Mechanical regulation of membrane potential in Non-excitable cell

Introduction: The cell membrane potential is a crucial regulator of electrical activities in excitable cells such as neurons and heart cells. Recently, its significance has been observed in non-excitable cells, including epithelial cells. This potential is generated by specific ion channels and transporters with distinct ion selectivity and permeability. Recent studies suggest that cytoskeleton-mediated protrusion during cell migration correlates with local ion charge changes. Yet, it remains uncertain whether these ion fluxes significantly change the membrane potential or how cells harness these dynamics to manage voltage regulation. Additionally, the complex interplay between membrane potential and cytoskeletal dynamics in controlling cell migration is not fully understood. In our study, we used the genetically encoded voltage indicator Jedi2p-CYOFP to monitor membrane voltage dynamics. We conducted a comprehensive investigation into the determinants of membrane potential and their interactions with cellular functions such as the cell cycle and migration under homeostatic conditions. Our results reveal that the membrane potential not only influences cellular functions in a significant manner but also orchestrates the interaction between the cytoskeleton and ion channels via the AKT pathway, affecting protrusion formation. This research advances our understanding of the regulatory role of membrane potential in cell function and the complex relationship between membrane potential and cytoskeletal dynamics in cancer metastasis. It also opens new avenues for therapeutic interventions targeting these essential pathways in cancer treatment.

Materials and

Methods: Transfection Method
To generate the permanent Jedi-Cyofp cell line, we employed the Piggy-bac transposon technique. To transfer the region of interest from its original plasmid to the Piggy-bac vector with puromycin resistance, we designed primers to trim the relevant sequence from the Jedi plasmid, followed by PCR amplification. Subsequently, the Gibson assembly method was utilized to integrate these components into the desired new plasmid seamlessly. The integrated plasmid was then transfected to cells using Lipofectamine 3000 Transfection Reagent (plasmid to transposon ratio 5:1). Puromycin was introduced for selection purposes.

Data Analysis
To analyze the membrane potential, we employ two main approaches: calculating the overall cell membrane potential and analyzing its distribution around the cell perimeter. We begin by defining the cell boundary through a masking process to identify each boundary pixel point. For each of these points, we construct a norm vector directed inward. Along this vector, we select three points and compute their average membrane potential. This average value represents the membrane potential at that specific boundary point. The overall membrane potential of the cell is then calculated as the mean of these average values from all boundary points. For the distribution analysis, we arrange the boundary pixel points, as previously calculated, in a clockwise direction from 0 degrees to 360 degrees. This organization allows us to map the membrane potential distribution along the cell perimeter. We then plot the ratio of membrane potential to degree, providing a visual representation of potential variation around the cell.

Results, Conclusions, and Discussions: Membrane potential is a cell cycle indicator.
In our investigation, we initially examined the behavior of membrane potential throughout the cell cycle, particularly from one division phase to the next. We observed that cells predominantly exhibit hyperpolarization during most of the cycle, with membrane potential normalizing during the mitotic phase. To further investigate changes in membrane potential during the cell cycle, we employed a CDK4 inhibitor and induced serum starvation to arrest cells at the late G1 and G0 phases. As we expected, our measurements revealed that the membrane potential does not fluctuate between hyperpolarization and depolarization during cell cycle arrest; instead, it remains stable. Additionally, we conducted a population analysis to compare the membrane potential of cells in a normal cycle with those in a cell cycle-arrested state. The analysis demonstrated that cells in the arrested state maintain a relatively depolarized membrane potential compared to those in the normal cycle. These findings collectively indicate that the membrane potential serves as an indicator of cell cycle progression, requiring specific patterns of hyperpolarization and depolarization to advance through different stages. This dynamic suggests a potential regulatory mechanism by which cells modulate their cycle progression through changes in electrical potential.

Membrane potential controls migration via NHE1 activation
In our investigation, we initially analyzed voltage distribution along polarized cells during migration. Our observations indicate that cell protrusions consistently correlate with voltage depolarization. Given the known activation of Na+/H+ exchanger 1 (NHE1) in the protrusion regions of polarized cells, we further explored how membrane potential and its distribution are altered by inhibiting NHE1 with EIPA. Interestingly, blocking NHE1 induced overall hyperpolarization in the cells. Specifically, the protrusion regions exhibited more pronounced hyperpolarization compared to lateral regions. This pattern suggests that during migration, NHE1 activation at the protrusions leads to localized depolarization, facilitating cell movement. To validate the association between cell protrusions and depolarization, we conducted experiments on cell spreading during detachment and reattachment, as well as during cell rounding in the division. In both scenarios, our results consistently showed that cell protrusions are accompanied by depolarization.

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