First person – Nevena Stajković Free

Nevena Stajković

How would you explain the main findings of your paper in lay terms?

Voltage-gated sodium channels (Na_V) are proteins that are abundant in a specific part of the neuron called the axon initial segment (AIS). In the AIS, the Na_V regulates the generation of electrical signals that are indispensable for communication between neurons. Changes in the structure or amount of Na_V and other AIS components have been linked to various neurological diseases. To design new treatments for such diseases, we need to better understand the organization of the AIS proteins and their behavior in living neurons. Such questions can be studied by fluorescence microscopy; however, for microscopy studies, we first need to fluorescently label AIS components. Conventional labeling approaches can affect protein function and introduce artefacts, so alternative labeling methods are needed. One such labeling approach (called click labeling) is based on modifying individual building blocks of proteins with the help of specific chemical reactions and small fluorescent dyes. In my work, I established and used this method to visualize AIS components, including disease-causing variants of Na_V in living neurons.

Were there any specific challenges associated with this project? If so, how did you overcome them?

Establishing click labeling of the voltage-gated sodium channel 1.6 isoform (Na_V1.6) in neurons was challenging on multiple levels. Owing to the low expression level of Na_V1.6 in neuronal cell lines, it was necessary to test different labeling positions and optimize click labeling directly in primary neurons. However, click labeling relies on a combination of advanced technologies (genetic code expansion and bioorthogonal click chemistry) that have only recently been established for the labeling of small proteins (∼60 kDa) in neurons. Establishing this labeling approach for the large (∼260 kDa) Na_V1.6, which has a more complex structural organization and compartment-specific expression, was challenging. Moreover, the complex structure of Na_V1.6 made it difficult to find a position for unnatural amino acid incorporation that would not affect the biophysical properties of Na_V1.6 and simultaneously allow highly efficient click labeling. For these reasons, establishing click labeling of the Na_V1.6 in neurons took longer than expected, and we were considering discontinuing the project. However, we overcame some of the challenges by focusing on NF186, an easier-to-express AIS component with a smaller and less complex structure.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

At the beginning of my PhD project, I had difficulties successfully expressing recombinant Na_V1.6 with incorporated unnatural amino acids for click labeling. I was trying different transfection protocols, expression conditions and cell lines for months. I remember going late in the afternoon to the microscope to check cells, not expecting positive results. Surprisingly, with one neuronal cell line, I observed bright Na_V1.6 expression. This experience was very important for my PhD journey as it taught me that sometimes it is necessary to stay motivated and persistent regardless of a lack of positive results and that hard work will eventually be rewarded.

Why did you choose Journal of Cell Science for your paper?

Journal of Cell Science covers a wide spectrum of topics in the field of cell biology and has a broad audience. Since we developed a new tool for labeling neuronal proteins that can be applied to other neuronal and non-neuronal targets, it was the perfect choice to publish our research in JCS.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

From a young age, I have been interested in biology and curious about nature. I used to read science books for kids about molecules and cells. Afterward, I would go to the garden and conduct my own experiments by looking at the plants and objects with a magnifying lens. Later in school, I learned more about DNA, proteins and cell biology. This prompted me to study molecular biology and physiology. During my Bachelor's and Master's studies, I became familiar with neurobiology and various microscopy methods, and learned how the latter can be used to study different diseases. I wanted to specialize in the cellular and molecular biology of neurons and study them in more detail with advanced microscopy methods, such as super-resolution microscopy. This led me to Tübingen and my current PhD lab, where I established a method for visualization of neuronal ion channels with live-cell and super-resolution microscopy.

Tell us something interesting about yourself that wouldn't be on your CV

In the realm of science and research, I am passionate about sodium channels, fluorescent dyes and microscopy. Outside of the lab, I am equally passionate about bouldering and climbing. What I find particularly exciting about these outdoor activities is their similarity to scientific projects. Deciphering a challenging bouldering or climbing route requires problem-solving skills and mental strength, much like in science. Furthermore, the thrill of completing a difficult route is as exciting as the results obtained from complex experiments.