ingest cdrApp 2017-07-06T11:49:18.518Z 082b3de9-6030-4a3e-a983-035a47fc699e modifyDatastreamByValue RELS-EXT fedoraAdmin 2017-07-06T11:56:37.085Z Setting exclusive relation modifyDatastreamByValue RELS-EXT fedoraAdmin 2017-07-06T11:56:46.060Z Setting exclusive relation addDatastream MD_TECHNICAL fedoraAdmin 2017-07-06T11:56:46.659Z Adding technical metadata derived by FITS modifyDatastreamByValue RELS-EXT fedoraAdmin 2017-07-06T11:57:02.499Z Setting exclusive relation addDatastream MD_FULL_TEXT fedoraAdmin 2017-07-06T11:57:11.230Z Adding full text metadata extracted by Apache Tika modifyDatastreamByValue RELS-EXT fedoraAdmin 2017-07-06T11:57:11.923Z Setting exclusive relation modifyDatastreamByValue RELS-EXT cdrApp 2017-07-06T12:27:29.043Z Setting exclusive relation modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-01-25T12:11:54.585Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-01-27T12:19:40.686Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-03-14T09:22:43.542Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-05-17T20:58:01.743Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-07-11T07:53:17.107Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-07-18T04:03:18.316Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-08-16T17:11:04.980Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-09-27T12:56:32.908Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-10-12T04:10:37.430Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2019-03-21T13:50:03.453Z Seth Zimmerman Author Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. Spring 2017 2017 Biochemistry Cellular biology Directional Migration, Fibronectin, GTPase, iLID, Light Induced Dimer, Optogenetics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. Spring 2017 2017 Biochemistry Cellular biology Directional Migration, Fibronectin, GTPase, iLID, Light Induced Dimer, Optogenetics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. Spring 2017 2017 Biochemistry Cellular biology Directional Migration, Fibronectin, GTPase, iLID, Light Induced Dimer, Optogenetics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. 2017-05 2017 Biochemistry Cellular biology Directional Migration, Fibronectin, GTPase, iLID, Light Induced Dimer, Optogenetics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. 2017 Biochemistry Cellular biology Directional Migration, Fibronectin, GTPase, iLID, Light Induced Dimer, Optogenetics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text 2017-05 Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. 2017 Biochemistry Cellular biology Directional Migration, Fibronectin, GTPase, iLID, Light Induced Dimer, Optogenetics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text 2017-05 Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. 2017 Biochemistry Cellular biology Directional Migration, Fibronectin, GTPase, iLID, Light Induced Dimer, Optogenetics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text 2017-05 Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. 2017 Biochemistry Cellular biology Directional Migration, Fibronectin, GTPase, iLID, Light Induced Dimer, Optogenetics eng Doctor of Philosophy Dissertation Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text 2017-05 University of North Carolina at Chapel Hill Degree granting institution Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. 2017 Biochemistry Cellular biology Directional Migration; Fibronectin; GTPase; iLID; Light Induced Dimer; Optogenetics eng Doctor of Philosophy Dissertation Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text 2017-05 University of North Carolina at Chapel Hill Degree granting institution Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine MOTIVATED BY LOV: DESIGN AND APPLICATION OF OPTOGENETIC TOOL REVEALS THAT CELLS LAY THEIR OWN TRACKS DURING DIRECTED MIGRATION A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. 2017 Biochemistry Cellular biology Directional Migration, Fibronectin, GTPase, iLID, Light Induced Dimer, Optogenetics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Biochemistry and Biophysics Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text 2017-05 Seth Zimmerman Creator Department of Biochemistry and Biophysics School of Medicine A cell’s ability to sense a gradient of extracellular cue and move in the direction of the gradient is central to many pathological and physiological conditions. For a cell to respond with directional migration it must translate the spatial and temporal information from the extracellular cue into intracellular signaling and a mechanical output. However, there is a lack of methodologies to directly probe these intracellular processes with the spatial and temporal precision that is necessary to fully understand how they translate into directed motility. We have therefore engineered a light inducible dimer (iLID) that provides spatial and temporal control of signaling by modulating protein-protein interactions. iLID was specifically engineered as a general use tool to control many aspects of biology. Therefore, we designed and characterized multiple variants with a broad range of binding affinities and kinetics. Furthermore, we benchmarked our dimer along with other light inducible dimers in the field. Rho family GTPases are one of the many downstream intracellular signaling nodes that spatially and temporally translate the extracellular cue into cytoskeletal remodeling, producing the protrusions and forces necessary for directional motility. Using iLID, we optogenetically controlled guanine nucleotide exchange factors (GEFs), specific for Rac or Cdc42. This approach allowed us to bypass extracellular signaling events and precisely manipulate localized GTPase activity. We first hypothesized and verified that cells expressing either optogenetic GEF, plated on fibronectin and exposed to a gradient of light would migrate up the gradient or “phototax”. Knowing that signals from integrin based adhesions can also regulate the actin cytoskeleton we were curious if these signals were necessary for phototaxis. We found that cells expressing the optogenetic GEF specific for Rac, plated on Poly-L-Lysine (abolishing integrin based adhesion) migrate randomly in a gradient of light. We provide evidence that integrin based adhesions provide signaling feedback within the optogenetically formed protrusions that reinforce the signals necessary for directed migration. Interestingly, we find that cells expressing the optogenetic GEF specific for Cdc42 move directionally in a gradient of light independent of a fibronectin substrate. Through further optogenetic experiments, we show that this is due to a Cdc42 dependent secretion of fibronectin under newly formed protrusions, stabilizing the lamellipodia and providing the necessary feedback for directional migration. 2017 Biochemistry Cellular biology Directional Migration; Fibronectin; GTPase; iLID; Light Induced Dimer; Optogenetics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Brian Kuhlman Thesis advisor James Bear Thesis advisor Jean Cook Thesis advisor John Sondek Thesis advisor Kevin Slep Thesis advisor text 2017-05 Zimmerman_unc_0153D_16776.pdf uuid:3a7e5c48-d246-40af-b9f0-c65d142b2fa3 proquest 2019-07-06T00:00:00 2017-04-27T01:34:06Z application/pdf 7204011 yes