Mobility Of Membrane Proteins Research Articles

Mechanical regulation of lymphocyte activation and function.

Mechanosensing, or how cells sense and respond to the physical environment, is crucial for many aspects of biological function, ranging from cell movement during development to cancer metastasis, the immune response and gene expression driving cell fate determination. Relevant physical stimuli include the stiffness of the extracellular matrix, contractile forces, shear flows in blood vessels, complex topography of the cellular microenvironment and membrane protein mobility. Although mechanosensing has been more widely studied in non-immune cells, it has become increasingly clear that physical cues profoundly affect the signaling function of cells of the immune system. In this Review, we summarize recent studies on mechanical regulation of immune cells, specifically lymphocytes, and explore how the force-generating cytoskeletal machinery might mediate mechanosensing. We discuss general principles governing mechanical regulation of lymphocyte function, spanning from the molecular scale of receptor activation to cellular responses to mechanical stimuli.

Forces and Flows at Cell Surfaces.

Flow is an important physiological signal and modulates a variety of cell functions. However, the molecular mechanisms that cells use to sense flow have remained surprisingly opaque. The first steps in flow sensing are likely to occur at the plasma membrane, the fluid barrier between the inside and outside of a cell. This membrane is an organized, two-dimensional molecular array that has both solid and fluid properties. The mobility of membrane proteins and lipids is constrained by complex interactions with the cytoskeletal protein network that supports the membrane. Physiologically relevant flows can only generate tiny forces on individual proteins, smaller than those from thermal noise. However, cells could overcome this problem by sensing micron-scale concentration gradients of extracellular membrane proteins. This is possible in cell plasma membranes because their particular physical state allows flow to sort membrane proteins laterally over the extracellular surface.

Effect of hydrophobic and hydrophilic surfaces on the morphological and physical properties of supported lipid membranes.

The characteristics of supported lipid bilayers (SLBs) have long been studied as SLBs are commonly accepted as basic model systems of cell membranes. Atomic force microscopy (AFM) has proved to be effective in characterising physical and thermodynamic properties of SLBs, owing to its high resolution and the ability of environmental control. Through study of these properties, information such as lipid phase transitions can be investigated. Lipid phase transitions, from solid-ordered to liquid-disordered states, are well known to vary depending upon temperature, pH and ionic strength etc., and play a substantial role into events such as membrane protein mobility. Although previously studied on hydrophilic substrates, knowledge on the impact of hydrophobic substrates, such as van der Waals (vdW) crystals, on lipid phase transition is still lacking. This is particularly important for developing new bio- and electrochemical sensing technologies, where conductive vdW materials are used as sensing electrodes. It is expected that the thermodynamic properties of SLBs affect their conductive and dielectric polarisation and in turn molecular charge distribution and dielectric properties are known to strongly influence hydration layers occurring at the interface of lipids. Here, we present the study of adsorption of lipid membranes on various substrates, where morphological and physical (i.e. thermodynamic, electrical, and interfacial) properties of the membrane were investigated using advanced AFM modes as a function of temperature. We find that the transition temperature of the lipid monolayers greatly differs depending upon the chosen substrate. We then probe interfacial properties of these electrically charged interfaces. Finally, we demonstrate that substrates with different chemical-physical properties used as a lipids support have a substantial effect on the physical properties of lipid monolayers.

Resolving the hierarchical network of receptor tyrosine kinase interactions and the effect of oncogenic mutations.

Cells have an array of protein receptors on the plasma membrane that help process environmental cues. The spatial and temporal arrangement of these receptors is critical to function, but the chemical forces driving this organization are not well understood. This talk will focus on receptor tyrosine kinases (RTKs), which are transmembrane proteins that regulate cell growth, proliferation, and differentiation. Several RTKs are oncogenic and are targeted in next generation anti-cancer drug development. The degree to which these oncogenic mutations affect the local network of interactions between RTKs has not been explored beyond a few high-profile examples. We use pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to resolve membrane protein mobility, concentration, and monomer/dimer/oligomer distributions. I will describe ongoing work in my group to resolve the network of interactions between EGFR, HER2, HER3 and HER4, and how this network responds to oncogenic mutations.

