T-cell receptor (TCR) signaling is tightly regulated by transmembrane phosphatase CD45, which functions as a molecular rheostat to maintain basal Lck activity and prevent spurious T-cell activation. The membrane partitioning of CD45 between raft-like and non-raft domains is regarded as a key regulatory mechanism; however, obtaining direct evidence has been limited by the lack of techniques for probing such transient, nanoscale interactions. Here, by integrating lipid domain-anchored tetrahedral probes and reversible DNA hybridization, we develop a Förster resonance energy transfer (FRET) nanoscopy to monitor the relative partitioning of membrane proteins between distinct lipid domains in live cells. Our results reveal that CD45 rapidly translocates from raft-like to non-raft domains upon TCR/CD3 engagement, mainly driven by cytoskeletal reorganization and lipid remodeling. Disruption of either actin or microtubule dynamics impairs both lipid domain compartmentation and CD45 redistribution, thereby suppressing TCR signaling. This work not only elucidates a key regulatory mechanism of T-cell activation but also provides a versatile platform for investigating membrane protein-lipid interactions─a fundamental yet underexplored layer of cellular regulation.
Zhu et al. (2026) studied this question.