Lateral Diffusion Of Membrane Proteins Research Articles

Colloidal and electrostatic interactions in free-standing membranes

Phospholipid bilayer elastic properties govern numerous biological processes, ranging from the deformation necessary during endocytosis and exocytosis, to the lateral diffusion of membrane proteins. As a result, there have been a multitude of studies using artificial techniques to reconstitute phospholipids into phospholipid bilayers and quantify membrane material properties. In this presentation, we will discuss recent advances in our lab applying a novel technique to create free-standing membranes to understand membrane elasticity and how membrane material properties dictate biological function.

Exploring Lipid and Membrane Protein Dynamics Using Lipid-Bilayer Nanodiscs and Solution-State NMR Spectroscopy.

The relationship of membrane protein function and the surrounding lipid bilayer goes far beyond simple hydrophobic interactions. At least from the 1980s, it is speculated that a certain fluid lipid state may be important not only for the lateral diffusion of membrane proteins (MPs) but also for modulating the catalytic activity of MPs (Lenaz. Bioscience Rep 7 (11):823-837, 1987). Indeed, acyl chain length, hydrophobic mismatch, and lipid headgroups are determinants for enzymatic and transport activities of MPs (Dumas et al. Biochemistry 39(16):4846-4854, 2000; Johannsson et al. Biochim Biophys Acta 641(2):416-421, 1981; Montecucco et al. FEBS Lett 144(1):145-148, 1982; Martens et al. Nat Struct Mol Biol 23(8):744-751, 2016). Moreover, it is speculated that changes in membrane lipid dynamics are important in the field of thermosensation (Vriens J, Nilius B, Voets T, Nat Rev Neurosci 15:573-589, 2014). Atomic insights into lipid-mediated modulation of membrane protein dynamics would therefore provide new insights with the potential to fundamentally extend our understanding on dynamic lipid-protein interdependencies.This chapter describes the expression and purification of nanodiscs assembled from membrane scaffold protein (MSP) as well as the expression and purification of the outer membrane protein X (OmpX). Subsequently, the incorporation of OmpX into MSP-derived nanodiscs is explained in detail. The chapter concludes with the setup of nuclear magnetic resonance (NMR) relaxation experiments and the extraction of relaxation rates for OmpX and the surrounding lipids.

Single-molecule visualization of dynamic transitions of pore-forming peptides among multiple transmembrane positions.

Research on the dynamics of single-membrane proteins remains underdeveloped due to the lack of proper approaches that can probe in real time the protein's insertion depth in lipid bilayers. Here we report a single-molecule visualization method to track both vertical insertion and lateral diffusion of membrane proteins in supported lipid bilayers by exploiting the surface-induced fluorescence attenuation (SIFA) of fluorophores. The attenuation follows a d−4 dependency, where d is the fluorophore-to-surface distance. The method is validated by observing the antimicrobial peptide LL-37 to transfer among five transmembrane positions: the surface, the upper leaflet, the centre, the lower leaflet and the bottom of the lipid bilayer. These results demonstrate the power of SIFA to study protein-membrane interactions and provide unprecedented in-depth understanding of molecular mechanisms of the insertion and translocation of membrane proteins.

Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and β-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete β-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for β-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics.

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.

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP

Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a “membrane fence” model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more “fluid” and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family.In conclusion: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R–Gq/G11α protein coupling.

Loss of hydroxyl groups from the ceramide moiety can modify the lateral diffusion of membrane proteins in S. cerevisiae

In the yeast Saccharomyces cerevisiae, structural diversities of complex sphingolipids [inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide] are often observed in the presence or absence of hydroxyl groups on the C-4 position of long-chain base (C4-OH) and the C-2 position of very long-chain fatty acids (C2-OH), but the biological significance of these groups remains unclear. Here, we evaluated cellular membrane fluidity in hydroxyl group-defective yeast mutants by fluorescence recovery after photobleaching. The lateral diffusion of enhanced green fluorescent protein-tagged hexose transporter 1 (Hxt1-EGFP) was influenced by the absence of C4-OH and/or C2-OH. Notably, the fluorescence recovery of Hxt1-EGFP was dramatically decreased in the sur2Δ mutant (absence of C4-OH) under the csg1Δcsh1Δ background, in which mannosylation of IPC is blocked leading to IPC accumulation, while the recovery in the scs7Δ mutant (absence of C2-OH) under the same background was modestly decreased. In addition, the amount of low affinity tryptophan transporter 1 (Tat1)-EGFP was markedly decreased in the sur2Δcsg1Δcsh1Δ mutant and accumulated in intracellular membranes in the scs7Δcsg1Δcsh1Δ mutant without altering its protein expression. These results suggest that C4-OH and C2-OH are most probably critical factors for maintaining membrane fluidity and proper turnover of membrane molecules in yeast containing complex sphingolipids with only one hydrophilic head group.

