Development and Characterization of Novel Rhodol Calcium Sensors for Investigating Neuronal Activity

Calcium ions mediate a multitude of biochemical and biophysical signaling processes in cells. Calcium imaging is the use of fluorescent probes and microscopy to monitor changes in intracellular calcium concentration, [Ca2+], to track calcium associated cellular processes. The development of fluorescent reporters of [Ca2+], genetically-encoded calcium indicators and chemical calcium indicators, has been paramount in increasing our understanding of the temporal and spatial dynamics of Ca2+ signaling. Coupling Ca2+-selective ligands to fluorescent reporters has provided a wealth of excellent indicators that span the visible excitation and emission spectrum and possess Ca2+ affinities suited to a variety of cellular contexts. One underdeveloped area is the use of rhodamine/fluorescein fluorophores, or rhodols, in the context of Ca2+ sensing. Rhodols are bright and photostable and have good two-photon absorption cross sections (σTPA), making them excellent candidates for incorporation into Ca2+-sensing scaffolds./nIn Chapter 1 of this dissertation, I provide an overview of calcium imaging including the commonly used calcium indicators and imaging techniques. In Chapter 2, I describe the design, synthesis, and application of a previously-published rhodol-based Ca2+ sensor RCS-1, a chlorinated pyrrolidinyl-rhodol calcium sensor. RCS-1 possesses a Ca2+ binding constant of 240 nM and a 10-fold turn-on response to Ca2+ ion binding. RCS-1 effectively absorbs infrared light and has a σTPA of 76 GM at 840 nm, 3-fold greater than that of its fluorescein-based counterpart. The acetoxy-methyl ester of RCS-1 stains the cytosol of live cells, enabling observation of Ca2+ fluctuations and cultured neurons using both one- and two-photon illumination. In Chapter 3, I present the design, synthesis, and application of four rhodol-based Ca2+ sensors: 5’- and 6’-isomers of a chlorinated pyrrolidine-based rhodol (RCS 1a and 1b, respectively) and a chlorinated azetidine-based rhodol (RCS 2a and 2b, respectively). The RCSs possess Ca2+ binding constants of 215 – 240 nM and 10-fold turn-on responses to Ca2+ ion binding. The pyrrolidinyl-rhodol Ca2+ sensors effectively absorb infrared light and have σTPAs of ~76 GM at 840 nm, 3-fold greater than that of its fluorescein-based counterpart. The azetidinyl-rhodol Ca2+ sensors also absorb infrared light and have σTPAs of ~44 GM at 840 nm, 2-fold greater than that of its fluorescein-based counterpart. The tetra-acetoxymethyl esters of RCS 1a, 1b, 2a, and 2b label the cytosol of live cells, enabling observation of histamine-induced Ca2+ fluctuations in HeLa cells, spontaneous activity in cultured hippocampal neurons, and retinal waves in developing retinal ganglion cells using both one- and two-photon illumination. Together, these results demonstrate the utility of rhodol-based scaffolds for Ca2+ sensing using two-photon illumination in neurons. In Chapter 4, I describe our attempt to utilize amino acid acyl ester salts to deliver negatively charged fluorescent probes to cells using carboxyfluorescein as a test probe. Using carboxyfluorescein as a test fluorophore, we were able to show that amino acyl esters of proline and beta-alanine increase cell permeability of negatively charged carboxyfluorescein, while the salts of the amino acyl esters retain solubility of the capped dye in aqueous solutions.

Advisor: Miller, Evan W