University of Tasmania
Devenish_whole_thesis.pdf (19.57 MB)

Optogenetic manipulation of calcineurin signalling

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posted on 2023-05-28, 09:18 authored by Devinish, EJ
Calcineurin (CaN) is a calcium (Ca\\(^{2+}\\)) dependent protein phosphatase that is crucial for mammalian development. The requirement for CaN signalling has been indicated by genetic and pharmacological experiments, and further insights into the spatial and temporal characteristics of CaN activity would be valuable in understanding its specific function in development. During development of the nervous system, axon outgrowth and pathfinding towards synaptic partners is guided by environmental cues detected by the growth cone, a navigational structure at the axon tip. Many guidance cues trigger Ca\\(^{2+}\\) influx that is asymmetric across the growth cone, and the guidance events that follow require CaN and Ca\\(^{2+}\\)/calmodulin dependent protein kinase II (CaMKII). It has been hypothesised that CaN signalling occurs in spatially restricted regions of the growth cone, following the patterns of \\(^{2+}\\) influx. Optogenetic techniques are uniquely positioned to probe the spatial and temporal patterns of protein signalling, because they use light to control protein activity. Modern optics can precisely control the illumination pattern in spatial and temporal dimensions. The aim of this project was to develop optogenetic tools to manipulate CaN signalling. This thesis will present approaches that can robustly inhibit CaN signalling in living cells using blue (<500 nm) or red (~650 nm) light. These approaches utilise reactive oxygen species, generated when certain chromophores are excited, to disrupt the phosphatase function of CaN. For the blue light version of the tool, the photosensitising fluorescent protein miniSOG is used, while the red light version utilises a novel photosensitising florescent dye (dibromo-JF649) that binds to the HaloTag protein domain. To target reactive oxygen species to endogenous CaN, the miniSOG or HaloTag domain is tethered to calcineurin B, an accessory subunit that incorporates into endogenous CaN complexes. Using both a live cell assay involving nuclear factor of activated T-cells (NFAT) in HEK293A cells, and biochemical measurement of CaN activity in cellular protein extracts, the data presented will show that CaN is selectively inhibited by these tools, and the extent of inhibition is dependent on the duration and intensity of the light. To activate CaN signalling using optogenetics, the protein must be modified to replace its regulatory mechanism with a light inducible module. Several approaches have been designed in this project, and the proteins that comprise each design have undergone initial screening. Data will be presented that demonstrate the potential of each approach, and where further development is required to achieve control of CaN signalling.


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