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In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (PR), a ligand-activated transcription factor. A small fraction of PR is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ERα). Upon hormone exposure, either directly or via interaction with ERα mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of PR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the PR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular PR. This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent of hormone binding to intracellular PR. We found that a membrane-attached progestin can activate mbPR, the ERK signalling pathway leading to PR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.

Liquid-liquid phase separation (LLPS) is emerging as a key physical principle for biological organization inside living cells, forming condensates that play important regulatory roles. Inside living nuclei, transcription factor (TF) condensates regulate transcriptional initiation and amplify the transcriptional output of expressed genes. However, the biophysical parameters controlling TF condensation are still poorly understood. Here we applied a battery of single-molecule imaging, theory, and simulations to investigate the physical properties of TF condensates of the progesterone receptor (PR) in living cells. Analysis of individual PR trajectories at different ligand concentrations showed marked signatures of a ligand-tunable LLPS process. Using a machine learning architecture, we found that receptor diffusion within condensates follows fractional Brownian motion resulting from viscoelastic interactions with chromatin. Interestingly, condensate growth dynamics at shorter times is dominated by Brownian motion coalescence (BMC), followed by a growth plateau at longer timescales that result in nanoscale condensate sizes. To rationalize these observations, we extended on the BMC model by including the stochastic unbinding of particles within condensates. Our model reproduced the BMC behavior together with finite condensate sizes at the steady state, fully recapitulating our experimental data. Overall, our results are consistent with condensate growth dynamics being regulated by the escaping probability of PR molecules from condensates. The interplay between condensation assembly and molecular escaping maintains an optimum physical condensate size. Such phenomena must have implications for the biophysical regulation of other nuclear condensates and could also operate in multiple biological scenarios. The research leading to these results has received funding from BIST-Ignite funding (PHASE-CHROM) (to C.R.-A. and J.A.T.-P.); the European Commission H2020 Program under grant agreement ERC Adv788546 (NANO-MEMEC) (to M.F.G.-P.), ERC AdG NOQIA and EU Horizon 2020 FET-OPEN OPTOlogic (Grant No 899794) (to M.L.), and ERC Synergy Grant 609989 (to M.B.); the Government of Spain (Severo Ochoa CEX2019-000910-S (to M.L. and to M.F.G.P.), JdC-IJCI-2017-33160 (to J.A.T.-P.), (PGC2018-097027-B-I00/10.13039/501100011033, CEX2019-000910-S/10.13039/501100011033 and QUSPIN RTC2019-007196-7) (to M.L.), the State Research Agency (FIDEUA PID2019-106901GB-I00/10.13039/501100011033) (to M.L.), PID2020-113068RB-I00/10.13039/501100011033 (to M.F.G.-P.) and (RYC-2017–22227 and PID2019-106232RB-I00/10.13039/501100011033) (to F.C.); QuantumCAT_U16-011424 (to M.L.), co-funded by the ERDF Operational Program of Catalonia 2014-2020; (QUANTERA MAQS (funded by the State Research Agency under PCI2019-111828-2/321 10.13039/501100011033) and QUANTERA DYNAMITE PCI2022-132919) (to M.L.); Barcelona Supercomputing Center MareNostrum (FI-2022-1-0042) (to M.L.); Obra Social La Caixa (LCF-ICFO) and the Austrian Science Fund (FWF) through SFB BeyondC F7102 (to G.M.-G.); National Science Centre, Poland (Symfonia Grant No. 2016/20/W/ST4/00314) (to M.L.); Fundació CELLEX (Barcelona); Fundació Mir-Puig; and the Generalitat de Catalunya through the CERCA program and AGAUR (grant no. 2017 SGR 1341 to M.L. and no. 2017SGR1000 to M.F.G.-P.).

Liquid-liquid phase separation (LLPS) is emerging as key physical principle for biological organization inside living cells, forming condensates that play important roles in the regulation of multiple functions. Inside living nuclei, transcription factor (TF) condensates regulate transcriptional initiation and amplify transcriptional output of expressed genes. Yet, the biophysical parameters controlling TF condensation are still poorly understood. Here we applied a battery of single molecule imaging tools, theory and simulations to investigate the physical properties of TF condensates of the Progesterone Receptor (PR) in vivo. Analysis of individual PR trajectories at different ligand concentrations showed marked signatures of a ligand-tunable and regulated LLPS process. Using a machine learning architecture, we uncovered that diffusion within condensates follows fractional Brownian motion, reflecting viscoelastic interactions between PR and chromatin within condensates. High density single molecule localization maps further revealed that condensate growth dynamics is dominated by Brownian motion coalescence (BMC) at shorter times, but deviate at longer timescales reaching a growth plateau with nanoscale condensate sizes. To understand our observations we developed an extension of the BMC model by including stochastic unbinding of particles within condensates. The model reproduced the BMC behavior together with finite condensate sizes a steady-state, fully recapitulating our experimental data. Our results are thus consistent with droplet growth dynamics being regulated by the escaping probability of TFs molecules from condensates. The interplay between condensation assembly and molecular escaping maintains an optimum physical condensate size. Such phenomena must have implications for the biophysical regulation of other TF condensates and could also operate in multiple biological scenarios.

