Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Bioengineering

First Advisor

Brian Y. Chow

Abstract

The Rho family of small GTPases coordinate actin cytoskeletal rearrangements underlying crucial cell processes including migration and mechanotransduction. Dysregulation in these signaling pathways has been associated with neurodegenerative disease and cancer. Rho GTPase signaling is tightly controlled in space and time: GTPases are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase accelerating proteins (GAPs) at the plasma membrane. To study Rho GTPase signaling, several optogenetic tools have been developed, most of which use light to induce a protein-protein interaction, recruiting a GTPase-activating GEF to the plasma membrane. Other optogenetic strategies involve the use of single-chain photoswitches sterically occluding a constitutively active GTPase, which can result in undesirable high dark-state activity of the tool. We sought to create single-component optogenetic tools to perturb Rho GTPase signaling at the GTPase, GEF, and GAP level, resulting in lower dark state activity and easier implementation in mammalian systems.In this work, we used BcLOV4, a fungal photoreceptor which directly binds membrane lipids in response to blue light inputs, to recruit Rho signaling proteins to the membrane, resulting in spatiotemporally precise signaling perturbation. We created BcLOV4 activation tools using the GTPase and GEF from the three best studied Rho GTPase pathways: RhoA, which induces cell contraction through stress fiber formation; Rac1, which induces sheet-like lamellipodial protrusions; and Cdc42, which induces spiky filopodial protrusions. Notably, we demonstrated that the BcLOV4 system is compatible with wildtype GTPases, resulting in lower unintended pathway activation in the dark state. We also report progress toward the creation of RhoA termination tools using GAP domains and dominant-negative GTPases, allowing for the induction of signaling activation and termination on the same optogenetic platform. Using structural knowledge we gained from Rho GTPase tool development, we created a plasmid set and cloning workflow to simplify BcLOV4 tool engineering for other signaling targets. Together, the BcLOV4 optogenetic toolbox will further the study of Rho GTPase signaling and enable others to use this technology for single-component optogenetic membrane recruitment.

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