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BE Doctoral Dissertation: “Biophysical dynamics of RGS-LOV proteins as systems for light-induced membrane recruitment” (Ivan Kuznetsov)
August 30 at 10:00 AM - 12:00 PM
The Department of Bioengineering at the University of Pennsylvania and
Dr. Brian Chow are pleased to announce the Doctoral Dissertation Defense of Ivan Kuznetsov.
Title: Biophysical dynamics of RGS-LOV proteins as systems for light-induced membrane recruitment
Date: August 30, 2021
Time: 10:00 AM
Location: Levine 307 and via zoom at the link below:
Light–oxygen–voltage sensitive (LOV) domains represent a ubiquitous family of blue-light photoreceptors. Recently, we reported BcLOV4, a LOV protein from B. cinerea, which demonstrates a light-induced protein-lipid interaction with the plasma membrane. Previous work demonstrated preliminary characterization of BcLOV4 but stopped short of demonstrating its applications as an optogenetic tool. Here, we report a 3D non-linear finite element model of the cell-wide spatiotemporal dynamics of BcLOV4 membrane recruitment. Inputs were data from single cells unique in morphology and initial absolute protein concentration, as opposed to fitting an idealized cell to population data. All necessary BcLOV4 biophysical parameters were measured with recombinant protein or in HEK cells. The model outperforms existing (2D and/or linear) approaches and recapitulates observed complexities, including cell-geometric effects on recruitment efficiency and inter-instrument diffractive effects. It also highlights underappreciated determinants of signaling magnitude, contrast, and spatial confinement – such as the nonlinearity introduced by finite membrane-binding sites, outsized role of rebinding kinetics, and impact of excitation volume on the signaling impulse response. By capturing both intrinsic biophysical contributions of the optogenetic tools and extrinsic contributions of the optical hardware, this framework improves understanding of how to spatiotemporally shape signaling.
The biophysical and computational study of BcLOV4 suggest that it has great utility as a generalizable modality for optogenetic tool development. We leverage this to construct a multitude of BcLOV4-based tools, focusing primarily on Rho GTPases, specifically Rac1 and RhoA. We demonstrate that these tools can generate physiologically relevant downstream signaling, including cell motility and, in the case of RhoA, YAP-TEAD-dependent transcriptional activity. This work sets the groundwork for a BcLOV4-based optogenetic toolkit for accurate dissection of mechanotransduction.