Micropatterning for quantitative analysis of protein-protein interactions in living cells

We present a method to identify and characterize interactions between a fluorophore-labeled protein ('prey') and a membrane protein ('bait') in live mammalian cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait extracellular domain. Bait-prey interactions are assayed through the redistribution of the fluorescent prey. We used the method to characterize the interaction between human CD4, the major co-receptor in T-cell activation, and human Lck, the protein tyrosine kinase essential for early T-cell signaling. We measured equilibrium associations by quantifying Lck redistribution to CD4 micropatterns and studied interaction dynamics by photobleaching experiments and single-molecule imaging. In addition to the known zinc clasp structure, the Lck membrane anchor in particular had a major impact on the Lck-CD4 interaction, mediating direct binding and further stabilizing the interaction of other Lck domains. In total, membrane anchorage increased the interaction lifetime by two orders of magnitude.

[1]  W. Knapp,et al.  GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. , 1991, Science.

[2]  Natasa Przulj,et al.  High-Throughput Mapping of a Dynamic Signaling Network in Mammalian Cells , 2005, Science.

[3]  M. Brameshuber,et al.  Thinning out clusters while conserving stoichiometry of labeling , 2005 .

[4]  J. Brogdon,et al.  CD4 Raft Association and Signaling Regulate Molecular Clustering at the Immunological Synapse Site1 , 2004, The Journal of Immunology.

[5]  N. Tinel,et al.  Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization , 2008, Nature Methods.

[6]  H. Craighead,et al.  Mast Cell Activation on Patterned Lipid Bilayers of Subcellular Dimensions , 2003 .

[7]  G. Schütz,et al.  Single-molecule microscopy reveals heterogeneous dynamics of lipid raft components upon TCR engagement. , 2007, International immunology.

[8]  I. Stagljar,et al.  A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Igor Stagljar,et al.  Analysis of membrane protein interactions using yeast-based technologies. , 2002, Trends in biochemical sciences.

[10]  P. Kavathas,et al.  Short related sequences in the cytoplasmic domains of CD4 and CD8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein kinase , 1990, Molecular and cellular biology.

[11]  S. Burakoff,et al.  Lipid Raft Distribution of CD4 Depends on its Palmitoylation and Association with Lck, and Evidence for CD4-Induced Lipid Raft Aggregation as an Additional Mechanism to Enhance CD3 Signaling1 , 2003, The Journal of Immunology.

[12]  A. Veillette,et al.  Enrichment of Lck in Lipid Rafts Regulates Colocalized Fyn Activation and the Initiation of Proximal Signals through TCRαβ1 , 2004, The Journal of Immunology.

[13]  Arup K Chakraborty,et al.  CD4 enhances T cell sensitivity to antigen by coordinating Lck accumulation at the immunological synapse , 2004, Nature Immunology.

[14]  Thomas J Magliery,et al.  Detecting protein-protein interactions with GFP-fragment reassembly , 2004, Nature Methods.

[15]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[16]  Akihiro Kusumi,et al.  GPI-anchored receptor clusters transiently recruit Lyn and Gα for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1 , 2007, The Journal of Cell Biology.

[17]  E. Isacoff,et al.  Subunit counting in membrane-bound proteins , 2007, Nature Methods.

[18]  Jason A. Papin,et al.  Reconstruction of cellular signalling networks and analysis of their properties , 2005, Nature Reviews Molecular Cell Biology.

[19]  A. Aronheim,et al.  The Ras recruitment system, a novel approach to the study of protein–protein interactions , 1998, Current Biology.

[20]  N. Lambert,et al.  Some G protein heterotrimers physically dissociate in living cells , 2006, Proceedings of the National Academy of Sciences.

[21]  Maïté Coppey-Moisan,et al.  Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments , 2005, Nature Methods.

[22]  Kai Simons,et al.  Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components , 1998, The Journal of cell biology.

[23]  B. Séraphin,et al.  The tandem affinity purification (TAP) method: a general procedure of protein complex purification. , 2001, Methods.

[24]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[25]  S. Ley,et al.  S‐acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes , 1997, The EMBO journal.

[26]  S. Blacklow,et al.  A Zinc Clasp Structure Tethers Lck to T Cell Coreceptors CD4 and CD8 , 2003, Science.

[27]  Barbara Baird,et al.  Visualization of plasma membrane compartmentalization with patterned lipid bilayers. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[28]  K. Mossman,et al.  Altered TCR Signaling from Geometrically Repatterned Immunological Synapses , 2005, Science.

[29]  Joachim P Spatz,et al.  Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly. , 2006, European journal of cell biology.

[30]  R. Perlmutter,et al.  Interaction of the unique N-terminal region of tyrosine kinase p56 lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs , 1990, Cell.