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.
Arachchige et al. (Sun,) studied this question.