Scanning Fluorescence Correlation Spectroscopy for Quantification of the Dynamics and Interactions in Tube Organelles of Living Cells.

Single-molecule spectroscopic quantification of protein-protein interactions directly in the organelles of living cells is highly desirable but remains challenging. Bulk methods, such as Förster resonance energy transfer (FRET), currently only give a relative quantification of the strength of protein-protein interactions. Here, we introduce tube scanning fluorescence cross-correlation spectroscopy (tubeSFCCS) for the absolute quantification of diffusion and complex formation of fluorescently labeled molecules in the mitochondrial compartments. We determined the extent of association between the apoptosis regulators Bcl-xL and tBid at the mitochondrial outer membrane of living cells and discovered that practically all mitochondria-bound Bcl-xL and tBid are associated with each other, in contrast to undetectable association in the cytosol. Furthermore, we show further applicability of our method to other mitochondrial proteins, as well as to proteins in the endoplasmic reticulum (ER) membrane.