TY - JOUR
T1 - Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane
AU - Sil, Parijat
AU - Mateos, Nicolas
AU - Nath, Sangeeta
AU - Buschow, Sonja
AU - Manzo, Carlo
AU - Suzuki, Kenichi G.N.
AU - Fujiwara, Takahiro
AU - Kusumi, Akihiro
AU - Garcia-Parajo, Maria F.
AU - Mayor, Satyajit
N1 - Funding Information:
We thank Rob Parton for sharing with us the CD44-GFP, CD44ECDTm-GFP, and CD44TmICD-GFP constructs, which were originally used in Mrass et al. (2008). The JF dyes used in SPT studies were a generous gift from Luke Lavis, HHMI Janelia Research Campus. We thank Suvrajit Saha for contributions to the initiation of the project. We thank the Central Imaging and Flow facility of the National Centre for Biological Science for enabling us to use their equipment and Divya Gowda and H. Krishnamurthy for their help. We thank Marcus J. Taylor for helping in reagent development pertaining to the SNAP-tagged constructs. We thank Chaitra Prabha-kara and Sanjeev Sharma for help with data representation, revision, and editing the manuscript. M.F.G.-P. acknowledges funding from the Fundació Privada Cellex, Generalitat de Catalunya through the Centres de Reserca de Catalunya program, Spanish Ministry of Economy and Competitiveness (“Severo Ochoa” Programme for Centres of Excellence in R&D [SEV – 2015 –0522] and FIS2017-89560-R) and from European Union H2020-ERC grant 788546-NANO-MEMEC. N.M. acknowledges funding from the European Union H2020 under Marie Sklodowska-Curie grant 754558-PREBIST. C.M. acknowledges funding from the Spanish Ministry of Economy and Competitiveness and the European Social Fund through the “Ramón y Cajal” program 2015 (grant RYC-2015-17896), the “Programa Estatal de I+D+i Orientada a los Retos de la Sociedad” (grant BFU2017-85693-R), and the Generalitat de Cata-lunya (AGAUR Grant 2017SGR940). A.K. acknowledges support in part by grants-in-aid for scientific research Kiban S (16H06386) from the Japan Society for the Promotion of Science. TK.F. acknowledges grants-in-aid for scientific research from the Japan Society for the Promotion of Science (Kiban B [16H04775)]. K.S. acknowledges grants-in-aid for scientific research from the Japan Society for the Promotion of Science (Kiban B [18H02401)]. S.M. acknowledges a JC Bose Fellowship from the Department of Science and Technology, Government of India, a collaborative grant from the Human Frontiers Science Program (RGP0027/2012 with M.F.G.-P.), and a Wellcome Trust–Department of Biotechnology, Alliance Marga-darshi fellowship (IA/M/15/1/502018).
Funding Information:
We thank Rob Parton for sharing with us the CD44-GFP, CD44ECDTm-GFP, and CD44TmICD-GFP constructs, which were originally used in Mrass et al. (2008). The JF dyes used in SPT studies were a generous gift from Luke Lavis, HHMI Janelia Research Campus. We thank Suvrajit Saha for contributions to the initiation of the project. We thank the Central Imaging and Flow facility of the National Centre for Biological Science for enabling us to use their equipment and Divya Gowda and H. Krishnamurthy for their help. We thank Marcus J. Taylor for helping in reagent development pertaining to the SNAP-tagged constructs. We thank Chaitra Prabhakara and Sanjeev Sharma for help with data representation, revision, and editing the manuscript. M.F.G.-P. acknowledges funding from the Fundaci? Privada Cellex, Generalitat de Catalunya through the Centres de Reserca de Catalunya program, Spanish Ministry of Economy and Competitiveness (?Severo Ochoa? Pro-gramme for Centres of Excellence in R&D [SEV ? 2015?0522] and FIS2017-89560-R) and from European Union H2020-ERC grant 788546-NANO-MEMEC. N.M. acknowledges funding from the European Union H2020 under Marie Sklodowska-Curie grant 754558-PREBIST. C.M. acknowledges funding from the Spanish Ministry of Economy and Competitiveness and the European Social Fund through the ?Ram?n y Cajal? program 2015 (grant RYC-2015-17896), the ?Programa Estatal de I+D+i Orientada a los Retos de la Sociedad? (grant BFU2017-85693-R), and the Generalitat de Catalunya (AGAUR Grant 2017SGR940). A.K. acknowledges support in part by grants-in-aid for scientific research Kiban S (16H06386) from the Japan Society for the Promotion of Science. TK.F. acknowledges grants-in-aid for scientific research from the Japan Society for the Promotion of Science (Kiban B [16H04775)]. K.S. acknowledges grants-in-aid for scientific research from the Japan Society for the Promotion of Science (Kiban B [18H02401)]. S.M. acknowledges a JC Bose Fellowship from the Department of Science and Technology, Government of India, a collaborative grant from the Human Frontiers Science Program (RGP0027/2012 with M.F.G.-P.), and a Wellcome Trust?Department of Biotechnology, Alliance Margadarshi fellowship (IA/M/15/1/502018).
Publisher Copyright:
© 2020 Sil, Mateos, et al.
PY - 2020/3
Y1 - 2020/3
N2 - Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular matrix (ECM) and the intracellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatiotemporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nanoclusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular domain. This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular domain and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nanoclusters localized to a meso-scale meshwork pattern.
AB - Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular matrix (ECM) and the intracellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatiotemporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nanoclusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular domain. This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular domain and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nanoclusters localized to a meso-scale meshwork pattern.
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U2 - 10.1091/MBC.E18-11-0715
DO - 10.1091/MBC.E18-11-0715
M3 - Article
C2 - 31577524
AN - SCOPUS:85082144178
SN - 1059-1524
VL - 31
SP - 561
EP - 579
JO - Molecular Biology of the Cell
JF - Molecular Biology of the Cell
IS - 7
ER -