Lateral Membrane Diffusion Corralled

Lateral Membrane Diffusion Corralled

Examining the Role of Glycolipids in Integrin and B Cell Receptor Activation

Glycolipids are an important component of the plasma membrane, and often play important roles in the spatial organization of membrane proteins. Our group is interested in the role of enzymes which alter the glycolipid composition of the plasma membrane in lymphocytes. Glycosyl hydrolase enzymes which catabolize membrane glycolipids have been proposed to regulate receptor signaling by altering membrane glycolipid composition. The human neuraminidase 3 (NEU3) is a membrane-associated enzyme that cleaves terminal neuraminic acid (also known as sialic acid) residues from membrane glycolipids, such as GM3. Cells treated with recombinant NEU3 show changes in their glycolipid composition, thus making NEU3 a powerful tool for probing the role of gangliosides in receptor biophysics. Using single dye tracking (SDT) of membrane receptors by total-internal reflection fluorescence (TIRF) microscopy, we have examined the influence of glycolipids on the lateral diffusion of membrane proteins on lymphocytes. We first examined the T cell integrin, LFA-1. SDT data show clear changes in LFA-1 diffusion, with an increased population of receptors with a large diffusion coefficient after NEU3 treatment. Imaging of LFA-1 by TIRF shows that NEU3 treatment results in co-localization of GM1 and LFA-1, which is distinct from its localization in cells activated with phorbol-12-myristate-13-acetate. To understand the generality of this observation, we examined the influence of NEU3 treatment on the B cell receptor (BCR) complex. NEU3 treatment again increased the proportion of mobile receptors in the B cell membrane relative to control. A similar increase in mobile receptors was observed when cells were activated with PMA. We will present our analysis of SDT and imaging data which support that altering the membrane composition of lymphocytes by NEU3 treatment results in changes to the lateral mobility and subcellular localization of specific receptors.

Ruffles limit diffusion in the plasma membrane during macropinosome formation

In murine macrophages stimulated with macrophage-colony-stimulating factor (M-CSF), signals essential to macropinosome formation are restricted to the domain of plasma membrane enclosed within cup-shaped, circular ruffles. Consistent with a role for these actin-rich structures in signal amplification, microscopic measures of Rac1 activity determined that disruption of actin polymerization by latrunculin B inhibited ruffling and the localized activation of Rac1 in response to M-CSF. To test the hypothesis that circular ruffles restrict the lateral diffusion of membrane proteins that are essential for signaling, we monitored diffusion of membrane-tethered, photoactivatable green fluorescent protein (PAGFP-MEM) in ruffling and non-ruffling regions of cells. Although diffusion within macropinocytic cups was not inhibited, circular ruffles retained photoactivated PAGFP-MEM inside cup domains. Confinement of membrane molecules by circular ruffles could explain how actin facilitates positive feedback amplification of Rac1 in these relatively large domains of the plasma membrane, thereby organizing the contractile activities that close macropinosomes.

Lateral Diffusion of Membrane Proteins: Consequences of Hydrophobic Mismatch and Lipid Composition

Biological membranes are composed of a large number lipid species differing in hydrophobic length, degree of saturation, and charge and size of the headgroup. We now present data on the effect of hydrocarbon chain length of the lipids and headgroup composition on the lateral mobility of the proteins in model membranes. The trimeric glutamate transporter (GltT) and the monomeric lactose transporter (LacY) were reconstituted in giant unilamellar vesicles composed of unsaturated phosphocholine lipids of varying acyl chain length (14–22 carbon atoms) and various ratios of DOPE/DOPG/DOPC lipids. The lateral mobility of the proteins and of a fluorescent lipid analog was determined as a function of the hydrophobic thickness of the bilayer (h) and lipid composition, using fluorescence correlation spectroscopy. The diffusion coefficient of LacY decreased with increasing thickness of the bilayer, in accordance with the continuum hydrodynamic model of Saffman-Delbrück. For GltT, the mobility had its maximum at diC18:1 PC, which is close to the hydrophobic thickness of the bilayer in vivo. The lateral mobility decreased linearly with the concentration of DOPE but was not affected by the fraction of anionic lipids from DOPG. The addition of DOPG and DOPE did not affect the activity of GltT. We conclude that the hydrophobic thickness of the bilayer is a major determinant of molecule diffusion in membranes, but protein-specific properties may lead to deviations from the Saffman-Delbrück model.

