Localised IP3 Photo-Uncaging in Endothelial Cells
Biologically active molecules can be engineered to contain a photolabile protecting group attached to a significant functional group, rendering the molecule biologically inert - these are known as "caged" compounds. Using light of an appropriate wavelength will remove these protecting groups, activating the molecule and permitting a high level of spatial and temporal control of the administration of the caged compound.
By bringing the light in through a fiberoptic cable independent of the imaging light source, specific cells or subsets of cells can be activated and the resulting response visualised.
In the McCarron lab, we are interested in using this technique to understand how calcium signaling in the endothelium propagates, and how IP3-stimulated responses are altered when different pharmacological interventions are used.
In the above video, 395nm light was used to simultaneously uncage IP3 within a subset of endothelial cells, and the subsequent calcium signal is visualised with Cal-520 live calcium indicator dye. The signal intensity profile is plotted for the dataset shown.
The video on the left shows an overlay of three repeat uncaging events. You can see that despite identical initial uncaging conditions, subsequent signal propagation quickly becomes much more random.
Since we know that all signals are a direct consequence of the initial uncaging event, this allows us to tease apart mechanisms of signal propagation.
Lab members working on localised photolysis: