{"id":{"repo_id":"penn","oai_identifier":"oai:repository.upenn.edu:20.500.14332/31857"},"canonical_url":"https://search.dev.ndltd.org/etd/penn/oai:repository.upenn.edu:20.500.14332/31857","repository":{"repo_id":"penn","name":"University of Pennsylvania","base_url":"https://repository.upenn.edu/server/oai/request"},"display":{"title":"Biophysical Dynamics Of Rgs-Lov Proteins As Systems For Light-Induced Membrane Recruitment","abstract":"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 and its utility as an optogenetic tool. In this thesis, I expand on those efforts, studying the dynamics of BcLOV4 in single cells and demonstrate its applications for controlling GTPase signaling. Here, I first 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. I leverage this to construct a multitude of BcLOV4-based tools, focusing primarily on Rho GTPases, specifically Rac1 and RhoA. I 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. In summary, here I have conducted an experimental and computational study of BcLOV4 optogenetic membrane recruitment, generating an accurate 3D FEM model which demonstrates the utility of BcLOV4 as an optogenetic tool and allows for more precise control and study of peripheral membrane protein signaling. Furthermore, I leverage BcLOV4 to make novel mechanotransductive tools, demonstrating how BcLOV4 as a protein technology is a versatile and powerful module for engineering chimeric optogenetic tools to control signaling of membrane-associated proteins.","abstract_html":"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 and its utility as an optogenetic tool. In this thesis, I expand on those efforts, studying the dynamics of BcLOV4 in single cells and demonstrate its applications for controlling GTPase signaling. Here, I first 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. I leverage this to construct a multitude of BcLOV4-based tools, focusing primarily on Rho GTPases, specifically Rac1 and RhoA. I 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. In summary, here I have conducted an experimental and computational study of BcLOV4 optogenetic membrane recruitment, generating an accurate 3D FEM model which demonstrates the utility of BcLOV4 as an optogenetic tool and allows for more precise control and study of peripheral membrane protein signaling. Furthermore, I leverage BcLOV4 to make novel mechanotransductive tools, demonstrating how BcLOV4 as a protein technology is a versatile and powerful module for engineering chimeric optogenetic tools to control signaling of membrane-associated proteins.","abstract_has_math":false,"creators":["Kuznetsov, Ivan"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Brian Y. Chow"],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021","date_published":"2021","updated_at":"2026-07-24T03:46:55Z","subjects":[],"languages":["en"],"rights":["Ivan Kuznetsov"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://repository.upenn.edu/handle/20.500.14332/31857","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Brian Y. Chow"]},{"key":"dc:creator","label":"Author","values":["Kuznetsov, Ivan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2023-05-18T03:27:06.000"]},{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2023-05-22T18:27:02Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-01-31T00:00:00Z"]},{"key":"dc:date.issued","label":"Date","values":["2021"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation/Thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Ivan Kuznetsov"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://repository.upenn.edu/handle/20.500.14332/31857"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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 and its utility as an optogenetic tool. In this thesis, I expand on those efforts, studying the dynamics of BcLOV4 in single cells and demonstrate its applications for controlling GTPase signaling. Here, I first 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. I leverage this to construct a multitude of BcLOV4-based tools, focusing primarily on Rho GTPases, specifically Rac1 and RhoA. I 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. In summary, here I have conducted an experimental and computational study of BcLOV4 optogenetic membrane recruitment, generating an accurate 3D FEM model which demonstrates the utility of BcLOV4 as an optogenetic tool and allows for more precise control and study of peripheral membrane protein signaling. Furthermore, I leverage BcLOV4 to make novel mechanotransductive tools, demonstrating how BcLOV4 as a protein technology is a versatile and powerful module for engineering chimeric optogenetic tools to control signaling of membrane-associated proteins."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Doctor of Philosophy (PhD)"]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Biophysical Dynamics Of Rgs-Lov Proteins As Systems For Light-Induced Membrane Recruitment"]}]}],"canonical_facts":{"dc:contributor.advisor":["Brian Y. Chow"],"dc:creator":["Kuznetsov, Ivan"],"dc:date":["2023-05-18T03:27:06.000"],"dc:date.accessioned":["2023-05-22T18:27:02Z"],"dc:date.available":["2025-01-31T00:00:00Z"],"dc:date.issued":["2021"],"dc:description.abstract":["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 and its utility as an optogenetic tool. In this thesis, I expand on those efforts, studying the dynamics of BcLOV4 in single cells and demonstrate its applications for controlling GTPase signaling. Here, I first 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. I leverage this to construct a multitude of BcLOV4-based tools, focusing primarily on Rho GTPases, specifically Rac1 and RhoA. I 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. In summary, here I have conducted an experimental and computational study of BcLOV4 optogenetic membrane recruitment, generating an accurate 3D FEM model which demonstrates the utility of BcLOV4 as an optogenetic tool and allows for more precise control and study of peripheral membrane protein signaling. Furthermore, I leverage BcLOV4 to make novel mechanotransductive tools, demonstrating how BcLOV4 as a protein technology is a versatile and powerful module for engineering chimeric optogenetic tools to control signaling of membrane-associated proteins."],"dc:description.degree":["Doctor of Philosophy (PhD)"],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["https://repository.upenn.edu/handle/20.500.14332/31857"],"dc:language":["en"],"dc:rights":["Ivan Kuznetsov"],"dc:title":["Biophysical Dynamics Of Rgs-Lov Proteins As Systems For Light-Induced Membrane Recruitment"],"dc:type":["Dissertation/Thesis"]},"updated_at":"2026-07-24T03:46:55Z"}