All cells maintain a membrane potential (Vm) that is critical for cell function and plays a role in a variety of cellular processes. Cell cycle regulation has been extensively studied due to its fundamental role in biology. Although preliminary evidence suggests that changes in membrane potential occur during the cell cycle, the possible role of Vm in cell cycle regulation is still unknown due to limitations in voltage imaging. The Miller lab has developed a series of VoltageFluors (VFs), which are voltage-sensitive fluorophores that localize to the plasma membrane. They respond to changes in Vm through a photoinduced electron transfer mechanism, resulting in modulation of fluorescence intensity and lifetime. Intensity-based voltage imaging is not ideal for measuring Vm during the cell cycle because it is dependent on fluorophore concentration, photobleaching, and illumination intensity. This makes quantification of Vm difficult. Fluorescence lifetime is affected only by the molecular environment of the probe, allowing us to quantify absolute Vm through fluorescence lifetime imaging (FLIM). This lets us develop a cell-type specific calibration between membrane potential and fluorescence lifetime. The VF-FLIM method enables non-invasive and high throughput optical determination of absolute membrane potential. Initial work focused on using VF-FLIM to measure Vm in MCF-7 breast cancer cells, which are known to undergo changes in membrane potential during the cell cycle. We successfully demonstrated that VF-FLIM reports differences in Vm between populations of MCF-7 cells synchronized in different phases of the cell cycle. Our current work aims to identify real-time changes in membrane potential for single cells as they progress through the cell cycle. We are utilizing the probe DHB-mVenus, which consists of a fragment of DNA Helicase B tagged to the fluorescent protein mVenus. DHB-mVenus localization shifts from the nucleus to the cytoplasm in response to CDK2 activity during the cell cycle. Tracking changes in DHB-mVenus localization and fluorescence intensity can thus be used to identify cell cycle phase in live cells. VF-FLIM in conjunction with DHB-mVenus will allow for simultaneous tracking of Vm and cell phase throughout the cell cycle. Future works aims to extend this method to other cell lines to offer better characterization of membrane potential throughout the cell cycle and further investigate the role of Vm in cell cycle regulation.

Optical determination of absolute membrane potential throughout the cell cycle

ACS Spring 2022

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