BPS2026 – Deciphering membrane dynamics and cellular communication through pan-membrane protein labeling

Membrane protein assemblies and their dynamic distribution are essential for regulating cellular communication. However, cell surface protein labeling methods that provide uniform labeling of membrane proteins, are compatible with live-cell imaging, universally applicable across diverse cell types, and easily adaptable for multiplexing are limited. We developed a simple and rapid approach to label live mammalian cell surface proteins using N-hydroxysuccinimide (NHS)-ester-based amine crosslinking of fluorescent dyes. Using mouse DC2.4 dendritic cells, human Jurkat T cells, and mouse primary lymphoma T cells as model systems, we revealed the membrane topology and membrane-associated cell communications previously challenging to capture by fluorescence microscopy techniques. In DC2.4 cells, we observed transient membrane protein accumulation at cell-cell contacts and their bidirectional migration patterns guided by membrane fibers. We further visualized the involvement of membrane fibers in the transfer of membrane-derived particles and ovalbumin, as well as caveolin-dependent endocytosis and subsequent transport of membrane labels in late endosomes and lysosomes. In addition, we demonstrate caveolin-1 phosphorylation-dependent insulin receptor endocytosis in HEK293T cells. Volumetric structured illumination microscopy revealed changes in the membrane topology of Jurkat T cells in response to the activating surface, generation of membrane-derived microvesicles, and phagocytosis of antigen mimicking CD3/CD28 Dynabeads on the glass surface. Finally, we demonstrate that the membrane labeling remains stable in the in vivo environment and evidence of intercellular membrane protein transfer from the donor T cell lymphoma SNF5 mouse splenocytes to the healthy splenocytes of C57/BL6 mouse 24 and 48 hours after cell transfer. The ultrahigh membrane labeling density, coupled with high multiplexity and rapid labeling of pan-membrane-protein labeling enables innovative fluorescence microscopy applications to dissect membrane and membrane-derived structures in intercellular and intracellular communication both in vitro and in vivo.