Nanoscale organization of the pathogen receptor DC-SIGN mapped by single-molecule high-resolution fluorescence microscopy.
DC-SIGN, a C-type lectin exclusively expressed on dendritic cells (DCs), plays an important role in pathogen recognition by binding with high affinity to a large variety of microorganisms. Recent experimental evidence points to a direct relation between the function of DC-SIGN as a viral receptor and its spatial arrangement on the plasma membrane. We have investigated the nanoscale organization of fluorescently labeled DC-SIGN on intact isolated DCs by means of near-field scanning optical microscopy (NSOM) combined with single-molecule detection. Fluorescence spots of different intensity and size have been directly visualized by optical means with a spatial resolution of less than 100 nm. Intensity- and size-distribution histograms of the DC-SIGN fluorescent spots confirm that approximately 80 % of the receptors are organized in nanosized domains randomly distributed on the cell membrane. Intensity-size correlation analysis revealed remarkable heterogeneity in the molecular packing density of the domains. Furthermore, we have mapped the intermolecular organization within a dense cluster by means of sequential NSOM imaging combined with discrete single-molecule photobleaching. In this way we have determined the spatial coordinates of 13 different individual dyes, with a localization accuracy of 6 nm. Our experimental observations are all consistent with an arrangement of DC-SIGN designed to maximize its chances of binding to a wide range of microorganisms. Our data also illustrate the potential of NSOM as an ultrasensitive, high-resolution technique to probe nanometer-scale organization of molecules on the cell membrane.
de Bakker
Bärbel I
BI
Applied Optics Group, Faculty of Science & Technology, MESA+ Institute for Nanotechnology, University of Twente, The Netherlands.
de Lange
Frank
F
Cambi
Alessandra
A
Korterik
Jeroen P
JP
van Dijk
Erik M H P
EM
van Hulst
Niek F
NF
Figdor
Carl G
CG
Garcia-Parajo
Maria F
MF
D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism; D006801:Humans; D037181:Lectins, C-Type / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D036103:Nanotechnology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D015815:Cell Adhesion Molecules; D037181:Lectins, C-Type; D011956:Receptors, Cell Surface; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin