{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/342743"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/342743","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"New roles of Rac-GEFs in Neutrophils and in Glucose Homeostasis","abstract":"Rac-GEFs (guanine-nucleotide exchange factors) are proteins that activate Rac GTPases, thereby enabling Rac-dependent cytoskeletal dynamics and cellular processes such as adhesion and migration. I used mice with genetically modified Rac-GEFs to identify new functional roles of these proteins in two distinct biological systems, neutrophil adhesion/migration and glucose homeostasis. In the first part of my thesis, I investigated cytoskeletal dynamics controlled by the Rac-GEF Tiam1 in neutrophil adhesion/migration. We previously found a paradoxical increase in the adhesion of Tiam1–/– neutrophils (unpublished). This was surprising, as deficiencies in other neutrophil Rac-GEFs impair adhesion. I showed deregulated neutrophil polarisation, Filamentous-actin (F-actin) polarity, F-actin dynamics and focal adhesion structures in Tiam1–/– neutrophils adhering to integrin ligands. I demonstrated increased integrin avidity in Tiam1–/– neutrophils stimulated with CXCL1, and increased migration of Tiam1–/– neutrophils under shear stress. These results contribute to our elucidation of the mechanisms underlying the paradoxical increase in the adhesion of Tiam1-deficient neutrophils. In the second part, I investigated spatiotemporal patterns of Rac activity generated during neutrophil adhesion/migration by several major neutrophil Rac-GEFs. The aim was to identify specific roles for these Rac-GEFs which all signal in response to the activation of GPCRs. I used our Rac activity FRET reporter mouse strain (RFC) (Johnsson, Dai et al. 2014), crossed to mice deficient in the Rac-GEFs P-Rex1/Vav1, DOCK2 or Tiam1. I demonstrated that Rac activity is increased in RFC Tiam1–/– neutrophils adhering to integrin ligands, which may explain the increased adhesion. In contrast, RFC DOCK2–/– and RFC P-Rex1–/– Vav1–/– neutrophils had reduced Rac activity, as expected for Rac-GEF deficient cells, confirming a unique and paradoxical role of Tiam1 in limiting Rac activity and Rac-dependent cell responses. The loss of Rac activity was global in RFC DOCK2–/– neutrophils but more localised in RFC P-Rex1–/– Vav1–/– cells. This project has identified specific roles of various Rac-GEFs in neutrophil adhesion and migration. In the third and final part, I investigated adaptor functions of P-Rex family Rac-GEFs P-Rex1 and P-Rex2 in glucose homeostasis. We previously showed that P-Rex1 and P-Rex2 deficient mice have accelerated glucose clearance during glucose challenge, along with low fasting blood glucose levels and altered insulin sensitivity (unpublished). In order to address the underlying mechanisms, I used mice with catalytically inactive P-Rex1 or P-Rex2 (GEF-dead mice) which we recently generated (unpublished). I demonstrated that the increased glucose clearance is an adaptor function of P-Rex Rac-GEFs, whereas fasting blood glucose levels and insulin sensitivity are Rac-GEF activity dependent. I showed increased plasma insulin levels in P-Rex1–/– and P-Rex2–/– mice upon glucose challenge and increased glucose-stimulated insulin secretion from P-Rex1–/– and P-Rex2–/– pancreatic islets. Use of P-Rex1 GEF-dead mice showed that these latter phenotypes were again adaptor functions, suggesting that these responses contribute to the accelerated glucose clearance in P-Rex-deficient mice. Combined, my work has provided a substantial body of data identifying unexpected novel roles for Rac-GEFs in both neutrophil biology and in glucose homeostasis, providing mechanistic insight in addition to new functions in both systems.","abstract_html":"Rac-GEFs (guanine-nucleotide exchange factors) are proteins that activate Rac GTPases, thereby enabling Rac-dependent cytoskeletal dynamics and cellular processes such as adhesion and migration. I used mice with genetically modified Rac-GEFs to identify new functional roles of these proteins in two distinct biological systems, neutrophil adhesion/migration and glucose homeostasis. In the first part of my thesis, I investigated cytoskeletal dynamics controlled by the Rac-GEF Tiam1 in neutrophil adhesion/migration. We previously found a paradoxical increase in the adhesion of Tiam1–/– neutrophils (unpublished). This was surprising, as deficiencies in other neutrophil Rac-GEFs impair adhesion. I showed deregulated neutrophil polarisation, Filamentous-actin (F-actin) polarity, F-actin dynamics and focal adhesion structures in Tiam1–/– neutrophils adhering to integrin ligands. I demonstrated increased integrin avidity in Tiam1–/– neutrophils stimulated with CXCL1, and increased migration of Tiam1–/– neutrophils under shear stress. These results contribute to our elucidation of the mechanisms underlying the paradoxical increase in the adhesion of Tiam1-deficient neutrophils. In the second part, I investigated spatiotemporal patterns of Rac activity generated during neutrophil adhesion/migration by several major neutrophil Rac-GEFs. The aim was to identify specific roles for these Rac-GEFs which all signal in response to the activation of GPCRs. I used our Rac activity FRET reporter mouse strain (RFC) (Johnsson, Dai et al. 2014), crossed to mice deficient in the Rac-GEFs P-Rex1/Vav1, DOCK2 or Tiam1. I demonstrated that Rac activity is increased in RFC Tiam1–/– neutrophils adhering to integrin ligands, which may explain the increased adhesion. In contrast, RFC DOCK2–/– and RFC P-Rex1–/– Vav1–/– neutrophils had reduced Rac activity, as expected for Rac-GEF deficient cells, confirming a unique and paradoxical role of Tiam1 in limiting Rac activity and Rac-dependent cell responses. The loss of Rac activity was global in RFC DOCK2–/– neutrophils but more localised in RFC P-Rex1–/– Vav1–/– cells. This project has identified specific roles of various Rac-GEFs in neutrophil adhesion and migration. In the third and final part, I investigated adaptor functions of P-Rex family Rac-GEFs P-Rex1 and P-Rex2 in glucose homeostasis. We previously showed that P-Rex1 and P-Rex2 deficient mice have accelerated glucose clearance during glucose challenge, along with low fasting blood glucose levels and altered insulin sensitivity (unpublished). In order to address the underlying mechanisms, I used mice with catalytically inactive P-Rex1 or P-Rex2 (GEF-dead mice) which we recently generated (unpublished). I demonstrated that the increased glucose clearance is an adaptor function of P-Rex Rac-GEFs, whereas fasting blood glucose levels and insulin sensitivity are Rac-GEF activity dependent. I showed increased plasma insulin levels in P-Rex1–/– and P-Rex2–/– mice upon glucose challenge and increased glucose-stimulated insulin secretion from P-Rex1–/– and P-Rex2–/– pancreatic islets. Use of P-Rex1 GEF-dead mice showed that these latter phenotypes were again adaptor functions, suggesting that these responses contribute to the accelerated glucose clearance in P-Rex-deficient mice. Combined, my work has provided a substantial body of data identifying unexpected novel roles for Rac-GEFs in both neutrophil biology and in glucose homeostasis, providing mechanistic insight in addition to new functions in both systems.","abstract_has_math":false,"creators":["Machin, Polly"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Welch, heidi"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-07-01","date_published":"2022-07-01","updated_at":"2026-07-22T22:23:59Z","subjects":["GEFs","Rac-GTPase","Glucose Homeostasis","Neutrophils"],"languages":["eng"],"rights":[],"rights_urls":["https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.90157","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Welch, heidi"]},{"key":"dc:creator","label":"Author","values":["Machin, Polly"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-07-01"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/342743"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["GEFs","Rac-GTPase","Glucose