LATERAL DIFFUSION OF MEMBRANE PROTEINS AT CELL MEMBRANE

The cell membrane is an important organ of living cells, which has a complex structure influenced by the interaction between membrane proteins and cell membrane. On the basis of fluid motion and diffusion interaction, a simple model is proposed to evaluate quantitatively the effects of the protein size and membrane fluid velocity on the lateral diffusion of membrane proteins at the cell membrane. The study shows that the diffusion coefficient is a dominant factor on the lateral diffusion.

Recent Applications of Fluorescence Recovery after Photobleaching (FRAP) to Membrane Bio-Macromolecules

This review examines some recent applications of fluorescence recovery after photobleaching (FRAP) to biopolymers, while mainly focusing on membrane protein studies. Initially, we discuss the lateral diffusion of membrane proteins, as measured by FRAP. Then, we talk about the use of FRAP to probe interactions between membrane proteins by obtaining fundamental information such as geometry and stoichiometry of the interacting complex. Afterwards, we discuss some applications of FRAP at the cellular level as well as the level of organisms. We conclude by comparing diffusion coefficients obtained by FRAP and several other alternative methods.

FFAT rescues VAPA-mediated inhibition of ER-to-Golgi transport and VAPB-mediated ER aggregation

The VAMP-associated proteins termed VAP are a small gene family of proteins characterised by the presence of an N-terminal major sperm protein (MSP) domain. The P56S mutation of the B isoform (VAPB) has been linked to late-onset amyotrophic lateral sclerosis (ALS8) and its expression causes formation of large ER aggregates. Overexpression of the wild-type A isoform (VAPA) but not the B isoform (VAPB), inhibited ER-to-Golgi transport of membrane proteins. This transport block by VAPA was primarily due to decreased segregation of membrane cargo into ER vesicles. We also found that VAPA inhibited lateral diffusion of membrane proteins, most likely through its stable association with microtubules. The MSP domain of VAP is known to interact with the FFAT motif (two phenylalanines in an acidic tract) of proteins involved in sterol regulation. Overexpression of FFAT restored ER-to-Golgi transport and lateral diffusion of membrane proteins, and resolved the large ER aggregates in VAPB-P56S. Application of a FFAT peptide restored in vitro ER vesicle budding and disrupted VAP-microtubule association. Thus, overexpression of the two VAP isoforms causes retention of ER membrane proteins by impeding lateral diffusion and their incorporation into transport vesicles. This inhibitory effect can be relieved by expression of the FFAT motif.

PEG molecular weight and lateral diffusion of PEG-ylated lipids in magnetically aligned bicelles

Lateral diffusion coefficients of PEG-ylated lipids with three different molecular weight PEG groups (1000, 2000 and 5000) were measured in magnetically-aligned bicelles using the stimulated echo (STE) pulsed field gradient (PEG) 1H nuclear magnetic resonance (NMR) method. At concentrations below the PEG “mushroom-to-brush” transition, all three PEG-ylated lipids exhibited unrestricted lateral diffusion, with lateral diffusion coefficients comparable to those of corresponding non-PEG-ylated lipids and independent of PEG molecular weight. At concentrations above this transition, lateral diffusion slowed progressively with increasing concentration of PEG-ylated lipid as a result of surface crowding. As well, the lateral diffusion coefficients exhibited a pronounced decrease with increasing PEG group molecular weight and a diffusion-time dependence indicative of obstructed diffusion. We conclude that, while lateral diffusion of PEG-ylated lipids within lipid bilayers is determined primarily by the hydrophobic anchoring group, when crowding at the lipid bilayer surface becomes significant, the size of the extra-membranous domain, in this case the PEG group, can influence lateral diffusion, leading to decreased diffusivity with increasing size and producing obstructed diffusion at high crowding. These findings imply that similar considerations will pertain to lateral diffusion of membrane proteins with large extra-membranous domains.

