Visualizing Calcium Stores
Using an engineered protein construct cloned into a carrier plasmid, I am able to transiently transfect target cells using a lipofection solution and visualize the compartmentalization of calcium in the ER.
The cameleon protein is calcium sensitive. When bound to calcium the two fluorescent moities undergo FRET. One excitation wavelength is used, and emitted light is collected at two wavelengths, corresponding to each fluorescent domain on the molecule. This ratiometric emission allows us to calculate the concentration of free calcium ions.
I aim to elucidate a model for the ER in amacrine cells, likely one of the following possibilities:
- The ER calcium store is continuous with that of the cell body and all other dendrites
- The ER calcium stores exist in discrete pockets controlled by specific types of calcium channels
- The ER is a dynamic system where pockets bud off and rejoin the larger ER
I also plan to measure spontaneous changes in calcium in the ER and calcium responses as a result of various stimuli.
Lipofection is known to be very efficient in chinese hamster ovary (CHO) cells. These cells also exhibit a very small number of endogenous receptors, making them a very simple model system. I use CHO cells to show that the cameleon DNA is effective in causing the expression of the cameleon protein. The below image shows the expected result: cameleon protein is strongly expressed when cells are transformed with the lipofection solution.
I have not yet acquired any images of amacrine cells expressing the cameleon protein. I am currently working on a protocol that will allow me to image amacrine cells with expressed cameleon at rest and under stimulus.