title: Nanomapping of CD1d-glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging.
abstract: CD1d molecule, a monomorphic major histocompatibility complex class I-like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d-glycolipid complex. THP1 cells were pulsed with three different glycolipids (α-GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d-α-GalCer and CD1d-C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm(2) (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm(2). Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d-glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions.
authors.affiliation: Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040, Linz, Austria.
authors.forename: Memed
authors.initials: M
authors.lastname: Duman
authors.forename: Lilia A
authors.initials: LA
authors.lastname: Chtcheglova
authors.forename: Rong
authors.initials: R
authors.lastname: Zhu
authors.forename: Bianca L
authors.initials: BL
authors.lastname: Bozna
authors.forename: Paolo
authors.initials: P
authors.lastname: Polzella
authors.forename: Vicenzo
authors.initials: V
authors.lastname: Cerundolo
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
mesh_terms: D055610:Antigens, CD1d / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D005699:Galactosylceramides / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods; D059010:Single-Cell Analysis / Q000379:methods
chemical_list: D055610:Antigens, CD1d; D005699:Galactosylceramides; D006017:Glycolipids
keywords: CD1d molecule; THP1 cell; atomic force microscopy; glycolipids; invariant natural killer T cell; nanomapping; simultaneous topography and recognition imaging; single molecule recognition