Spatial triple-correlation spectroscopy reveals heterotrimer dynamics in live cells

Heterotrimeric protein complexes are central regulators of intracellular signaling, yet their dynamic assembly and transport in living cells remain difficult to resolve. Here, we present spatial triple-correlation spectroscopy (S3CS), a fluorescence fluctuation method that integrates three-channel line scan microscopy with a spatial triple-correlation function to directly detect fluorescent heterotrimers and map their movement relative to subcellular architecture. Simulations establish that S3CS quantitatively captures heterotrimer formation, local diffusion, and long-range transport, while live-cell experiments confirm its specificity for following fluorescent ternary assemblies in the presence of free independent subunits. Applying S3CS to the importin-α/importin-β/NLS cargo complex revealed directional, irreversible nuclear import, whereas analysis of the NF-Y transcription factor showed that heterotrimer assembly precedes chromatin engagement. By selectively resolving the dynamics of fluorescent ternary assemblies at intracellular boundaries, S3CS provides a versatile platform to dissect how heterotrimeric signaling complexes employ molecular interactions to navigate the dynamic structural framework of the living cell.