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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Poster Abstracts
114
32-POS
Board 16
Activation Time Distribution of Membrane Recruitment Process Revealed by First-Passage
Analysis of Full-Length SOS
William Y. C. Huang
1
, Steven Alvarez
1
, Young Kwang Lee
1
, Yasushi Kondo
2
, Jean K. Chung
1
,
Hiu Yue Monatrice Lam
1
, John Kuriyan
1,2
, Jay T. Groves
1
.
1
University of California, Berkeley, Berkeley, CA, USA,
2
University of California, Berkeley,
Berkeley, CA, USA.
Many cytosolic signaling proteins are autoinhibited in the cytosol and only activate upon
membrane recruitment. Release of autoinhibition generally involves structural rearrangements
within the protein and therefore leads to a time interval between initial recruitment and
activation. The physical mechanism of activation establishes both the mean time to activation as
well as distribution of activation times. The form of the activation time distribution, not its mean,
plays a critical role in enabling regulatory processes such as kinetic proof reading. Here, we
develop a single-molecule assay to temporally map the activation process of the Ras guanine
nucleotide exchange factor SOS on membrane surfaces. Simultaneous imaging of individual
SOS molecules and localized Ras activation on supported membrane microarrays reveals both
the activation time and rejection time distributions resulting from Grb2-mediated membrane
recruitment of SOS. The gamma-like shape of the activation time distribution indicates the
existence of rate-limiting kinetic intermediates in the release of autoinhibition, and establishes a
basis for kinetic proofreading in the activation of Ras. Using an analytical kinetic model, we
demonstrate how competition between the SOS activation pathway and dissociation from the
membrane together shape the Ras activation response to receptor-mediated SOS recruitment.
Finally, we proposed a framework to reconstruct signaling timescale of proximal signaling from
single-molecule activation timing.