T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

ACRF Department of Cancer Biology and Therapeutics The John Curtin School of Medical Research The Australian National University 131 Garran Road Canberra 2601 Australia

ARC Centre of Excellence in Advanced Molecular Imaging The University of New South Wales Sydney 2052 Australia

Department of Biochemistry and Molecular Biology Bio21 Institute University of Melbourne Melbourne VIC 3010 Australia

EMBL Australia Node in Single Molecule Science School of Medical Sciences The University of New South Wales Sydney 2052 Australia

IMCF at BIOCEV Faculty of Science Charles University Průmyslová 595 25250 Vestec Czech Republic

Research School of Electrical Energy and Materials Engineering College of Engineering and Computer Science The Australian National University 31 North Road Canberra 2601 Australia

School of Physics University of Melbourne Melbourne VIC 3010 Australia

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