Directed Migration Achieved in Mammalian Cells via Ca2+ Signal Rewiring in Synthetic Protein Chimeras

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.

Department dc:contributor.department

Biomedical Engineering

Year dc:date.issued

2019

Author dc:creator

• Qudrat, Anam

Advisor dc:contributor.advisor

• Truong, Kevin

• calcium signaling
• chimeras
• nano bodies
• synthetic biology
• TNF alpha
• VEGFR2

Handle dc:identifier.uri

http://hdl.handle.net/1807/94088

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/94088