Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes

doi:10.1073/pnas.0710517105

Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes

*Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147;
^‡National Institute of Dental and Craniofacial Research,
^§National Institute of Neurological Disorders and Stroke, and
^¶National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and
^‖National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, FL 32310

Communicated by Charles V. Shank, Janelia Farm Research Campus, Ashburn, VA, November 6, 2007 (received for review September 23, 2007)

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 (≈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 ≈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.

Footnotes

^†To whom correspondence should be addressed. E-mail: shroffh{at}janelia.hhmi.org
Author contributions: H.S., C.G.G., J.A.G., M.W.D., and E.B. designed research; H.S., C.G.G., J.A.G., H.W., J.G., S.O., and E.B. performed research; S.O. and M.W.D. contributed new reagents/analytic tools; H.S., C.G.G., J.A.G., and E.B. analyzed data; and H.S., C.G.G., J.A.G., M.W.D., and E.B. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0710517105/DC1.
Freely available online through the PNAS open access option.