapter is to provide the reader with the necessary information and some practical guidelines to perform FCS studies on artificial and cellular membranes.

The Development and Enhancement of FRAP as a Key Tool for Investigating Protein Dynamics

The saga of fluorescence recovery after photobleaching (FRAP) illustrates how disparate technical developments impact science. Starting with the classic 1976 Axelrod et al. work in Biophysical Journal, FRAP (originally fluorescence photobleaching recovery) opened the door to extraction of quantitative information from photobleaching experiments, laying the experimental and theoretical groundwork for quantifying both the mobility and the mobile fraction of a labeled population of proteins. Over the ensuing years, FRAP’s reach dramatically expanded, with new developments in GFP technology and turn-key confocal microscopy, which enabled measurement of protein diffusion and binding/dissociation rates in virtually every compartment within the cell. The FRAP technique and data catalyzed an exchange of ideas between biophysicists studying membrane dynamics, cell biologists focused on intracellular dynamics, and systems biologists modeling the dynamics of cell activity. The outcome transformed the field of cellular biology, leading to a fundamental rethinking of long-held theories of cellular dynamism. Here, we review the pivotal FRAP studies that made these developments and conceptual changes possible, which gave rise to current models of complex cell dynamics.

The Ins and Outs of Lipid Flip-Flop.

Our current view of cellular membranes centers on the fluid-mosaic model, which envisions the cellular membrane as a "liquidlike" bilayer of lipids, cholesterol, and proteins that freely diffuse in two dimensions. In stark contrast, the exchange of materials between the leaflets of a bilayer was presumed to be prohibited by the large enthalpic barrier associated with translocating hydrophilic materials, such as a charged lipid headgroup, through the hydrophobic membrane core. This static picture with regard to lipid translocation (or "flip-flop" as it is affectionately known) has been a long-held belief in the study of membrane dynamics. The current accepted membrane model invokes specific protein flippase (inward moving), floppase (outward moving), and scramblase (bidirectional) enzymes that assist in the movement of lipids between the leaflets of cellular membranes. The low rate of protein-free lipid flip-flop has also been a cornerstone of our understanding of the bilateral organization of cellular membrane components, specifically the asymmetric distribution of lipid species found in the luminal and extracellular leaflets of the plasma membrane of eukaryotic cells. Much of the previous work contributing to our current understanding of lipid flip-flop has utilized fluorescent- or spin-labeled lipids. However, there is growing evidence that these lipid probes do not accurately convey the dynamics and thermodynamics of native (unlabeled) lipid motion. This Account summarizes our research efforts directed toward developing a deep physical and chemical understanding of protein-free lipid flip-flop in phospholipid membrane models using sum-frequency vibrational spectroscopy (SFVS). Our use of SFVS enables the direct measurement of native lipid flip-flop in model membranes. In particular, we have explored the kinetic rates and activation thermodynamics of lipid translocation as a means of deciphering the underlying chemical and physical directors governing this process. By means of transition state theory, the contributions from enthalpy and entropy on the activation energy barrier to lipid flip-flop have been explored in detail for a variety of lipid species and membrane compositions. Specifically, the effect of lipid structure and packing and the inclusion of cholesterol and transmembrane peptides on the rates and thermodynamics of lipid translocation have been investigated in detail. It is our hope that these studies will provide a new perspective on lipid translocation in biological membranes and the role of lipid flip-flop in generating and maintaining cell membrane lipid asymmetry.

Membrane dynamics during cellular wound repair.

Cells rapidly reseal after damage, but how they do so is unknown. It has been hypothesized that resealing occurs due to formation of a patch derived from rapid fusion of intracellular compartments at the wound site. However, patching has never been directly visualized. Here we study membrane dynamics in wounded Xenopus laevis oocytes at high spatiotemporal resolution. Consistent with the patch hypothesis, we find that damage triggers rampant fusion of intracellular compartments, generating a barrier that limits influx of extracellular dextrans. Patch formation is accompanied by compound exocytosis, local accumulation and aggregation of vesicles, and rupture of compartments facing the external environment. Subcellular patterning is evident as annexin A1, dysferlin, diacylglycerol, active Rho, and active Cdc42 are recruited to compartments confined to different regions around the wound. We also find that a ring of elevated intracellular calcium overlaps the region where membrane dynamics are most evident and persists for several minutes. The results provide the first direct visualization of membrane patching during membrane repair, reveal novel features of the repair process, and show that a remarkable degree of spatial patterning accompanies damage-induced membrane dynamics.