Homeostasis","Neutrophils"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.90157"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/5cbeb935-fad0-4eab-b1e5-60474eb88768/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Rac-GEFs (guanine-nucleotide exchange factors) are proteins that activate Rac GTPases, thereby enabling Rac-dependent cytoskeletal dynamics and cellular processes such as adhesion and migration. I used mice with genetically modified Rac-GEFs to identify new functional roles of these proteins in two distinct biological systems, neutrophil adhesion/migration and glucose homeostasis. In the first part of my thesis, I investigated cytoskeletal dynamics controlled by the Rac-GEF Tiam1 in neutrophil adhesion/migration. We previously found a paradoxical increase in the adhesion of Tiam1–/– neutrophils (unpublished). This was surprising, as deficiencies in other neutrophil Rac-GEFs impair adhesion. I showed deregulated neutrophil polarisation, Filamentous-actin (F-actin) polarity, F-actin dynamics and focal adhesion structures in Tiam1–/– neutrophils adhering to integrin ligands. I demonstrated increased integrin avidity in Tiam1–/– neutrophils stimulated with CXCL1, and increased migration of Tiam1–/– neutrophils under shear stress. These results contribute to our elucidation of the mechanisms underlying the paradoxical increase in the adhesion of Tiam1-deficient neutrophils. In the second part, I investigated spatiotemporal patterns of Rac activity generated during neutrophil adhesion/migration by several major neutrophil Rac-GEFs. The aim was to identify specific roles for these Rac-GEFs which all signal in response to the activation of GPCRs. I used our Rac activity FRET reporter mouse strain (RFC) (Johnsson, Dai et al. 2014), crossed to mice deficient in the Rac-GEFs P-Rex1/Vav1, DOCK2 or Tiam1. I demonstrated that Rac activity is increased in RFC Tiam1–/– neutrophils adhering to integrin ligands, which may explain the increased adhesion. In contrast, RFC DOCK2–/– and RFC P-Rex1–/– Vav1–/– neutrophils had reduced Rac activity, as expected for Rac-GEF deficient cells, confirming a unique and paradoxical role of Tiam1 in limiting Rac activity and Rac-dependent cell responses. The loss of Rac activity was global in RFC DOCK2–/– neutrophils but more localised in RFC P-Rex1–/– Vav1–/– cells. This project has identified specific roles of various Rac-GEFs in neutrophil adhesion and migration. In the third and final part, I investigated adaptor functions of P-Rex family Rac-GEFs P-Rex1 and P-Rex2 in glucose homeostasis. We previously showed that P-Rex1 and P-Rex2 deficient mice have accelerated glucose clearance during glucose challenge, along with low fasting blood glucose levels and altered insulin sensitivity (unpublished). In order to address the underlying mechanisms, I used mice with catalytically inactive P-Rex1 or P-Rex2 (GEF-dead mice) which we recently generated (unpublished). I demonstrated that the increased glucose clearance is an adaptor function of P-Rex Rac-GEFs, whereas fasting blood glucose levels and insulin sensitivity are Rac-GEF activity dependent. I showed increased plasma insulin levels in P-Rex1–/– and P-Rex2–/– mice upon glucose challenge and increased glucose-stimulated insulin secretion from P-Rex1–/– and P-Rex2–/– pancreatic islets. Use of P-Rex1 GEF-dead mice showed that these latter phenotypes were again adaptor functions, suggesting that these responses contribute to the accelerated glucose clearance in P-Rex-deficient mice. Combined, my work has provided a substantial body of data identifying unexpected novel roles for Rac-GEFs in both neutrophil biology and in glucose homeostasis, providing mechanistic insight in addition to new functions in both systems."