Optogenetics has emerged as a branch of biotechnology that combines genetic engineering with optics to observe intracellular changes as well as control cellular function. Despite recent progress, there still remains the need for an optogenetic tool that can specifically control Ca2+. Such a tool would greatly facilitate the study of highly Ca2+-dependent cellular processes that are regulated both spatially and temporally. Ca2+ signaling regulates many cellular processes in both healthy and diseased cells. The ability to modulate the shape, duration, and amplitude of Ca2+ signaling is important for elucidating mechanisms by which endogenous Ca2+ concentrations are maintained. In this thesis, we used optogenetic approaches to explore a number of strategies to control Ca2+ influx through store-operated Ca2+ entry (SOCE) mediated by Stim1 and Orai1.
To better study Ca2+ signaling in live cells, protein-based biosensors can be developed to monitor intracellular Ca2+ changes. To aid in this, we developed a computational modeling tool called FPMOD to improve both new and existing biosensor designs. Although FPMOD was initially intended for evaluating biosensor designs, other research groups have since used it to construct models of other proteins to answer questions related to protein conformation.
We next studied the modulation of SOCE by using drug-inducible fusion proteins to study the regulation of Stim1 puncta formation. Interestingly, recruiting a Ca2+-buffering protein to Stim1 led to puncta formation, a previously unknown means of inducing puncta. These results suggest Stim1 may additionally be regulated by cytoplasmic Ca2+ levels.
Finally, we developed LOVS1K, an optogenetic tool to directly activate Orai1 channels and specifically control Ca2+ influx. Photo-sensitive LOVS1K was used to generate both local Ca2+ influx at the membrane and global cytoplasmic Ca2+ signals. As proof of concept, LOVS1K was further used to modulate engineered Ca2+-dependent proteins.
Ca2+ is a remarkably versatile intracellular messenger. The combination of high spatiotemporal control of irradiation and the ability of LOVS1K to generate both local and global Ca2+ changes provides a promising platform to study cellular processes that are highly dependent on different Ca2+ signals. Together, biosensors and engineered Ca2+-modulating tools can be used to study the many different aspects of Ca2+ signaling and controllably manipulate endogenous Ca2+ signaling pathways.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/31898 |
| Date | 11 January 2012 |
| Creators | Pham, Elizabeth |
| Contributors | Truong, Kevin |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis, Video |
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