Newly developed tool makes cell membrane lipids easier to visualize

A research group consisting of Professor Taki Nishimura of the Institute for Protein Research at the University of Osaka, Lecturer Kotaro Tsuboyama of the Institute of Industrial Science at the University of Tokyo, Project Researcher Yuki Ishino and Associate Professor Nozomu Kono of the Graduate School of Pharmaceutical Sciences at the University of Tokyo, Assistant Professor Shiou-Ling Lu and Professor Takeshi Noda of the Graduate School of Dentistry at the University of Osaka, Yuki Nakagaki of the Graduate School of Science and Technology at Keio University (at the time of research), Associate Professor Eiji Yamamoto of the Graduate School of Science and Technology at Keio University, and Professor Noboru Mizushima of the Graduate School of Medicine at the University of Tokyo has developed a new experimental system called the CLiB (Cell surface Liposome Binding) assay to examine interactions between proteins and lipids. The CLiB assay not only allows the analysis of protein-lipid binding with few experimental steps but also enables high-throughput analysis targeting thousands of types of proteins when combined with next-generation sequencing analysis. The study was published online in Nature Cell Biology.

Lipid molecules constituting biological membranes play crucial roles in determining cellular functions and states. However, over a thousand types of lipid species exist within a cell. It is not easy to capture where and how specific lipid species are distributed inside cells.

To track the intracellular behavior of lipid molecules, proteins that selectively bind to specific lipid species (lipid probes) are generally utilized. However, many lipid probes currently in use merely repurpose naturally occurring lipid-binding domains. No general method existed to generate lipid probes with required properties according to specific objectives.

The research group first succeeded in constructing an experimental system that reproduces protein-lipid binding on the surface of yeast cells using the yeast display method. With the developed CLiB assay, selective binding between proteins and lipids can be rapidly investigated in approximately two hours. It also has excellent compatibility with next-generation sequencing analysis, allowing individual lipid-binding data of clones to be obtained even from samples containing a mixture of multiple clones. Utilizing this mechanism, they also succeeded in acquiring binding data between target lipids and more than 6,000 different proteins at once.

Next, using the CLiB assay, they worked on the artificial modification of lipid-binding domains. While PX-SnxA was known to bind to the phosphatidylinositol bisphosphate PI(3,5)P₂, it had the problem of weak binding affinity. Therefore, by combining directed evolution methods with efficient screening via the CLiB assay, they isolated a clone with enhanced binding affinity, PX-SnxA^GV, and clarified its binding mode through molecular dynamics (MD) simulations. When PX-SnxA^GV was expressed in cells as a GFP fusion protein, it was observed to localize to membrane structures rich in PI(3,5)P₂. Furthermore, they clarified that under specific stress conditions, membrane structures enriched with PI(3,5)P₂ appeared on the vacuolar membrane of yeast cells and on the lysosome-like organelle membrane of macrophage-like cultured cells.

This development opens the path to designing and developing lipid probes tailored to research objectives and is expected to significantly advance the understanding of the diversity of intracellular membrane lipid environments.

Nishimura commented: "By using the CLiB assay, it has become possible to efficiently develop lipid probes targeting various membrane lipids. Beyond its utility in basic research, such as visualizing diverse membrane lipid environments and predicting lipid-binding affinity, it holds promising potential for applications in AI-driven drug discovery, including the development of VHH antibodies that target specific lipids."

Journal Information
Publication: Nature Cell Biology
Title: Cell surface liposome binding (CLiB) allows lipid-binding probe engineering via high-throughput screening
DOI: 10.1038/s41556-026-01996-8