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["4839583520fa64c44a13d7e625e22785"]},{"key":"dc:title","label":"Title","values":["New roles of Rac-GEFs in Neutrophils and in Glucose Homeostasis"]}]}],"canonical_facts":{"dc:contributor.advisor":["Welch, heidi"],"dc:creator":["Machin, Polly"],"dc:date.issued":["2022-07-01"],"dc:description.abstract":["Rac-GEFs (guanine-nucleotide exchange factors) are proteins that activate Rac GTPases, thereby enabling Rac-dependent cytoskeletal dynamics and cellular processes such as adhesion and migration. I used mice with genetically modified Rac-GEFs to identify new functional roles of these proteins in two distinct biological systems, neutrophil adhesion/migration and glucose homeostasis. In the first part of my thesis, I investigated cytoskeletal dynamics controlled by the Rac-GEF Tiam1 in neutrophil adhesion/migration. We previously found a paradoxical increase in the adhesion of Tiam1–/– neutrophils (unpublished). This was surprising, as deficiencies in other neutrophil Rac-GEFs impair adhesion. I showed deregulated neutrophil polarisation, Filamentous-actin (F-actin) polarity, F-actin dynamics and focal adhesion structures in Tiam1–/– neutrophils adhering to integrin ligands. I demonstrated increased integrin avidity in Tiam1–/– neutrophils stimulated with CXCL1, and increased migration of Tiam1–/– neutrophils under shear stress. These results contribute to our elucidation of the mechanisms underlying the paradoxical increase in the adhesion of Tiam1-deficient neutrophils. In the second part, I investigated spatiotemporal patterns of Rac activity generated during neutrophil adhesion/migration by several major neutrophil Rac-GEFs. The aim was to identify specific roles for these Rac-GEFs which all signal in response to the activation of GPCRs. I used our Rac activity FRET reporter mouse strain (RFC) (Johnsson, Dai et al. 2014), crossed to mice deficient in the Rac-GEFs P-Rex1/Vav1, DOCK2 or Tiam1. I demonstrated that Rac activity is increased in RFC Tiam1–/– neutrophils adhering to integrin ligands, which may explain the increased adhesion. In contrast, RFC DOCK2–/– and RFC P-Rex1–/– Vav1–/– neutrophils had reduced Rac activity, as expected for Rac-GEF deficient cells, confirming a unique and paradoxical role of Tiam1 in limiting Rac activity and Rac-dependent cell responses. The loss of Rac activity was global in RFC DOCK2–/– neutrophils but more localised in RFC P-Rex1–/– Vav1–/– cells. This project has identified specific roles of various Rac-GEFs in neutrophil adhesion and migration. In the third and final part, I investigated adaptor functions of P-Rex family Rac-GEFs P-Rex1 and P-Rex2 in glucose homeostasis. We previously showed that P-Rex1 and P-Rex2 deficient mice have accelerated glucose clearance during glucose challenge, along with low fasting blood glucose levels and altered insulin sensitivity (unpublished). In order to address the underlying mechanisms, I used mice with catalytically inactive P-Rex1 or P-Rex2 (GEF-dead mice) which we recently generated (unpublished). I demonstrated that the increased glucose clearance is an adaptor function of P-Rex Rac-GEFs, whereas fasting blood glucose levels and insulin sensitivity are Rac-GEF activity dependent. I showed increased plasma insulin levels in P-Rex1–/– and P-Rex2–/– mice upon glucose challenge and increased glucose-stimulated insulin secretion from P-Rex1–/– and P-Rex2–/– pancreatic islets. Use of P-Rex1 GEF-dead mice showed that these latter phenotypes were again adaptor functions, suggesting that these responses contribute to the accelerated glucose clearance in P-Rex-deficient mice. Combined, my work has provided a substantial body of data identifying unexpected novel roles for Rac-GEFs in both neutrophil biology and in glucose homeostasis, providing mechanistic insight in addition to new functions in both systems."],"dc:format.checksum.md5":["4839583520fa64c44a13d7e625e22785"],"dc:identifier.doi":["10.17863/CAM.90157"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/5cbeb935-fad0-4eab-b1e5-60474eb88768/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/342743"],"dc:rights":["https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["GEFs","Rac-GTPase","Glucose Homeostasis","Neutrophils"],"dc:title":["New roles of Rac-GEFs in Neutrophils and in Glucose Homeostasis"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:23:59Z"}