High throughput platform to engineer light-controlled inhibitors against guanine exchange factors of the Dbl family

ABSTRACT
Signaling networks that control cellular behavior are highly dynamic and precisely coordinated in space and
time. Rho family GTPases regulate diverse biological processes such as cell migration, proliferation and
immune activation. The activity of these molecules is tightly controlled at the subcellular level and is observed
with precise timing only in discrete regions of the cell. The Dbl family of guanine exchange factors (GEFs) are
the main activators of RhoA GTPases. There are typically multiple GEFs present in a cell that can act on the
same GTPase, and certain GEFs can interact with different GTPases. Recently, it has been shown that the
activity of Dbl GEFs is also distributed at discrete regions in the cell and regulated with precise kinetics.
Therefore, GEFs and GTPases form complex signaling networks that are tightly controlled in space and time.
Traditional GEF studies typically rely on depletion, by knock down or knock out, or augmentation, by
overexpression, of specific GEF activities. While informative, these approaches lack spatiotemporal resolution
and could introduce biological artifacts due to possible compensatory effects in connected GEF/GTPase
signaling networks. Therefore, to fully understand the biological roles of GEFs, new molecular tools are needed
that allow the rapid and precise control of their activity in living cells. The goal of this proposal is to develop a
high throughput platform that can be readily applied to engineer light-controlled inhibitors against the Dbl family
of GEFs. These inhibitors will make possible the reversible inhibition of endogenous GEFs with second-level
kinetics and at micron resolution in living cells. In Aim 1, three different approaches, that rely on computational
modeling and high throughput library screening, will be tested to engineer molecules that bind with high affinity
and specificity to Dbl GEFs and prevent their GTPase association. In Aim 2, engineered inhibitors will be fused
to known optogenetic modules in order to allow the precise control of their activity by irradiation. In Aim 3, the
optogenetic inhibitors will be studied by live cell microscopy to determine the experimental parameters that
need to be fine-tuned in order to achieve efficient GEF inhibition in vivo. The utility of this platform will be
demonstrated by engineering optogenetic inhibitors against three different Dbl GEFs that target the three major
RhoA GTPases, Rac1, RhoA and Cdc42. The platform developed here is general and could be readily applied
to develop molecular tools for the study of other Dbl GEFs. This proposal will thus facilitate the study of Dbl
GEFs at unprecedent spatial and temporal resolution across diverse biological systems.

Grant Number: 5R01GM144632-04
NIH Institute/Center: NIH
Principal Investigator: Mihai Azoitei