Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution

ELife
Scott Coyle, Wendell A Lim

Abstract

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping ...Continue Reading

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Methods Mentioned

BETA
GTPases
GTPase
super-resolution microscopy
FRET
nucleotide exchange
gel-filtration
Nucleotide
PCR
electron microscopy

Software Mentioned

Kintek
Matlab
shell script
RasGRF
MultiStackReg
Genevestigator
Kintek Student Explorer
SOScat
ImageJ
GREP

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