Authors

Sherman, E., Barr, V. A., & Samelson, L. E.

Affiliations

Laboratory of Cellular and Molecular Biology, CCR, NCI, NIH, Bethesda, MD, USA 20892.

Application Area

Biophotonics

PALM

Abstract

Multi-molecular protein complexes are critical to many cellular functions, including signalling, 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 photo-activated localization microscopy (PALM) to resolve individual proteins with a resolution down to 20nm in intact cells, and second-order statistics to study the spatial interactions of the proteins. We demonstrate the feasibility of this technique by studying signalling complexes that form in activated T cells. We first use single colour 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 colour 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 multi-molecular protein complexes and their significance to cell function.

Extract

… “light emitting diode (LED) source (a blue light source, CoolLED, PE-100”…

Product Associated Features

pE-100: A range of compact, simple-to-use, single wavelength illumination systems for screening fluorescence.

Diascopic Technique

-

Live Cell Issues

Photobleaching

Product Type

pE-100

Journal

Methods

Year of Publication

2013

Country of Publication

USA