Capacitive Membrane Activity in Isolated Cardiac Myocytes

Cardiac myocytes are cells with complex membrane structure of unknown dynamics. A good physical measure of the state of the sarcolemma is membrane capacitance. High-resolution capacitance measurements were instrumental in disclosing secretory and endocytotic mechanisms in simpler cells. We used a new method of membrane capacitance recording based on deconvolution of current responses to square wave voltage stimuli (MAT-MECAS: https://sourceforge.net/projects/mat-mecas/) to study membrane dynamics of isolated cardiac myocytes; specifically, the spontaneous and stimulated membrane capacitance changes. We observed spontaneous discrete capacitance events of 1 − 20 fF that may correspond to fusion and fission of single vesicles of 180 − 800 nm in diameter. In addition, large spontaneous stepwise increases of membrane capacitance of 80 − 250 fF were also observed. These large capacitance increases were occasionally preceded by a transient opening of a fusion pore with a conductance of several hundreds of pS that lasted for a few hundreds of milliseconds. Such large stepwise changes of membrane capacitance might be related to the opening of the mouth of transversal tubules. Discrete capacitance increases were also observed after stimulation of myocytes by a train of calcium currents. In these experiments, the discrete capacitance increases of 50 − 160 fF occurred 3 to 20 seconds after stimulation. This might reflect calcium induced fusion of intracellular membrane bodies with the surface membrane. In conditions of permanently raised intracellular concentration of calcium ions, membrane capacitance showed increased activity of step-like capacitance events. These observations support the role of intracellular calcium in regulation of membrane dynamics in cardiac myocytes. Altogether, these findings provide the first evidence of vesicle fusion events related to physiologically relevant membrane activity in cardiac myocytes.Supported by: APVV-0721-10, VEGA 2/0147/14.

Lipid Diffusion in Supported Lipid Bilayers: A Comparison between Line-Scanning Fluorescence Correlation Spectroscopy and Single-Particle Tracking.

Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of DFCS ~ 3 μm2 · s−1 and DSPT ~ 2 μm2 · s−1, respectively. These comparable, yet statistically different values are used to highlight the main message of the paper, namely that the two considered methods give access to distinctly different dynamic ranges: D ≳ 1 μm2 · s−1 for FCS and D ≲ 5 μm2 · s−1 for SPT (with standard imaging conditions). In the context of membrane diffusion, this means that FCS allows studying lipid diffusion in fluid membranes, as well as the diffusion of loosely-bound proteins hovering above the membrane. SPT, on the other hand, is ideal to study the motions of membrane-inserted proteins, especially those presenting different conformations, but only allows studying lipid diffusion in relatively viscous membranes, such as supported lipid bilayers and cell membranes.

Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane--a minimally invasive investigation by STED-FCS.

Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

Exploiting Conjugated Polyelectrolyte Photophysics toward Monitoring Real-Time Lipid Membrane-Surface Interaction Dynamics at the Single-Particle Level.

Herein we report the real-time observation of the interaction dynamics between cationic liposomes flowing in solution and a surface-immobilized charged scaffolding formed by the deposition of conjugated polyanion poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene (MPS-PPV) onto 100-nm-diameter SiO2 nanoparticles (NPs). Contact of the freely floating liposomes with the polymer-coated surfaces led to the formation of supported lipid bilayers (SLBs). The interaction of the incoming liposomes with MPS-PPV adsorbed on individual SiO2 nanoparticles promoted the deaggregation of the polymer conformation and led to large emission intensity enhancements. Single-particle total internal reflection fluorescence microscopy studies exploited this phenomenon as a way to monitor the deformation dynamics of liposomes on surface-immobilized NPs. The MPS-PPV emission enhancement (up to 25-fold) reflected on the extent of membrane contact with the surface of the NP and was correlated with the size of the incoming liposome. The time required for the MPS-PPV emission to reach a maximum (ranging from 400 to 1000 ms) revealed the dynamics of membrane deformation and was also correlated with the liposome size. Cryo-TEM experiments complemented these results by yielding a structural view of the process. Immediately following the mixing of liposomes and NPs the majority of NPs had one or more adsorbed liposomes, yet the presence of a fully formed SLB was rare. Prolonged incubation of liposomes and NPs showed completely formed SLBs on all of the NPs, confirming that the liposomes eventually ruptured to form SLBs. We foresee that the single-particle studies we report herein may be readily extended to study membrane dynamics of other lipids including cellular membranes in live cell studies and to monitor the formation of polymer-cushioned SLBs.

Focus on photonics, biomedical optics, and microscopy

Focus on photonics, biomedical optics, and microscopy

Dynamic speckle illumination wide-field reflection phase microscopy.

We demonstrate a quantitative reflection-phase microscope based on time-varying speckle-field illumination. Due to the short spatial coherence length of the speckle field, the proposed imaging system features superior lateral resolution, 520 nm, as well as high-depth selectivity, 1.03 μm. Off-axis interferometric detection enables wide-field and single-shot imaging appropriate for high-speed measurements. In addition, the measured phase sensitivity of this method, which is the smallest measurable axial motion, is more than 40 times higher than that available using a transmission system. We demonstrate the utility of our method by successfully distinguishing the motion of the top surface from that of the bottom in red blood cells. The proposed method will be useful for studying membrane dynamics in complex eukaryotic cells.

Depolarization Laplace Transform Analysis of Exchangeable Hyperpolarized 129Xe for Detecting Ordering Phases and Cholesterol Content of Biomembrane Models

We present a highly sensitive nuclear-magnetic resonance technique to study membrane dynamics that combines the temporary encapsulation of spin-hyperpolarized xenon (129Xe) atoms in cryptophane-A-monoacid (CrAma) and their indirect detection through chemical exchange saturation transfer. Radiofrequency-labeled Xe@CrAma complexes exhibit characteristic differences in chemical exchange saturation transfer-driven depolarization when interacting with binary membrane models composed of different molecular ratios of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). The method is also applied to mixtures of cholesterol and POPC. The existence of domains that fluctuate in cluster size in DPPC/POPC models at a high (75–98%) DPPC content induces up to a fivefold increase in spin depolarization time τ at 297 K. In POPC/cholesterol model membranes, the parameter τ depends linearly on the cholesterol content at 310 K and allows us to determine the cholesterol content with an accuracy of at least 5%.

Fluorescence correlation and lifetime correlation spectroscopy applied to the study of supported lipid bilayer models of the cell membrane

Supported Lipid Bilayers (SLBs) are versatile models capable of mimicking some of the key properties of the cell membrane, including for example lipid fluidity, domain formation and protein support, without the challenging complexity of the real biological system. This is important both from the perspective of understanding the behaviour and role of the lipid membrane in cell structure and signalling, as well as in development of applications of lipid membranes across domains as diverse as sensing and drug delivery. Lipid and protein diffusion within the membrane is vital to its function and there are several key experimental methods used to study membrane dynamics. Amongst the optical methods are Fluorescence Recovery After Photobleaching (FRAP), single particle tracking and Fluorescence Correlation (and Fluorescence Lifetime Correlation) Spectroscopy (FCS/FLCS). Each of these methods can provide different and often complementary perspectives on the dynamics of the fluid membrane. Although FCS is well established, FLCS is a relatively new technique and both methods have undergone a number of extensions in recent years which improve their precision and accuracy in studying supported lipid bilayers, most notably z-scan methods. This short review focusses on FCS and FLCS and their recent applications, specifically to artificial lipid bilayer studies addressing key issues of cell membrane behaviour.

Reduction of Lectin Valency Drastically Changes Glycolipid Dynamics in Membranes but Not Surface Avidity

Multivalency is proposed to play a role in the strong avidity of lectins for glycosylated cell surfaces and also in their ability to affect membrane dynamics by clustering glycosphingolipids. Lectins with modified valency were designed from the β-propeller fold of Ralstonia solanacearum lectin (RSL) that presents six fucose binding sites. After identification of key amino acids by molecular dynamics calculations, two mutants with reduced valency were produced. Isothermal titration calorimetry confirmed the loss of three high affinity binding sites for both mutants. Crystal structures indicated that residual low affinity binding occurred in W76A but not in R17A. The trivalent R17A mutant presented unchanged avidity toward fucosylated surfaces, when compared to hexavalent RSL. However, R17A is not able anymore to induce formation of membrane invaginations on giant unilamellar vesicules, indicating the crucial role of number of binding sites for clustering of glycolipids. In the human lung epithelial cell line H1299, wt-RSL is internalized within seconds whereas the kinetics of R17A uptake is largely delayed. Neolectins with tailored valency are promising tools to study membrane dynamics.

Crumbs Affects Protein Dynamics In Anterior Regions Of The Developing Drosophila Embryo

Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.