Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

Signal integration between activating Fc receptors and inhibitory signal regulatory protein &agr; (SIRP&agr;) controls macrophage phagocytosis. Here, using dual-color direct stochastic optical reconstruction microscopy, we report that Fc&ggr; receptor I (Fc&ggr;RI), Fc&ggr;RII, and SIRP&agr; are not homogeneously distributed at macrophage surfaces but are organized in discrete nanoclusters, with a mean radius of 71 ± 11 nm, 60 ± 6 nm, and 48 ± 3 nm, respectively. Nanoclusters of Fc&ggr;RI, but not Fc&ggr;RII, are constitutively associated with nanoclusters of SIRP&agr;, within 62 ± 5 nm, mediated by the actin cytoskeleton. Upon Fc receptor activation, Src-family kinase signaling leads to segregation of Fc&ggr;RI and SIRP&agr; nanoclusters to be 197 ± 3 nm apart. Co-ligation of SIRP&agr; with CD47 abrogates nanocluster segregation. If the balance of signals favors activation, Fc&ggr;RI nanoclusters reorganize into periodically spaced concentric rings. Thus, a nanometer- and micron-scale reorganization of activating and inhibitory receptors occurs at the surface of human macrophages concurrent with signal integration.

[1]  F. Batista,et al.  New insights into the early molecular events underlying B cell activation. , 2008, Immunity.

[2]  S. Wright,et al.  Macrophages form circular zones of very close apposition to IgG-coated surfaces. , 1990, Cell motility and the cytoskeleton.

[3]  Michael Loran Dustin,et al.  What is the importance of the immunological synapse? , 2004, Trends in immunology.

[4]  Ken Jacobson,et al.  Nanoclustering as a dominant feature of plasma membrane organization , 2014, Journal of Cell Science.

[5]  R. Geha,et al.  Physical and functional association of the high affinity immunoglobulin G receptor (Fc gamma RI) with the kinases Hck and Lyn , 1994, The Journal of experimental medicine.

[6]  S. Grinstein,et al.  Receptor mobility, the cytoskeleton, and particle binding during phagocytosis. , 2011, Current opinion in cell biology.

[7]  B. Ripley.  Modelling Spatial Patterns , 1977 .

[8]  T. Matozaki,et al.  Negative Regulation of Phagocytosis in Macrophages by the CD47-SHPS-1 System1 , 2005, The Journal of Immunology.

[9]  Dylan M Owen,et al.  Superresolution Microscopy Reveals Nanometer-Scale Reorganization of Inhibitory Natural Killer Cell Receptors upon Activation of NKG2D , 2013, Science Signaling.

[10]  Andreas Bruckbauer,et al.  The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling. , 2013, Immunity.

[11]  Rajat Varma,et al.  Supported Planar Bilayers for Study of the Immunological Synapse , 2007, Current protocols in immunology.

[12]  S. Latour,et al.  High Expression of Inhibitory Receptor SHPS-1 and Its Association with Protein-tyrosine Phosphatase SHP-1 in Macrophages* , 1998, The Journal of Biological Chemistry.

[13]  Valentin Jaumouillé,et al.  The cell biology of phagocytosis. , 2012, Annual review of pathology.

[14]  J. Orange.  Formation and function of the lytic NK-cell immunological synapse , 2008, Nature Reviews Immunology.

[15]  John Q. Davies,et al.  Isolation and culture of human macrophages. , 2005, Methods in molecular biology.

[16]  Tetsuo Yamazaki,et al.  T cell receptor ligation induces the formation of dynamically regulated signaling assemblies , 2002, The Journal of cell biology.

[17]  Astrid Magenau,et al.  Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events , 2011, Nature Immunology.

[18]  Daniel M. Davis,et al.  The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling , 2016, Cell reports.

[19]  George L. W. Perry,et al.  SpPack: spatial point pattern analysis in Excel using Visual Basic for Applications (VBA) , 2004, Environ. Model. Softw..

[20]  J. Franklin,et al.  Second-Order Neighborhood Analysis of Mapped Point Patterns , 1987 .

[21]  P. Verveer,et al.  Coordinate-based colocalization analysis of single-molecule localization microscopy data , 2011, Histochemistry and Cell Biology.

[22]  B. Hinz,et al.  Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis , 2016, Cell.

[23]  G. J. Cannon,et al.  The macrophage capacity for phagocytosis. , 1992, Journal of cell science.

[24]  C. Lagenaur,et al.  Role of CD47 as a marker of self on red blood cells. , 2000, Science.

[25]  Raibatak Das,et al.  Actin cytoskeleton reorganization by Syk regulates Fcγ receptor responsiveness by increasing its lateral mobility and clustering. , 2014, Developmental cell.

[26]  C. Lagenaur,et al.  Integrin-associated Protein Is a Ligand for the P84 Neural Adhesion Molecule* , 1999, The Journal of Biological Chemistry.

[27]  Guy M. Hagen,et al.  ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging , 2014, Bioinform..

[28]  Mark Bates,et al.  Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes , 2007, Science.

[29]  H. Gresham,et al.  Cd47-Signal Regulatory Protein α (Sirpα) Regulates Fcγ and Complement Receptor–Mediated Phagocytosis , 2001, The Journal of experimental medicine.

[30]  M. Kasuga,et al.  A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion , 1996, Molecular and cellular biology.

[31]  M. Heilemann,et al.  Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. , 2008, Angewandte Chemie.

[32]  P. Bruhns,et al.  Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. , 2009, Blood.

[33]  Gerhard J Schütz,et al.  Varying label density allows artifact-free analysis of membrane-protein nanoclusters , 2016, Nature Methods.

[34]  Mark Bates,et al.  Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy , 2008, Science.

[35]  M. Reth,et al.  B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk , 2014, eLife.

[36]  J. Bolen,et al.  Physical and functional association of Src-related protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells. , 1994, The Journal of biological chemistry.

[37]  Štefan Bálint,et al.  Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections , 2013, Proceedings of the National Academy of Sciences.

[38]  Mark M Davis,et al.  TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation , 2010, Nature Immunology.

[39]  J. Michl,et al.  Studies of the Mechanism of Phagocytosis , 1981 .

[40]  K. Kawauchi,et al.  Myosin II‐dependent exclusion of CD45 from the site of Fcγ receptor activation during phagocytosis , 2012, FEBS letters.

[41]  Astrid Magenau,et al.  PALM imaging and cluster analysis of protein heterogeneity at the cell surface , 2010, Journal of biophotonics.

[42]  Mark Bates,et al.  Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection , 2011, Chemphyschem : a European journal of chemical physics and physical chemistry.

[43]  S. Silverstein,et al.  Studies on the mechanism of phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane , 1975, The Journal of experimental medicine.

[44]  D. Klenerman,et al.  Initiation of T cell signaling by CD45 segregation at ‘close-contacts’ , 2016, Nature Immunology.

[45]  Alexandre F. Carisey,et al.  The central role of the cytoskeleton in mechanisms and functions of the NK cell immune synapse , 2013, Immunological reviews.

[46]  Inhibition of “self” engulfment through deactivation of myosin-II at the phagocytic synapse between human cells , 2008 .

[47]  H. Gresham,et al.  CD 47-Signal Regulatory Protein a ( SIRP a ) Regulates Fc g and Complement Receptor – mediated Phagocytosis , 2001 .