Resolving multi-molecular protein interactions by photoactivated localization microscopy

Eilon Sherman, Valarie A. Barr, Lawrence E. Samelson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

Multi-molecular protein complexes are critical to many cellular functions, including signaling, DNA transcription and enzymatic reactions. In spite of their importance, current research techniques such as biochemistry and diffraction-limited microscopy cannot resolve the heterogeneity and nanoscale organization of protein complexes in intact cells. Here we describe a technique that enables the study of multi-molecular protein complexes at the single molecule level in intact cells. The technique uses photoactivated localization microscopy (PALM) to resolve individual proteins with a resolution down to 20. nm in intact cells, and second-order statistics to study the spatial interactions of the proteins. We demonstrate the feasibility of this technique by studying signaling complexes that form in activated T cells. We first use single color PALM imaging and univariate second-order statistics to resolve the clustering of Linker for Activation of T cells (LAT) at the plasma membrane (PM) of the cells. We then use two color PALM and bivariate second-order statistics to resolve the interaction of LAT with key interacting proteins. We discuss potential caveats in studying molecular clustering and the robustness of the technique to study bimolecular interactions. Our proposed technique, combined with older techniques, could help shed new light on the nature of multimolecular protein complexes and their significance to cell function.

Original languageAmerican English
Pages (from-to)261-269
Number of pages9
JournalMethods
Volume59
Issue number3
DOIs
StatePublished - 1 Mar 2013
Externally publishedYes

Bibliographical note

Funding Information:
The authors would like to thank Subhadra Banerjee and Barbara J Taylor at the FACS CORE Facility (NIH, NCI), Zeiss, Harald Hess (HHMI, Janelia Farm) for providing the PALM software, Wolfgang Losert (UMD) for multiple discussions on data analyses, Thorstan Wiegand (Helmholtz Centre for Environmental Research - UFZ) for providing us his point-pattern analyses software and Connie Sommers (NIH, NCI) for her comments on the manuscript. This research was supported by the Intramural Research Programs of the National Cancer Institute (The Center for Cancer Research).

Keywords

  • Microclusters
  • Photoactivated localization microscopy
  • Protein interactions
  • Second order statistics
  • Single molecule
  • Super resolution microscopy
  • T cell activation

Fingerprint

Dive into the research topics of 'Resolving multi-molecular protein interactions by photoactivated localization microscopy'. Together they form a unique fingerprint.

Cite this