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Single particle tracking experiments have recently uncovered that the motion of cell membrane components can undergo changes of diffusivity as a result of the heterogeneous environment, producing subdiffusion and nonergodic behavior. In this paper, we show that an autoregressive fractionally integrated moving average (ARFIMA) with noise given by generalized autoregressive conditional heteroscedasticity (GARCH) can describe inhomogeneous diffusion in the cell membrane, where the ARFIMA process models anomalous diffusion and the GARCH process explains a fluctuating diffusion parameter.

A current challenge in cell motility studies is to understand the molecular and physical mechanisms that govern chemokine receptor nanoscale organization at the cell membrane, and their influence on cell response. Using single-particle tracking and super-resolution microscopy, we found that the chemokine receptor CXCR4 forms basal nanoclusters in resting T cells, whose extent, dynamics, and signaling strength are modulated by the orchestrated action of the actin cytoskeleton, the co-receptor CD4, and its ligand CXCL12. We identified three CXCR4 structural residues that are crucial for nanoclustering and generated an oligomerization-defective mutant that dimerized but did not form nanoclusters in response to CXCL12, which severely impaired signaling. Overall, our data provide new insights to the field of chemokine biology by showing that receptor dimerization in the absence of nanoclustering is unable to fully support CXCL12-mediated responses, including signaling and cell function in vivo, This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF 2014-53416-R, SAF 2017-82940-R AEI/FEDER, EU) and the RETICS Program (RD16/0012/0006; RIER), Ministry of Economy and Competitiveness, Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2013-0347; SEV-2015-0522), and Fundación Privada Cellex and Generalitat de Catalunya (CERCA program). L.M.-M. is supported by the COMFUTURO program of the Spanish Research Council General Foundation.

Invariant Natural killer T (iNKT) cells are a subset of lipid-specific T cells, restricted by the MHC-I class like molecule CD1d. A recent study showed a correlation between the co-localization of lipid-loaded CD1d molecules with lipid rafts on the membrane of antigen presenting cells, and their capacity of eliciting secretion of Th1-cytokines by stimulated iNKT cells [1]. Thus, this study suggested that not only the structure of CD1d bound to an exogenous lipid could influence Cd1d-mediated immunity, but also its partitioning on the membrane. Here, we address the spatiotemporal behaviour of α-Galactosylceramide loaded CD1d complexes on the cell membrane of human myeloid cells using multiple colour high-speed single-particle tracking (100 Hz) combined with an iNKT T Cell Receptor-Qdot conjugate as imaging probe. Furthermore, we complement these studies using STED nanoscopy to obtain nanoscale images of CD1d spatial organization. Our results indicate a direct role of the actin cytoskeleton in actively segregating CD1d nanoclusters on the cell membrane resulting in an inhibition of the activation of iNKT cells [2]. As a whole, our work proposes a new paradigm of biophysical interaction between CD1d presenting cells and NKT cells which deviates significantly from classical MHCI/II complexes and CD8/CD4-T Cells interactions.[1] J. S. Im et al., Immunity, 30, 888, 2009.[2] J. A. Torreno-Pina et al., in preparation.

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters.

Glycan-protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. We applied a combination of super-resolution nanoscopy and single molecule-based approaches to study the role of glycan-based interactions on the dynamics of the glycosylated pathogen recognition receptor DC-SIGN, at the nano- and micrometer scale. We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN in nanoclusters but restrict the mobility of the receptor to distinct micron-size membrane regions. These meso-scale regions are in turn enriched by the endocytic protein clathrin, thereby dynamically promoting DC-SIGN transient nano-scale arrest and interaction with clathrin. Disruption of glycan-based connectivity leads to larger membrane exploration, reduced clathrin interaction and compromised clathrin-dependent internalization of virus-like particles. Therefore, our work uncovers a novel mechanism through which glycan-protein interactions act as decision-makers in fine-tuning membrane-related functions by dynamically coupling micro- and nanoscale receptor lateral mobility, thus adding a new layer of regulation to the hierarchical spatiotemporal organization of the cell membrane.

Molecular transport in living systems regulates numerous processes underlying biological function. Although many cellular components exhibit anomalous diffusion, only recently has the subdiffusive motion been associated with nonergodic behavior. These findings have stimulated new questions for their implications in statistical mechanics and cell biology. Is nonergodicity a common strategy shared by living systems? Which physical mechanisms generate it? What are its implications for biological function? Here, we use single particle tracking to demonstrate that the motion of DC-SIGN, a receptor with unique pathogen recognition capabilities, reveals nonergodic subdiffusion on living cell membranes. In contrast to previous studies, this behavior is incompatible with transient immobilization and therefore it can not be interpreted according to continuous time random walk theory. We show that the receptor undergoes changes of diffusivity, consistent with the current view of the cell membrane as a highly dynamic and diverse environment. Simulations based on a model of ordinary random walk in complex media quantitatively reproduce all our observations, pointing toward diffusion heterogeneity as the cause of DC-SIGN behavior. By studying different receptor mutants, we further correlate receptor motion to its molecular structure, thus establishing a strong link between nonergodicity and biological function. These results underscore the role of disorder in cell membranes and its connection with function regulation. Due to its generality, our approach offers a framework to interpret anomalous transport in other complex media where dynamic heterogeneity might play a major role, such as those found, e.g., in soft condensed matter, geology and ecology., 27 pages, 5 figures

The spatial organization of membrane receptors at the nanoscale has major implications in cellular function and signaling. The advent of super-resolution techniques has greatly contributed to our understanding of the cellular membrane. Yet, despite the increased resolution, unbiased quantification of highly dense features, such as molecular aggregates, remains challenging. Here we describe an algorithm based on Bayesian inference of the marker intensity distribution that improves the determination of molecular positions inside dense nanometer-scale molecular aggregates. We tested the performance of the method on synthetic images representing a broad range of experimental conditions, demonstrating its wide applicability. We further applied this approach to STED images of GPI-anchored and model transmembrane proteins expressed in mammalian cells. The analysis revealed subtle differences in the organization of these receptors, emphasizing the role of cortical actin in the compartmentalization of the cell membrane.

