{"id":{"repo_id":"toronto-retro","oai_identifier":"oai:utoronto.scholaris.ca:1807/94088"},"canonical_url":"https://search.dev.ndltd.org/etd/toronto-retro/oai:utoronto.scholaris.ca:1807/94088","repository":{"repo_id":"toronto-retro","name":"University of Toronto","base_url":"https://utoronto.scholaris.ca/server/oai/request"},"display":{"title":"Directed Migration Achieved in Mammalian Cells via Ca2+ Signal Rewiring in Synthetic Protein Chimeras","abstract":"Synthetic biology achieves control over cellular behavior by endogenous re-wiring. Ca2+ signals allow cells to regulate diverse processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g. VEGFR2), Ca2+ signals are generated upon ligand binding (e.g. VEGF-A). Here, firstly we engineered fusion proteins that generate a Ca2+ signal upon ligand binding by creating fusions of domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we re-wired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Next, we validated this design strategy in two systems, i.e., inflammatory cytokines such as TNFα and antibodies. A system of proteins was used: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca2+-activated RhoA (named CaRQ), VSVG and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs allowing migration towards the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, post-ganciclovir treatment, cells undergo death via the thymidine kinase suicide mechanism. Hence, this forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low pH microenvironment. Lastly, we exploited antibodies to bind their antigens exclusively in combination with the ability of the cytoplasmic domain of VEGFR2 to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e. nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumour necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state but instead, only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively.","abstract_html":"Synthetic biology achieves control over cellular behavior by endogenous re-wiring. Ca2+ signals allow cells to regulate diverse processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g. VEGFR2), Ca2+ signals are generated upon ligand binding (e.g. VEGF-A). Here, firstly we engineered fusion proteins that generate a Ca2+ signal upon ligand binding by creating fusions of domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we re-wired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Next, we validated this design strategy in two systems, i.e., inflammatory cytokines such as TNFα and antibodies. A system of proteins was used: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca2+-activated RhoA (named CaRQ), VSVG and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs allowing migration towards the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, post-ganciclovir treatment, cells undergo death via the thymidine kinase suicide mechanism. Hence, this forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low pH microenvironment. Lastly, we exploited antibodies to bind their antigens exclusively in combination with the ability of the cytoplasmic domain of VEGFR2 to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e. nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumour necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state but instead, only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively.","abstract_has_math":false,"creators":["Qudrat, Anam"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Biomedical Engineering","school":null,"contributors":[],"advisors":["Truong, Kevin"],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-03","date_published":"2019-03","updated_at":"2026-07-27T21:27:52Z","subjects":["calcium signaling","chimeras","nano bodies","synthetic biology","TNF alpha","VEGFR2"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1807/94088","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Truong, Kevin"]},{"key":"dc:contributor.department","label":"Department","values":["Biomedical Engineering"]},{"key":"dc:creator","label":"Author","values":["Qudrat, Anam"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2019-03"]},{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2019-03-25T12:00:56Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2019-03-25T12:00:56Z"]},{"key":"dc:date.issued","label":"Date","values":["2019-03"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["calcium signaling","chimeras","nano bodies","synthetic biology","TNF alpha","VEGFR2"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1807/94088"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Synthetic biology achieves control over cellular behavior by endogenous re-wiring. Ca2+ signals allow cells to regulate diverse processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g. VEGFR2), Ca2+ signals are generated upon ligand binding (e.g. VEGF-A). Here, firstly we engineered fusion proteins that generate a Ca2+ signal upon ligand binding by creating fusions of domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we re-wired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Next, we validated this design strategy in two systems, i.e., inflammatory cytokines such as TNFα and antibodies. A system of proteins was used: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca2+-activated RhoA (named CaRQ), VSVG and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs allowing migration towards the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, post-ganciclovir treatment, cells undergo death via the thymidine kinase suicide mechanism. Hence, this forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low pH microenvironment. Lastly, we exploited antibodies to bind their antigens exclusively in combination with the ability of the cytoplasmic domain of VEGFR2 to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e. nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumour necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state but instead, only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["Directed Migration Achieved in Mammalian Cells via Ca2+ Signal Rewiring in Synthetic Protein Chimeras"]}]}],"canonical_facts":{"dc:contributor.advisor":["Truong, Kevin"],"dc:contributor.department":["Biomedical Engineering"],"dc:creator":["Qudrat, Anam"],"dc:date":["2019-03"],"dc:date.accessioned":["2019-03-25T12:00:56Z"],"dc:date.available":["2019-03-25T12:00:56Z"],"dc:date.issued":["2019-03"],"dc:description.abstract":["Synthetic biology achieves control over cellular behavior by endogenous re-wiring. Ca2+ signals allow cells to regulate diverse processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g. VEGFR2), Ca2+ signals are generated upon ligand binding (e.g. VEGF-A). Here, firstly we engineered fusion proteins that generate a Ca2+ signal upon ligand binding by creating fusions of domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we re-wired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Next, we validated this design strategy in two systems, i.e., inflammatory cytokines such as TNFα and antibodies. A system of proteins was used: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca2+-activated RhoA (named CaRQ), VSVG and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs allowing migration towards the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, post-ganciclovir treatment, cells undergo death via the thymidine kinase suicide mechanism. Hence, this forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low pH microenvironment. Lastly, we exploited antibodies to bind their antigens exclusively in combination with the ability of the cytoplasmic domain of VEGFR2 to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e. nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumour necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state but instead, only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively."],"dc:description.degree":["Ph.D."],"dc:identifier.uri":["http://hdl.handle.net/1807/94088"],"dc:subject":["calcium signaling","chimeras","nano bodies","synthetic biology","TNF alpha","VEGFR2"],"dc:title":["Directed Migration Achieved in Mammalian Cells via Ca2+ Signal Rewiring in Synthetic Protein Chimeras"],"dc:type":["Thesis"]},"updated_at":"2026-07-27T21:27:52Z"}