12th Carl Zeiss sponsored workshop on ... - Institut Fresnel

Quantitative Analyses of Protein Interaction Networks
Yi-Da
Chung,
Ivo R.
Ruttekolk,
Petra H.
Bovée-Geurts,
Michael D.
Sinzinger,
Roland Brock
Dept. of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University
Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands
r.brock@ncmls.ru.nl
Quantitative analyses still are a major challenge in cell biology. In order to understand how an
extracellular signal is translated into a cellular response, quantitative knowledge on the
number of signaling molecules inside a cell, the fraction of molecules that actually participate
in signal transduction and the temporal and spatial organization of the signaling machinery is
required.
Here, we present a combination of fluorescence correlation spectroscopy (FCS) in cell lysates
and in intact cells, confocal laser scanning microscopy and quantitative Western Blots to
address this problem. We focused our analyses on interactions of the transmembrane
scaffolding protein Linker for the Activation of T cells (LAT) and down-stream signaling
proteins interacting with LAT. These complexes form upon activation of T cell receptors in T
lymphocytes and are linked to the initiation of an immune response.
For the quantification of intracellular protein expression we developed a protocol in which
transiently expressed fusion proteins with fluorescent proteins serve as an internal standard. In
cell lysates, fusion proteins are quantitated with FCS and the relative amount of fusion protein
and endogenous protein determined by Western Blots. Furthermore, we account for different
folding efficiencies of individual fluorescent protein fusion proteins. This protocol overcomes
the requirement for the purification of recombinant proteins associated with standard
quantitative Western Blots.
In cells expressing a LAT-GFP construct and a red fluorescent mCherry-fusion protein of a
down-stream signaling protein next to the non-fluorescent endogenous proteins, stimulationdependent
interactions were quantitated through a combination of confocal laser scanning
microscopy using single photon detectors for fluorescence detection, fluorescence correlation
spectroscopy and cross-correlation spectroscopy. Owing to the sensitivity of the detection
methods, interactions could be quantitated for cells expressing only low levels of fusion
proteins, thereby avoiding potential artifacts associated with the overexpression of fusion
proteins. Through integration of optical tweezers, contacts of T cells with antigen-presenting
cells could be established in a highly controlled fashion, enabling these measurements with
precise timing control for preselected cells.
In summary, the results present an answer to the experimental challenges associated with
quantitative intracellular analyses. This approach critically depends on the ability of FCS to
determine absolute concentrations of fluorescent molecules, both in vitro and in cells.
12 th <strong>Zeiss</strong> <strong>sponsored</strong> FCS <strong>workshop</strong> – Cargèse, Corsica, France – <strong>12th</strong> -16th October 2009