Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane

Abstract

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

Toward a new picture of the living plasma membrane

“…Note the transient confinement zones and regions of free diffusion (marked by arrowheads) of one such protein, CD44 (b), which provides an appreciation of the influence of the multiple scales and modes of diffusion. The trajectory has been adapted from Reference 13…”

“…Recent studies on the cell adhesion protein, CD44 have revealed such a hierarchical structuring of the membrane 13,61 . The extra‐cellular domain of CD44 binds the ECM component hyaluronic acid, 62 while its cytoplasmic domain binds to the actin cytoskeleton.…”

“…The extra‐cellular domain of CD44 binds the ECM component hyaluronic acid, 62 while its cytoplasmic domain binds to the actin cytoskeleton. Several studies have demonstrated the existence of CD44 nanoclusters at the plasma membrane using a multitude of localization based super‐resolution microscopy 63 as well as FRET‐based studies 13,64 . CD44 forms nanoclusters in a Galectin‐3 dependent manner on mouse embryonic fibroblast cell membrane, and secreted Galectins are known to be associated with the ECM.…”

“…FRET‐based measurements identified the existence of much higher density clustered distribution of the protein, indicative of proteins at <5 nm distances from each other. These studies found that the cytoplasmic domain‐based interactions, especially with the cytoskeletal adaptor, Ezrin, is key for clustering of CD44 in an actin‐dependent manner 13,64 . Ezrin is a member of the ERM family of actin binding proteins.…”

“…It binds to CD44 by its ERM binding domain, and with a high affinity to the filamentous actin via its actin binding domain 65 . SPT studies showed that CD44 exhibits non‐Brownian diffusion at the plasma membrane with frequent temporal and spatial confinements subject to its ability to interact with the actin cytoskeleton via Ezrin, thereby acting as membrane pickets of the picket fence model of the plasma membrane proposed by Kusumi and colleagues 13,61 . This function of CD44 enabled the restriction of FcγR1 mobility and thereby promoting phagocytic function in macrophages.…”