4.3 Article

Determination of binding curves via protein micropatterning in vitro and in living cells

Journal

CYTOMETRY PART A
Volume 83, Issue 9, Pages 847-854

Publisher

WILEY-BLACKWELL
DOI: 10.1002/cyto.a.22225

Keywords

Micropatterning; plasma membrane; CD4; Lck; single molecule microscopy; equilibrium binding constant

Funding

  1. GEN-AU Project of the Austrian Federal Ministry for Science and Research
  2. Austrian Science Fund [Y250-B03, I301-B12]
  3. DOC-fellowship of the Austrian Academy of Sciences at the Institute of Biophysics
  4. Austrian Science Fund (FWF) [I 301] Funding Source: researchfish

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Quantification of protein interactions in living cells is of key relevance for understanding cellular signaling. With current techniques, however, it is difficult to determine binding affinities and stoichiometries of protein complexes in the plasma membrane. We introduce here protein micropatterning as a convenient and versatile method for such investigations. Cells are grown on surfaces containing micropatterns of capture antibody to a bait protein, so that the bait gets rearranged in the live cell plasma membrane. Upon interaction with the bait, the fluorescent prey follows the micropatterns, which can be readout with fluorescence microscopy. In this study, we addressed the interaction between Lck and CD4, two central proteins in early T-cell signaling. Binding curves were recorded using the natural fluctuations in the Lck expression levels. Surprisingly, the binding was not saturable up to the highest Lck expression levels: on average, a single CD4 molecule recruited more than nine Lck molecules. We discuss the data in view of protein- and lipid-mediated interactions. (c) 2012 International Society for Advancement of Cytometry

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