To measure the membrane potentials of cell organelles, current technology requires destroying the plasma membrane. Evan W. Miller, University of California, Berkeley, USA, and colleagues have developed the first non-invasive optical sensor system targeting organelle membranes. It records changes in membrane potential using variations in fluorescence and, in combination with a patch-clamp procedure, can be used to investigate electrophysiological relationships between plasma membranes and organelles.

 

How Healthy Is a Cell?

One way to explore the health of a cell is by measuring its membrane potential. A depolarized plasma membrane, for example, indicates that the membrane is leaky and that, therefore, the cell is unhealthy. Membrane potentials can be measured with the patch-clamp technique, where an electrode grabs onto a small spot on the membrane and records its inner and outer potentials.

However, patch-clamp techniques are less helpful if the health of a cell organelle—mitochondria, nucleus, endoplasmatic reticulum (ER), etc.—is of interest. Any attempt to access the membrane of the desired organelle would mean breaking through the plasma membrane and disrupting the cell.

 

Non-Invasive Methods

Another way to detect membrane potentials involves the use of voltage-sensitive fluorescent dyes. These organic dyes respond to potential differences across a membrane by fluorescing either more or less brightly. They probe the charged states on both sides of a membrane, which affects the electronic distribution within the fluorophore, either quenching its fluorescence or promoting it.

Using a rational design, the team created a fluorescent dye consisting of two parts: a “silent” fluorophore, which is activated in the targeted cell compartment, and a second molecule, which directs the dye to its site.

As the fluorophore, the team chose a rhodamine unit, which gives purple fluorescence. To enable voltage sensing across the membrane, they attached a molecular “wire” to it that reaches deep into the membrane. They also attached a tetrazine unit to the rhodamine, which acted as an inherent quenching unit, keeping the fluorophore silent.

 

Bioorthogonal Targeting and Activation

Bioorthogonal chemistry then activated the silent fluorophore in the ER membranes. In this approach, the full, functional version of a molecule is not available until two functional units have reacted at the site of interest. In this case, one functional unit, a trans-cyclooctene, targeted the ER membrane and was anchored there using a ceramide tail. The other functional unit was the tetrazine-quenched rhodamine: Its tetrazine unit reacted with the cyclooctene in a click reaction, unlocking the fluorescence.

The researchers propose that other subcellular locations could also be targeted using this approach. The click reaction partner could be modified to target other organelles such as mitochondria, Golgi apparatus, etc.

 

Sensing Potentials

Once assembled, the sensor indicated potential differences between the inside of the ER (lumen) and the outside of the ER (cytosol) by dimmed or brightened fluorescence. To establish a test system, the team soaked living HeLa cells (a human cell line) in high or low potassium ion concentrations. After ion channels were activated by molecular substances, a fluorescence peak indicated a potassium ion influx into the ER lumen. The team then set out to measure real cases, with the aim of discovering if the ER membrane is electronically coupled to the plasma membrane. Clear-cut proof of such a relationship has not yet been found.

They installed a dual measurement system consisting of a patch-clamp unit and the fluorescence sensor. When the patch-clamp electrode applied a depolarization voltage to the plasma membrane, the researchers observed a parallel jump in fluorescence in the ER. This means the ER membrane followed plasma membrane depolarization—the first direct evidence of electronic coupling between both membranes.

And there may be more to see: The researchers want to dig deeper into the coupling of the plasma membrane and the ER membrane, and they believe this non-invasive optical method could be a practical means to detect ER membrane functionality in unprecedented levels of detail. It can complement the existing electrophysiological tool suite for measuring the health of cells.

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• Bioorthogonal, Fluorogenic Targeting of Voltage-Sensitive Fluorophores for Visualizing Membrane Potential Dynamics in Cellular Organelles,
  Pavel E. Z. Klier, Anneliese M. M. Gest, Julia G. Martin, Ryan Roo, Marisol X. Navarro, Lauren Lesiak, Parker E. Deal, Neville Dadina, Jonathan Tyson, Alanna Schepartz, Evan W. Miller,
  J. Am. Chem. Soc. 2022, 144, 12138–12146.
  https://doi.org/10.1021/jacs.2c02664