Calcium: The Missing Link in Dead Cell Clean-up by Bridging Membrane Domains

Activation of scramblase causes changes in membrane tension. When considering how to demonstrate calcium binding to Xkr4, we got one idea by measuring the membrane tension in cells. Membrane tension is measured by applying physical force to pull beads attached to the cell membrane surface. However, since I used Baf3 cells, which are suspension cells, a challenge arose as they could not be attached to the dish surface tightly, leading to cells being easy to detach during prolonged measurement. It took over six months just to optimize the conditions for analysis before eventually getting the results. During this experiment I learned that success often requires great patience and perseverance in overcoming challenges.

The most challenging point of this study was understanding how calcium binding activates Xkr4. To measure the interaction of Xkr4 with calcium, I tried to purify Xkr4, but Xkr4 is unstable and difficult to purify! Eventually, I found one very cool approach. Our studies found that calcium was expected to bind to the negatively charged residues on transmembrane 1 and 3, functioning as a bridge to connect the two transmembrane domains. To emulate this calcium-mediated connection, I made mutations of these calcium-interacting residues. For instance, I utilized Cys mutations to introduce a disulfide bond, or positively charged Lys/Arg mutations to introduce a salt bridge with the calcium-interacting residue (the negatively charged residues: Asp/Glu). Using the artificial crosslinker, we successfully activated Xkr4 even in the absence of calcium. This method allows us to show that the calcium bindings to Xkr4 function as a molecular glue, facilitating the connection of the transmembrane domain in activated Xkr4.

Since joining the Suzuki Lab in 2018 for my PhD, I have been focused on studying scramblase to understand the connection between cell death and human health. This is a very interesting and important field, so I will still work in this field for my future research. Working in Suzuki Lab has provided me with invaluable experience in cell biology which will significantly help my future studies. Protein structure analysis has captivated me during my time as a master's student. Through the development of Cryo-electron microscopy, we can observe how proteins undergo changes and perform functions crucial for sustaining life. Next, I intend to explore membrane protein structure based on the knowledge which I learned here.

I have moved to Faculty of Science, Hokkaido University as a Postdoc since Apr 2024. iCeMS is an outstanding international research platform that gathers researchers from around the globe, providing us with a great research environment and abundant resources. In addition, the on-site labs offer us more opportunities to collaborate with scholars from different countries. I'm grateful for the chance to visit the Center for Integrated Biosystems, iCeMS on-site lab in Taiwan. During my five years at iCeMS, a crucial phase in my research career as a doctoral candidate, I received comprehensive training and learned to approach my research with an international perspective. I believe that the experiences I gained here have equipped me with the necessary skills and confidence for my research career.

Take things step by step and think by yourself, but do not hesitate to communicate with others, who you might get a good idea from.