Technical Reference #1704

1704.

Stoichiometry of molecular complexes at adhesions in living cells Michelle A. Digman;ab Paul W. Wiseman;b Colin Choi;c Alan R. Horwitz;c and Enrico Grattona1, University of California, PNAS, 106(1704), (2009)
Link To Paper

Abstract:
We describe a method to detect molecular complexes and measure their stoichiometry in living cells from simultaneous fluctuations of the fluorescence intensity in two image channels each detecting a different kind of protein. The number and brightness (N&B) analysis namely the use of the ratio between the variance and the average intensity to obtain the brightness of molecules is extended to the cross-variance of the intensity fluctuations in two channels. We apply the cross-variance method to determine the stoichiometry of complexes containing paxillin and vinculin or focal adhesion kinase (FAK) in disassembling adhesions in mouse embryo fibroblasts expressing FAK vinculin and paxillin-tagged with EGFP and mCherry. We found no complexes of these proteins in the cytoplasm away from the adhesions. However at the adhesions large aggregates leave forming a hole during their disassembly. This hole shows cross-correlation between FAK and paxillin and vinculin and paxillin. From the amplitude of the correlated fluctuations we determine the composition of the aggregates leaving the adhesions. These aggregates disassemble rapidly in the cytoplasm because large complexes are found only in very close proximity to the adhesions or at their borders.

Keywords:
brightness analysis; confocal microscopy; cross-correlation

Materials & Methods:
Cell Culture and Protein Transfection. Mouse embryonic fibroblasts were cultured at 37 °C in a 5%CO2 humidified incubator. After trypsinization cells were subcultured and transferred from a 35-mm tissue culture flask to a 25-mm 6-well Falcon tissue culture (Becton-Dickinson). Cells were then grown to 50–80% confluence transfected with 1 g of DNA (0.5 g of DNA per protein for cotransfections) and 5 g of Lipofectamine 2000 (Invitrogen). Vn FAK the FAK mutant I937E/I999E and Pax cDNA were ligated to EGFP or mCherry at the C terminus (7) as described. After 24 h of transfection cells were plated by using high-glucose DMEM supplemented with 10% FBS and penicillin/streptomycin (Hyclone) on MatTek imaging dishes coated with 3 g of fibronectin from Sigma–Aldrich 1 h before imaging.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
MEF