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.

Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species.

Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in <1 min.

Toxicity of an α-pore-forming toxin depends on the assembly mechanism on the target membrane as revealed by single molecule imaging.

α-Pore-forming toxins (α-PFTs) are ubiquitous defense tools that kill cells by opening pores in the target cell membrane. Despite their relevance in host/pathogen interactions, very little is known about the pore stoichiometry and assembly pathway leading to membrane permeabilization. Equinatoxin II (EqtII) is a model α-PFT from sea anemone that oligomerizes and forms pores in sphingomyelin-containing membranes.

Scanning fluorescence correlation spectroscopy on biomembranes.

Fluorescence correlation spectroscopy (FCS) is a powerful quantitative method to study dynamical properties of biophysical systems. It exploits the temporal autocorrelation of fluorescence intensity fluctuations originating from a tiny volume (~fL). A theoretical model function can be then fitted to the measured auto-correlation curve to obtain physical parameters such as local concentration and diffusion time.

Automated analysis of giant unilamellar vesicles using circular Hough transformation.

Motivation: In order to obtain statistically relevant results, the study of membrane effects at the single-vesicle level requires the analysis of several hundreds of giant unilamellar vesicles (GUVs), which becomes a very time-consuming task if carried out manually. Complete and user-friendly software for fast and bias-free automated analysis has not been reported yet.

Results: We developed a framework for the automated detection, tracking and analysis of individual GUVs on digital microscopy images.