In Situ Hydrogelation of Cellular Monolayers Enables Conformal Biomembrane Functionalization for Xeno-Free Feeder Substrate Engineering.

The intricate functionalities of cellular membranes have inspired strategies for deriving and anchoring cell-surface components onto solid substrates for biological studies, biosensor applications, and tissue engineering. However, introducing conformal and right-side-out cell membrane coverage onto planar substrates requires cumbersome protocols susceptible to significant device-to-device variability. Here, a facile approach for biomembrane functionalization of planar substrates is demonstrated by subjecting confluent cellular monolayer to intracellular hydrogel polymerization. The resulting cell-gel hybrid, herein termed GELL (gelated cell), exhibits extraordinary stability and retains the structural integrity, membrane fluidity, membrane protein mobility, and topology of living cells. In assessing the utility of GELL layers as a tissue engineering feeder substrate for stem cell maintenance, GELL feeder prepared from primary mouse embryonic fibroblasts not only preserves the stemness of murine stem cells but also exhibits advantages over live feeder cells owing to the GELL's inanimate, non-metabolizing nature. The preparation of a xeno-free feeder substrate devoid of non-human components is further shown with HeLa cells, and the resulting HeLa GELL feeder effectively sustains the growth and stemness of both murine and human induced pluripotent stem cells. The study highlights a novel bio-functionalization strategy that introduces new opportunities for tissue engineering and other biomedical applications.

Computation of the inverse Mittag–Leffler function and its application to modeling ultraslow dynamics

The inverse Mittag–Leffler function has been used to model the logarithmic growth of the mean squared displacement in anomalous diffusion, the restricted mobility of membrane proteins, and the slow viscoelastic creep observed in glasses. These applications are hindered because the inverse Mittag–Leffler function has no explicit form and cannot be approximated by existing methods in the domain $$x \in (0,+\infty )$$ . This study proposes a conversion method to compute the inverse Mittag–Leffler function in terms of the Mittag–Leffler function. The new method uses the one- and two-parameter Mittag–Leffler function to compute the inverse Mittag–Leffler function in the target domain. We apply this method to fit data collected in studies of: (i) the ultraslow mobility of beta-barrel proteins in bacterial membranes, (ii) the ultraslow creep observed in high strength self-compacting concrete, and (iii) the ultraslow relaxation seen in various glasses. The results show that the inverse Mittag–Leffler function can capture ultraslow dynamics in all three cases. This method may also be extended to other generalized logarithmic laws.

Diffusion State Transitions in Single-Particle Trajectories of MET Receptor Tyrosine Kinase Measured in Live Cells

Single-particle tracking enables the analysis of the dynamics of biomolecules in living cells with nanometer spatial and millisecond temporal resolution. This technique reports on the mobility of membrane proteins and is sensitive to the molecular state of a biomolecule and to interactions with other biomolecules. Trajectories describe the mobility of single particles over time and provide information such as the diffusion coefficient and diffusion state. Changes in particle dynamics within single trajectories lead to segmentation, which allows to extract information on transitions of functional states of a biomolecule. Here, mean-squared displacement analysis is developed to classify trajectory segments into immobile, confined diffusing, and freely diffusing states, and to extract the occurrence of transitions between these modes. We applied this analysis to single-particle tracking data of the membrane receptor MET in live cells and analyzed state transitions in single trajectories of the un-activated receptor and the receptor bound to the ligand internalin B. We found that internalin B-bound MET shows an enhancement of transitions from freely and confined diffusing states into the immobile state as compared to un-activated MET. Confined diffusion acts as an intermediate state between immobile and free, as this state is most likely to change the diffusion state in the following segment. This analysis can be readily applied to single-particle tracking data of other membrane receptors and intracellular proteins under various conditions and contribute to the understanding of molecular states and signaling pathways.

Aggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells.

To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.

Cell-Free Synthesis of a Transmembrane Mechanosensitive Channel Protein into a Hybrid-Supported Lipid Bilayer.

Supported lipid bilayers (SLBs) hold tremendous promise as cellular-mimetic structures that can be readily interfaced with analytical and screening tools. The incorporation of transmembrane proteins, a key component in biological membranes, is a significant challenge that has limited the capacity of SLBs to be used for a variety of biotechnological applications. Here, we report an approach using a cell-free expression system for the cotranslational insertion of membrane proteins into hybrid-supported lipid bilayers (HSLBs) containing phospholipids and diblock copolymers. We use cell-free expression techniques and a model transmembrane protein, the large conductance mechanosensitive channel (MscL), to demonstrate two routes to integrate a channel protein into a HSLB. We show that HSLBs can be assembled with integrated membrane proteins by either cotranslational integration of protein into hybrid vesicles, followed by fusion of these proteoliposomes to form a HSLB, or preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB. Both approaches lead to the assembly of HSLBs with oriented proteins. Notably, using single-particle tracking, we find that the presence of diblock copolymers facilitates membrane protein mobility in the HSLBs, a critical feature that has been difficult to achieve in pure lipid SLBs. The approach presented here to integrate membrane proteins directly into preformed HSLBs using cell-free cotranslational insertion is an important step toward enabling many biotechnology applications, including biosensing, drug screening, and material platforms requiring cell membrane-like interfaces that bring together the abiotic and biotic worlds and rely on transmembrane proteins as transduction elements.

Alterations in the Properties of Red Blood Cells in Men with Coronary Artery Diseases after Comprehensive Cardiac Rehabilitation.

Purpose Comprehensive cardiac rehabilitation (CCR) is a complex program aimed at improving the health status of patients with coronary artery disease (CAD), especially those who have been subjected to cardiac interventions (PCI and CABG).The aim of this study was to measure the changes in the properties of red blood cells (RBCs) in men with CAD after cardiac intervention and after participation in CCR program. Methods In this study, we have investigated the influence of the physical training-based CCR program in 12 men with CAD, after PCI or CABG. The characteristics of RBCs including the basic morphology of RBCs, the conformational state of RBC membrane protein and hemoglobin, acetylcholinesterase activity, membrane fluidity, the osmotic fragility, and thiol concentration in membrane and in hemolysate were measured. Ascorbate concentration and reduced glutathione were also determined. The analysis was performed in men, before and after participation in CCR. The properties of RBCs were observed in connection with the exercise test, and parameters were evaluated before, immediately after, and 1 hour after the exercise test. Results After CCR, a decrease in the mobility of erythrocyte membrane proteins was observed, which was accompanied by a decrease in lipid fluidity. In addition, immediately after the exercise test and 1 hour later, we measured a decrease in thiol level in hemolysate, but not in the plasma membrane. Unexpectedly, an increase in reduced glutathione concentration one hour after the exercise test after completing comprehensive cardiac rehabilitation was observed. Conclusion CCR in men with CAD after cardiac intervention is connected with decreased membrane fluidity and decreased membrane protein mobility, which indicates that reduction of oxidative changes in these components occurs.

Resolving Membrane Protein-Protein Interactions in Live Cells with Pulsed Interleaved Excitation Fluorescence Cross-Correlation Spectroscopy.

The cell plasma membrane (PM) contains thousands of proteins that sense and respond to the outside environment. These proteins have evolved sensitivity to a wide variety of physical and chemical signals and act as a delivery system across the PM. Membrane proteins are critical for information flow and decision making in the cell and thus are important targets in drug development. A critical aspect of membrane protein function is the way they interact with other proteins, often through the formation of dimers or small oligomers that regulate function at the protein, cell, and organism levels. Resolving membrane protein interactions in a live cell environment is challenging because of the chemical diversity and spatial heterogeneity of the PM. In this Account, we describe a fluorescence technique called pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) that is ideally suited to quantify membrane associations in live cells. PIE-FCCS is a two-color fluorescence fluctuation method that can simultaneously measure the concentration, mobility, proximity, and oligomerization state of membrane proteins in situ. It has several advantages over two related approaches, single-molecule tracking (SMT) and Förster resonance energy transfer (FRET), including that it measures all of the properties listed above in a single measurement. Another advantage is that PIE-FCCS is most sensitive at the physiological expression levels for many membrane proteins rather than the very low or high levels typical in other techniques. Here, we review the history of FCCS as it has been applied to study membrane protein interactions in cells. We also describe PIE-FCCS and the advantages it has over biochemical approaches like coimmunoprecipitation (co-IP) and proximity ligation assays (PLA). Finally, we review two classes of membrane proteins that have been studied with FCCS and PIE-FCCS: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). For RTKs, ligand induced dimerization directly regulates the catalytic activity of the kinase, but higher order oligomerization and ligand-independent dimerization can complicate this historically simple paradigm. PIE-FCCS data have resolved a low population of EGFR dimers under basal conditions and assembly into multimers when stimulated with ligand. While GPCRs function primarily as monomers, dimerization has been hypothesized to regulate function for some receptors. PIE-FCCS data have established the dimerization potential of rhodopsin at low densities and were critical for the discovery of a novel dimerization interface in human cone opsins. This Account describes the how FCCS and PIE-FCCS can reveal the details of quaternary interactions in each of these receptor systems.