Phase separation is emerging as key principle in the spatiotemporal organization of living cells. Given its relevance in the regulation of numerous biological functions, including gene transcription and chromatin architecture, modeling biomolecular condensation is gaining interest. Yet, most models developed so far rely on specific descriptions and/or experimentally inaccessible properties. Here we propose a theoretical model, where phase separation is explained by means of interaction probabilities between particles. With minimum model requirements, particle condensates emerge above a critical interaction probability. We tested the model predictions with single molecule experiments of tunable transcription factor condensates in the nucleus of living cells. Phase separation, condensate sizes, diffusion behavior, and mobility parameters, quantified by data analysis and machine learning, are fully recapitulated by our model. Our combined theoretical and experimental approach provides a general framework to investigate the biophysical parameters controlling phase separation in living cells and in other soft matter-based interacting systems.

Sanjuan Nandin et al. describe an innovative approach based on antigen-dependent activation of human memory B cells in culture. It results in the rapid generation of human antibodies against infectious agents and offers the potential for therapeutic antibody production and vaccine development., Vaccines remain the most effective tool to prevent infectious diseases. Here, we introduce an in vitro booster vaccination approach that relies on antigen-dependent activation of human memory B cells in culture. This stimulation induces antigen-specific B cell proliferation, differentiation of B cells into plasma cells, and robust antibody secretion after a few days of culture. We validated this strategy using cells from healthy donors to retrieve human antibodies against tetanus toxoid and influenza hemagglutinin (HA) from H1N1 and newly emergent subtypes such as H5N1 and H7N9. Anti-HA antibodies were cross-reactive against multiple subtypes, and some showed neutralizing activity. Although these antibodies may have arisen as a result of previous influenza infection, we also obtained gp120-reactive antibodies from non–HIV-infected donors, indicating that we can generate antibodies without prior antigenic exposure. Overall, our novel approach can be used to rapidly produce therapeutic antibodies and has the potential to assess the immunogenicity of candidate antigens, which could be exploited in future vaccine development.

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.

Nonergodicity observed in single-particle tracking experiments is usually modeled by transient trapping rather than spatial disorder. We introduce models of a particle diffusing in a medium consisting of regions with random sizes and random diffusivities. The particle is never trapped, but rather performs continuous Brownian motion with the local diffusion constant. Under simple assumptions on the distribution of the sizes and diffusivities, we find that the mean squared displacement displays subdiffusion due to non-ergodicity for both annealed and quenched disorder. The model is formulated as a walk continuous in both time and space, similar to the L\'{e}vy walk., Comment: 6 pages, 2 figures; removed spurious pi from (13), and k from inline eqn preceding (11). Fixed other typos

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.

Fluorescence nanoscopy, both in the far-field (STED) and near-field version (NSOM) allows imaging of biological samples with exquisite resolution and high signal-to-noise ratio, representing a powerful tool to address biological questions at the nanometer scale. Yet, despite the highly improved resolution and quality in comparison with conventional confocal microscopy, Poisson noise, background noise and cell autofluorescence are limiting factors for the automated quantification of molecular spatial organization as well as for the recognition of distinct features such as clusters and aggregates. For these reasons, the analysis of nanoscopy images, if at all, is often performed in a semi-automatic fashion, requiring the manual identification of image features and the supply of user-defined parameters. As such, these approaches are time-consuming and might suffer from non-objective evaluation, which are critical parameters in the analysis of samples with high emitters density.Here we propose a method that allows fully automated reconstruction of fluorescence nanoscopy images and quantitative analysis of protein spatial organization. Our approach is based on the combination of a maximum likelihood algorithm and an expectation-maximization step, by means of which the image is de-noised and decomposed into point-spread functions. The further application of a local maxima identifying routine and Voronoi tessellation, allow us to extract the maximum information from the fluorescence images without impacting on the optical resolution. The performance of the method has been tested on simulated images at varying emitter density and signal-to-noise ratio, showing its applicability to standard STED and NSOM images. Furthermore, we have successfully applied this algorithm to quantitatively analyze fluorescence nanoscopy images of high densely packed membrane receptors on mammalian cells. Our results show faithful retrieval of receptor positions, stoichiometry and their lateral distribution of receptors on the cell membrane.

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.