Cyclodextrins but not Compactin Inhibit the Lateral Diffusion of Membrane Proteins Independent of Cholesterol

Cholesterol and glycosphingolipid-enriched membrane domains, termed lipid rafts, were proposed to play important roles in trafficking and signaling events. These functions are inhibited following putative disruption of rafts by cholesterol depletion, commonly induced by treatment with methyl-beta-cyclodextrin (MbetaCD). However, several studies showed that the lateral diffusion of membrane proteins is inhibited by MbetaCD, suggesting that it may have additional effects on membrane organization unrelated to cholesterol removal. Here, we investigated this possibility by comparison of the effects of cholesterol depletion by MbetaCD and by metabolic inhibition (compactin), and of treatment with alpha-CD, which does not bind cholesterol. The studies employed two series of proteins (Ras and influenza hemagglutinin), each containing as internal controls related mutants that differ in raft association. Mild MbetaCD treatment retarded the lateral diffusion of both raft and non-raft mutants, whereas similar cholesterol reduction (30-33%) by metabolic inhibition enhanced selectively the diffusion of the raft-associated mutants. Moreover, alpha-CD also inhibited the diffusion of raft and non-raft mutants, despite its lack of effect on cholesterol content. These findings suggest that the widely used treatment with CD to reduce cholesterol has additional, cholesterol-independent effects on membrane protein mobility, which do not necessarily distinguish between raft and non-raft proteins.

Lateral Diffusion of Membrane Proteins in the Presence of Static and Dynamic Corrals: Suggestions for Appropriate Observables

We consider the possibility of inferring the nature of cytoskeletal interaction with transmembrane proteins via optical experiments such as single-particle tracking (SPT) and near-field scanning optical microscopy (NSOM). In particular, we demonstrate that it may be possible to differentiate between static and dynamic barriers to diffusion by examining the time-dependent variance and higher moments of protein population inside cytoskeletal “corrals.” Simulations modeling Band 3 diffusion on the surface of erythrocytes provide a concrete demonstration that these statistical tools might prove useful in the study of biological systems.

Chapter 10 Application of Laser Tweezers to Studies of the Fences and Tethers of the Membrane Skeleton that Regulate the Movements of Plasma Membrane Proteins

This chapter discusses the application of laser tweezers to studies of the fences and tethers of the membrane skeleton that regulate the movements of plasma membrane proteins. The cellular mechanisms that control the movement and assembly of membrane proteins in the plasma membrane are currently undergoing rapid evolution. The chapter illustrates the application of laser tweezers to studies of the mechanisms by which cells regulate the movement and lateral diffusion of membrane proteins. In these experiments, gold or latex particles are attached to membrane proteins, and the particle-membrane protein complex is captured by laser tweezers and moved laterally along the plasma membrane by scanning the trapping laser beam. Two types of major interactions between the membrane skeleton and membrane proteins have been found: One is the binding of the membrane proteins to the membrane skeleton, and the other is the corralling or fence effects of the membrane skeleton on the lateral diffusion of membrane proteins.

Single-particle tracking: effects of corrals

Structural proteins of the membrane skeleton are thought to form "corrals" at the membrane surface, and these corrals may restrict lateral diffusion of membrane proteins. Recent experimental developments in single-particle tracking and laser trapping make it possible to examine the corral model in detail. Techniques to interpret these experiments are presented. First, escape times for a diffusing particle in a corral are obtained from Monte Carlo calculations and analytical solutions for various corral sizes, shapes, and escape probabilities, and reduced to a common curve. Second, the identification of corrals in tracking experiments is considered. The simplest way to identify corrals is by sight. If the walls are impermeable enough, a trajectory fills the corral before the diffusing particle escapes. The fraction of distinct sites visited before escape is calculated for corrals of various sizes, shapes, and escape probabilities, and reduced to a common curve. This fraction is also a measure of the probability that the diffusing species will react with another species in the corral before escaping. Finally, the effect of the sampling interval on the measurement of the short-range diffusion coefficient is examined.

Patches and fences: probing for plasma membrane domains.

Cell plasma membranes appear to be composed of domains, patches whose composition and function differ from the average for an entire membrane surface. Proteins and lipids may be segregated into domains by different mechanisms. Some of these mechanisms are discussed, followed by a summary of the evidence for membrane domains obtained in my laboratory. This evidence is largely based on measurements of the lateral diffusion of membrane proteins and lipids. Recent new approaches to the interpretation of lateral diffusion measurements, consideration of so-called fractal or long time-tails promise to give new insights into the stability and lifetime of membrane domains.