Optogenetic control and monitoring of ion transport in cells

Abstract

Ion transport is a dynamic process that is important in signaling both between and within cells. Fluorescence-based measurements are well suited for investigations of ion transport due to their high sensitivity and high spatiotemporal resolution. Optogenetic actuators can locally perturb ion concentrations via combined optical and genetic targeting. Here we combine spectrally separate optogenetic control and monitoring to probe ion transport within cells. We apply these tools in neurons where ionic signals play many important roles across a wide range of space and time scales. In Chapter 2 I investigate the effect on the intracellular pH of repeated neuronal stimulation with a blue-shifted optogenetic actuator, CheRiff, as measured with a red-shifted fluorescent pH sensor, pHoran4. We found that repeated CheRiff stimulation acidified neurons. The acidification was due to the channelrhodopsin CheRiff passing a proton current into the cell. To reduce this acidification, we characterized new channelrhodopsin variants with low proton permeability, and showed that they reduced the neuronal acidification and are viable alternatives for neuron stimulation. In Chapter 3 I investigate diffusion of Ca2+ in dendrites, by combining a blue-shifted Ca2+ selective channelrhodopsin, CapChR2, with a red shifted fluorescent Ca2+ sensor, FR-GECO1c. Using patterned optical stimulation, we created small Ca2+ point sources in the dendrites of cultured neurons and imaged the diffusion of the Ca2+ ions along the dendrite. After the system reached steady-state characterized the length constants of the Ca2+ spread through the dendrites. We then did a paired measurement mapping the kinetic responses of the neuron cell body and dendrites to short wide-area Ca2+ perturbations. By combining this spatial and kinetic data, we estimated the effective diffusion coefficient for Ca2+ in dendrites. We then investigated the effects of Ca2+ efflux on Ca2+ spread and kinetics. We added a pharmacological blocker of NCX1 sodium-calcium exchanger and repeated the Ca2+ transport and homeostasis measurements. These experiments showed that the NCX1 exchanger had a larger effect on calcium handling in fine dendrite than near the cell body, an effect that could be explained by the difference in surface-to-volume ratio of these structures. Finally, I discuss other optogenetic actuators and reporters that could be combined for control and monitoring of different signals of interest. I discuss the factors for choosing what tools to use together and how to ensure the proper functioning and interpretation. As more optogenetic actuators and reporters are created, there are ever more possibilities for probing important signaling pathways. However, it is also important to make sure that the molecular and optical tools are well understood and validated, as there can be artifacts which can affect cellular state and which may confound results, as seen in Chapter 2.