Interferometric Scattering Microscopy Reveals Microsecond Nanoscopic Protein Motion on a Live Cell Membrane

Tracking the diffusive mobility of individual membrane proteins within the cell offers a route to unravel how protein function is sculpted by the architecture of the membrane and its constituents. To meet this challenge fully, however, demands a high resolution in both space and time which exceeds the capabilities of fluorescence microscopies. Interferometric scattering (iSCAT) microscopy is an emerging fluorescence-free technique highly suited to the sensitive detection and precise tracking of nanoscopic biological entities, including viruses, vesicles, and individual proteins, both in artificial and live-cell contexts. In this presentation, we demonstrate successful implementation of iSCAT microscopy to track proteins within the plasma membrane of the living cell to nanometric precision in all three-dimensions whilst at fast microsecond speeds. We will discuss our recent results resolving nanoscopic membrane confinement, uptake and prolonged directed diffusion along a filopodium for the epidermal growth factor receptor protein. In addition, we will also discuss the future prospects and challenges for iSCAT microscopy in exploring single-molecule dynamics within living cells.

Determination of δ-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis

Fluorescence recovery after photobleaching (FRAP) is the preferred method for analyzing the lateral mobility of fluorescently-tagged proteins in the plasma membranes (PMs) of live cells. FRAP experiments are described as being easy to perform; however, the analysis of the acquired data can be difficult. The evaluation procedure must be properly combined with the imaging setup of the confocal microscope to provide unbiased results.With the aim of increasing the accuracy of determining the diffusion coefficient (D) and mobile fraction (Mf) of PM proteins, we developed a novel method for FRAP analysis in the equatorial plane of the cell. This method is based on the calculation of photobleaching characteristics, derived from the light intensity profile and optical parameters of the confocal microscope, and on the model of fluorescent molecule diffusion in PM regions outside of the focal plane. Furthermore, cell movement artifacts in the FRAP data are ameliorated by using a region of interest, which is not fixed but instead moves adaptively in coordination with the movement of cells.When this method was used to determine the mobility of the δ-opioid receptor-eYFP in HEK293 cells, a highly significant decrease in receptor mobility was detected in cholesterol-depleted cells. This decrease was fully reversible by the replenishment of cholesterol levels. Our results demonstrate the crucial role played by cholesterol in the dynamic organization of δ-opioid receptors in the PM under in vivo conditions. Our method may be applied for the determination of the D and Mf values of other PM proteins.

Intracellular hydrogelation preserves fluid and functional cell membrane interfaces for biological interactions

Cell membranes are an intricate yet fragile interface that requires substrate support for stabilization. Upon cell death, disassembly of the cytoskeletal network deprives plasma membranes of mechanical support and leads to membrane rupture and disintegration. By assembling a network of synthetic hydrogel polymers inside the intracellular compartment using photo-activated crosslinking chemistry, we show that the fluid cell membrane can be preserved, resulting in intracellularly gelated cells with robust stability. Upon assessing several types of adherent and suspension cells over a range of hydrogel crosslinking densities, we validate retention of surface properties, membrane lipid fluidity, lipid order, and protein mobility on the gelated cells. Preservation of cell surface functions is further demonstrated with gelated antigen presenting cells, which engage with antigen-specific T lymphocytes and effectively promote cell expansion ex vivo and in vivo. The intracellular hydrogelation technique presents a versatile cell fixation approach adaptable for biomembrane studies and biomedical device construction.

GABAAR isoform and subunit structural motifs determine synaptic and extrasynaptic receptor localisation

GABAA receptors (GABAARs) are the principal inhibitory neurotransmitter receptors in the central nervous system. They control neuronal excitability by synaptic and tonic forms of inhibition mostly mediated by different receptor subtypes located in specific cell membrane subdomains. A consensus suggests that α1-3βγ comprise synaptic GABAARs, whilst extrasynaptic α4βδ, α5βγ and αβ isoforms largely underlie tonic inhibition. Although some structural features that enable the spatial segregation of receptors are known, the mobility of key synaptic and extrasynaptic GABAARs are less understood, and yet this is a key determinant of the efficacy of GABA inhibition. To address this aspect, we have incorporated functionally silent α-bungarotoxin binding sites (BBS) into prominent hippocampal GABAAR subunits which mediate synaptic and tonic inhibition. Using single particle tracking with quantum dots we demonstrate that GABAARs that are traditionally considered to mediate synaptic or tonic inhibition are all able to access inhibitory synapses. These isoforms have variable diffusion rates and are differentially retained upon entering the synaptic membrane subdomain. Interestingly, α2 and α4 subunits reside longer at synapses compared to α5 and δ subunits. Furthermore, a high proportion of extrasynaptic δ-containing receptors exhibited slower diffusion compared to δ subunits at synapses. A chimera formed from δ-subunits, with the intracellular domain of γ2L, reversed this behaviour. In addition, we observed that receptor activation affected the diffusion of extrasynaptic, but not of synaptic GABAARs. Overall, we conclude that the differential mobility profiles of key synaptic and extrasynaptic GABAARs are determined by receptor subunit composition and intracellular structural motifs.This article is part of the special issue entitled ‘Mobility and trafficking of neuronal membrane proteins’.

Array of Freestanding Planar Lipid Bilayers for Parallel Optical and Electrical Recordings

Here we report on the design and validation of an array platform for parallel high-resolution electrophysiological and optical recordings. The recording chip is based on a SU-8 coated glass slide (ca. 200 μm thickness) containing 4 cavities with individual ring-shaped Ag/AgCl-microelectrodes as well as a common ground electrode. Four suspended lipid bilayers can be self-assembled on the chip surface over the cavities. The electrodes are connected to a multichannel amplifier capable of simultaneous electrical recording with high-bandwidth and low noise (<1pA [email protected]). The ring shape of the electrodes creates an optical window allowing access with a high-NA oil or water-immersion objective on an inverted microscope. This allows for e.g. single-molecule fluorescence measurements from one of the four bilayers using time-correlated-single-photon-counting (TCSPC). Fluorescently labeled alpha-hemolysin for optical and electrical measurements was expressed in-vitro and purified. BODIPY at position 131 was incorporated during the translation in a site-directed manner via stop or four-base codon suppression using pre-charged tRNAs. A series of characterization and validation experiments performed with the system include: (1)Studies of diffusion rates of fluorescently labelled Hemolysin and lipid molecules in the bilayer using fluorescence correlation spectroscopy (FCS); (2)Monitoring of alpha-hemolysin membrane insertion using fluorescence lifetime imaging (FLIM) and (3)Visualization of fusion of labeled proteoliposomes resulting in membrane channel insertion. In the two latter cases, changes in fluorescence intensity were monitored simultaneously with alterations in membrane conductance. This system allows for rapid membrane array formation and by providing ready combination of fluorescence microscopy with high-resolution electrophysiology, enables correlated optical and electrical characterization and monitoring of the interactions of channel forming proteins in artificial lipid bilayers on a single-molecule level. The technology can be further implemented for studies of membrane protein folding and mobility as well as of membrane transport.

Effect of the volatile anesthetic agent isoflurane on lateral diffusion of cell membrane proteins.