Item does not contain fulltext Glycan-protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN-clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.

Lipid rafts, cell membrane domains with unique composition and properties, modulate the membrane distribution of receptors and signaling molecules facilitating the assembly of active signaling platforms. However, the underlying mechanisms that link signal transduction and lipid rafts are not fully understood, mainly because of the transient nature of these membrane assemblies. Several methods have been used to study the association of membrane receptors with lipid rafts. In the first part of this chapter, a description of how biochemical methods such as raft disruption by cholesterol depletion agents are useful in qualitatively establishing protein association with lipid rafts is presented. The second part of this chapter is dedicated to imaging techniques used to study membrane receptor organization and lipid rafts. We cover conventional approaches such as confocal microscopy to advanced imaging techniques such as homo-FRET microscopy and superresolution methods. For each technique described, their advantages and drawbacks are discussed.

The plasma membrane of eukaryotic cells is responsible for a myriad of functions that regulate cell physiology and plays a crucial role in a multitude of processes that include adhesion, migration, signaling recognition and cell–cell communication. This is accomplished by specific interactions between different membrane components such as lipids and proteins on the lipid bilayer but also through interactions with the underlying cortical actin cytoskeleton on the intracellular side and the glycocalyx matrix in close proximity to the extracellular side. Advanced biophysical techniques, including single particle tracking (SPT) have revealed that the lateral diffusion of molecular components on the plasma membrane represents a landmark manifestation of such interactions. Indeed, by studying changes in the diffusivity of individual membrane molecules, including sub-diffusion, confined diffusion and/or transient arrest of molecules in membrane compartments, it has been possible to gain insight on the nature of molecular interactions and to infer on its functional role for cell response. In this review, we will revise some exciting results where SPT has been crucial to reveal homo- and hetero-interactions on the cell membrane.

Contains fulltext : 109735.pdf (Publisher’s version ) (Open Access) The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length scale. Insight into how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection.

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small ( ca. 5%) subset of conformational-active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca 2+ resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.

Contains fulltext : 139177.pdf (Publisher’s version ) (Open Access) Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at nanoscale lengths. In this Commentary, we present selected examples of glycosylphosphatidyl-anchored proteins, Ras family members and several immune receptors that provide evidence for nanoclustering. We advocate the view that nanoclustering is an important part of the hierarchical organization of proteins in the plasma membrane. According to this emerging picture, nanoclusters can be organized on the mesoscale to form microdomains that are capable of supporting cell adhesion, pathogen binding and immune cell-cell recognition amongst other functions. Yet, a number of outstanding issues concerning nanoclusters remain open, including the details of their molecular composition, biogenesis, size, stability, function and regulation. Notions about these details are put forth and suggestions are made about nanocluster function and why this general feature of protein nanoclustering appears to be so prevalent.

Receptor nanoclustering prior to ligand activation is rapidly emerging as an essential feature common to most cell membranes. Yet, the mechanisms governing this distinct spatial patterning and functional role are still poorly understood. We used a combination of biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, to investigate the spatiotemporal organization of DC-SIGN, a pathogen recognition receptor that homo-oligomerizes in-vitro. We found an intrinsic nanoclustering capacity of DC-SIGN (ca. 180 nm in size) far beyond basal tetramerization, which strictly depended on its molecular structure. DC-SIGN nanoclusters exhibited Brownian diffusion on the cell membrane with values of the order of 10−2 μm2/s. Truncation of the neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution compromised DC-SIGN binding to nanoscale size pathogens. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length-scale. We suggest that protein-protein interactions facilitated by structural molecular motifs might represent a so far underestimated but general mechanism to pre-organize receptors on the cell membrane for efficient action under stimulated conditions.

The application of techniques such as fluorescence correlation spectroscopy and single particle tracking to the plasma membrane of living cells continuously evidences subdiffusive motion of proteins and lipids. This anomalous motion is generally associated to the interplay of molecular crowding, diffusion barriers and specific interactions. Addressing the cause of subdiffusion is essential for understanding molecular mechanisms underlying cellular function, such as target search, kinetics of transport-limited reactions, trafficking and signalling. Recently, the subdiffusive motion of some cellular and membrane components has been associated to weak ergodicity breaking and attributed to transient immobilization caused by interactions with microtubules or actin cytoskeleton. These works have opened new questions about the role of nonergodic subdiffusion in the dynamics of living systems.By means of single particle tracking experiments, we show that the dynamics of DC-SIGN, a transmembrane receptor with unique pathogen recognition capabilities, also reveals subdiffusion with signatures of weak ergodicity breaking and aging. In contrast to other biological systems, we demonstrate that DC-SIGN nonergodic subdiffusion cannot be explained by transient immobilization, but rather is compatible with dynamical heterogeneity induced by spatiotemporal changes of diffusivity. While nonergodic diffusion due to long-lived immobilization events can be understood within the framework of continuous-time random walk, our experimental data are accurately interpreted through a new theoretical model describing anomalous transport in biological systems and complex media. A comprehensive analysis of mutated forms of the receptor allowed us to establish a link between receptor molecular structure, nonergodic diffusion and function. Our results underscore the role of spatiotemporal disorder in the dynamics of cell membrane receptors. In addition, they have broad implications to other heterogeneous systems where the occurrence of nonergodicity remains unexplored.