title: Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes.
abstract: Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics ( approximately 200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of approximately 20-30 nm at acquisition times of 5-30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.
authors: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147, USA. shroffh@janelia.hhmi.org
authors: Hari
authors: H
authors: Shroff
authors: Catherine G
authors: CG
authors: Galbraith
authors: James A
authors: JA
authors: Galbraith
authors: Helen
authors: H
authors: White
authors: Jennifer
authors: J
authors: Gillette
authors: Scott
authors: S
authors: Olenych
authors: Michael W
authors: MW
authors: Davidson
authors: Eric
authors: E
authors: Betzig
mesh_terms: D002448:Cell Adhesion; D002460:Cell Line; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D015335:Molecular Probes / Q000032:analysis* / Q000235:genetics; D049329:Nanostructures / Q000032:analysis; D038761:Photobleaching; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins