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

Abstract

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 Fö rster resonance energy transfer (FRET), currently only give a relative quantification of the strength of protein-protein interactions. Here, we introduce tube scanning fluorescence crosscorrelation 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 mitochondriabound 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.[a] J.

Mitochondrial outer membrane permeabilization at the single molecule level

“…With this in mind, big efforts have been put in the implementation of FCS methods to membranes of living cells [ 80 ]. Recently, tube scanning fluorescence cross-correlation spectroscopy was introduced for the absolute quantification of diffusion and binding of fluorescently labeled proteins in the mitochondrial compartments [ 100 ]. Based on the tBid/Bcl-xL complex as a reference system, results in cells compared to in vitro studies confirming that all mitochondria-bound tBid and Bcl-xL were associated with each other, while their interaction in the cytosol was undetectable.…”

Single molecule imaging of protein aggregation in Dementia: Methods, insights and prospects

“…FCS has extensively been utilized to quantity molecular interactions in vitro and in vivo ( Schwille et al, 1999 ; Banks and Fradin, 2005 ; Eggeling et al, 2009 ; Petrášek and Schwille, 2008 ) as well as used for studying the growth kinetics of amyloid aggregates (see Section 3 ). The distinct advantage of FCS over SMLM is its ability to quantify molecular dynamics in real-time and in solution or inside the tubular and lamellar organelles of the cell ( J R et al, 2009 ; Unsay et al, 2018 ) and using a wide array of labelling and immunostaining techniques. Combined with its cost-effectiveness and ease of assembly ( Ambrose et al, 2020 ), confocal FCS is a viable solution for answering some questions related to the kinetics of aggregation.…”