Weak ergodicity breaking of receptor motion in living cells stemming from random diffusivity

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.Comment: 27 pages, 5 figure

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

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

Stochastic particle unbinding modulates growth dynamics and size of transcription factor condensates in living cells

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.)

Novel in vitro booster vaccination to rapidly generate antigen-specific human monoclonal antibodies

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