The volatile anesthetic isoflurane (ISO) has previously been shown to increase the fluidity of artificial lipid membranes, but very few studies have used biological cell membranes. Therefore, to investigate whether ISO affects the mobility of membrane proteins, fluorescence‐labeled transferrin receptor (TfR) and glycosylphosphatidylinositol (GPI)‐anchored protein were expressed in human embryonic kidney 293T cells and neural cells and lateral diffusion was examined using fluorescence recovery after photobleaching. Lateral diffusion of the TfR increased with ISO treatment. On the other hand, there was no effect on GPI‐anchored protein. We also used GC/MS to confirm that there was no change in the concentration of ISO due to vaporization during measurement. These results suggest that ISO affects the mobility of transmembrane protein molecules in living cells.

A Minimal Coarse-Grained Molecular Dynamics Model of Axon Plasma Membrane with its Implication on the Diffusion Behavior of Axon Membrane Proteins

The axon initial segment (AIS) in the neuron has two crucial functions: maintaining the polarity of neurons and initiation of action potentials. One extraordinary feature of the AIS is the separation of somatodendritic and axonal domains which was confirmed by measuring the trajectory of beads attached to various axonal membrane proteins that there exists a diffusion barrier in the AIS restricting the motion of membrane proteins and lipids. According to the “fences and pickets” model, the periodic cytoskeleton and the highly concentrated transmembrane proteins in the AIS are capable of hindering the diffusion of other proteins and lipids through steric interaction and hydrodynamic friction effect. In this work, we established the first coarse-grained molecular dynamics model of axon plasma membrane which includes both axon membrane skeleton and axonal membranes to investigate the diffusion behavior of membrane proteins and lipids in the AIS from a mechanics aspect. First, we found that transmembrane proteins and proteins in the inner lipid leaflet are confined within the areas between the actin rings. We suspected and proved from the computational model that the size of actin associated protein junctions, which connected to the lipid bilayers may restrict the motion of proteins in the outer leaflet. Then we showed that the spectrin-lipid attraction level will affect the circumferential mobility of membrane proteins. Finally, the highly dense membrane proteins slowed down the motion of all membrane proteins even the lipids. We conclude that actin rings and spectrin filaments restrict the motion of membrane proteins along and around the axon, respectively. However, the main mechanism forming the diffusion barrier in the AIS is the accumulation of concentrated transmembrane proteins.

Dynamic imaging of mitochondrial membrane proteins in specific sub-organelle membrane locations.

Mitochondria are cellular organelles with multifaceted tasks and thus composed of different sub-compartments. The inner mitochondrial membrane especially has a complex nano-architecture with cristae protruding into the matrix. Related to their function, the localization of mitochondrial membrane proteins is more or less restricted to specific sub-compartments. In contrast, it can be assumed that membrane proteins per se diffuse unimpeded through continuous membranes. Fluorescence recovery after photobleaching is a versatile technology used in mobility analyses to determine the mobile fraction of proteins, but it cannot provide data on subpopulations or on confined diffusion behavior. Fluorescence correlation spectroscopy is used to analyze single molecule diffusion, but no trajectory maps are obtained. Single particle tracking (SPT) technologies in live cells, such as tracking and localization microscopy (TALM), do provide nanotopic localization and mobility maps of mitochondrial proteins in situ. Molecules can be localized with a precision of between 10 and 20nm, and single trajectories can be recorded and analyzed; this is sufficient to reveal significant differences in the spatio-temporal behavior of diverse mitochondrial proteins. Here, we compare diffusion coefficients obtained by these different technologies and discuss trajectory maps of diverse mitochondrial membrane proteins obtained by SPT/TALM. We show that membrane proteins in the outer membrane generally display unhindered diffusion, while the mobility of inner membrane proteins is restricted by the inner membrane architecture, resulting in significantly lower diffusion coefficients. Moreover, tracking analysis could discern proteins in the inner boundary membrane from proteins preferentially diffusing in cristae membranes, two sub-compartments of the inner mitochondrial membrane. Thus, by evaluating trajectory maps it is possible to assign proteins to different sub-compartments of the same membrane.

Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane

The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.