Membrane potential is a crucial signaling factor in many biological processes and understanding these processes requires measurements of membrane potential in a normal physiological environment. Voltage-sensitive fluorophores provide a non-invasive tool for high throughput optical measurements of membrane potential. In the Miller lab, we focus on a class of voltage-sensitive fluorophores known as VoltageFluors which rapidly modulate their fluorescence in response to changes in membrane potential via changes in the rate of photoinduced electron transfer from an electron-rich aniline. Prior to this work, VoltageFluors had only been targeted to the extracellular surface of plasma membranes. This work reports the development of new strategies for targeting of VoltageFluors to specific membranes including those of subcellular organelles. Chapter 1 provides background material on methods of targeting fluorescent probes to organelles. Chapter 2 describes the targeting and methods for use of a novel VoltageFluor, SPIRIT RhoVR, that localizes to the mitochondrial inner membrane via electrostatic accumulation followed by unmasking of an ester for retention in mitochondria. Chapter 3 describes a tetrazine-transcyclooctene click chemistry strategy for targeting of a novel VoltageFluor, LUnAR RhoVR, to multiple cellular locations with the primary focus being the endoplasmic reticulum (ER). Using LUnAR RhoVR, we were able to monitor ER membrane potential during calcium release and establish the directionality of ER-plasma membrane electrical coupling. Chapter 4 focuses on the physics behind energy transfer in fluorescent dyes, provides a computational description of VoltageFluor sensitivity, and proposes an approach for using VoltageFluors as ratiometric sensors of membrane potential. This dissertation follows this main body of work with two appendices describing initial steps towards synthesizing water-soluble and enzymatically uncageable dyes that would enhance targeting of fluorescent dyes to specific membranes in complex samples.
