We show a hierarchical organization of the adhesion receptor CD44 at multiple-length scales on the plasma membrane by studying the diffusion and clustering behavior of the protein.

23 24 25 26 27 28 29 2 Keywords: actomyosin, cartography, CD44, fluorescence emission anisotropy, formin, homo-30 FRET, meshwork, meso-scale organization, nanoclustering, nano-scale organization, plasma 31 membrane, single particle tracking 32 Abbreviations: ECD-extra-cellular domain; ICD-intra-cellular domain; ECM-extra-cellular 33 matrix; FRET -Forster"s resonance energy transfer; HA-hyaluronic acid; SPT-single particle 34tracking; DC-SPT-dual color single particle tracking 35 36 37 Abstract: 38Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with 39 modules to interact with the extracellular-matrix(ECM) and the intra-cellular cytoskeletal 40 machinery. CD44 has been recently shown to compartmentalize the membrane into domains by 41 acting as membrane pickets, facilitating the function of signaling receptors. While spatial 42 organization and diffusion studies of membrane proteins are usually conducted separately, here 43we combine observations of organization and diffusion by using high spatio-temporal resolution 44imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-45 scale meshwork pattern where it exhibits enhanced confinement and is enriched in nano-clusters 46 of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin 47 dynamics. Interaction with actin is mediated by specific segments of the intracellular-48 domain(ICD). This influences the organization of the protein at the nano-scale, generating a 49 selective requirement for formin over Arp2/3-based actin-nucleation machinery. The 50 extracellular-domain(ECD) and its interaction with elements of ECM do not influence the meso-51 scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken 52 together, our results capture the hierarchical nature of CD44 organization at the cell surface, with 53 active cytoskeleton-templated nano-clusters localized to a meso-scale meshwork pattern. 54 55 56 57 58 59 60 61 3 Introduction 62

Significance New imaging-based approaches are incorporating new concepts to our knowledge of biological processes. The analysis of receptor dynamics involved in cell movement using single-particle tracking demonstrates that cells require chemokine-mediated receptor clustering to sense appropriately chemoattractant gradients. Here, we report that this process does not occur in T cells expressing CXCR4 R334X , a mutant form of CXCR4 linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis). The underlaying molecular mechanism involves inappropriate actin cytoskeleton remodeling due to the inadequate β-arrestin1 activation by CXCR4 R334X , which alters its lateral mobility and spatial organization. These defects, associated to CXCR4 R334X expression, contribute to the retention of hematopoietic precursors in bone marrow niches and explain the severe immunological symptoms associated with WHIM syndrome.

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.