UC Berkeley

Membrane potential (Vm) is a critical biophysical property in electrically excitable and non-electrically excitable cells. Established methods for measuring Vm successfully report the large, rapid changes that trigger key signaling pathways in electrically excitable cells but lack the capabilities to record over extended time periods or reliably quantify smaller changes in Vm. Emerging evidence suggests that smaller fluctuations in membrane potential occurring over seconds to hours may have a regulatory role in fundamental physiological processes in non-electrically excitable cells, such as cell cycle progression. However, robust characterization of membrane potential dynamics and possible mechanisms of bioelectric control has been limited by insufficient techniques for quantifying Vm over these physiologically relevant timescales. Fluorescence lifetime imaging with VoltageFluor dyes (VF-FLIM) provides a new approach for making minimally invasive and high-throughput measurements of absolute membrane potential over extended timescales.

This dissertation demonstrates the application of VF-FLIM to quantify membrane potential and track changes in non-electrically excitable cells. Chapter 1 introduces techniques for measuring membrane potential and summarizes evidence for membrane potential dynamics and bioelectric signaling during cell cycle progression. Chapter 2 presents initial work using VF-FLIM to identify differences in membrane potential across cell cycle phases, revealing a ~10 mV depolarization in G2/M phase cells compared to cells in G1 and S phase. Chapter 3 expands on this work and describes efforts towards monitoring real-time changes in Vm in live cells with development of a stable cell line expressing the sensor DHB-mVenus to track cell cycle progression, combined with a red-shifted VoltageFluor. Chapter 4 explores characterization of membrane potential dynamics induced by epidermal growth factor receptor signaling (EGFR), spanning four different EGFR ligands across two cell lines showing disparate responses to EGFR stimulation. A general protocol for VF-FLIM experiments in provided in Chapter 5, and Chapter 6 details essential considerations for data acquisition and analysis. Together, this work highlights the utility of VF-FLIM as an approach for optically recording Vm over extended time scales and offers new insights into membrane potential dynamics during cell cycle progression and epidermal growth factor receptor signaling in non-electrically excitable cells.