Thinning Out Clusters while Conserving the Stoichiometry of Labeling (TOCCSL)

During their random motion, biomolecules experience a manifold of interactions that transiently conjoin their paths. It is extremely difficult to measure such binding events directly in the context of a living cell: interactions may be short lived, they may affect only a minority fraction of molecules, or they may not lead to a macroscopically observable effect. We developed a single molecule imaging method that allows for detecting and quantifying associations of mobile molecules. By “thinning out clusters while conserving the stoichiometry of labeling” (TOCCSL) we can virtually dilute the probe directly in the cell, without affecting the fluorescence labeling of single clusters.

Essentially, an analysis region is created within the cell by photobleaching; this region is devoid of active probe. Brownian motion or other transport processes lead to reentry of active probe molecules into the analysis region. At the onset of the recovery process, single spots can be resolved as well separated, diffraction-limited signals. Standard single molecule microscopy then allows for characterizing the spots in terms of their composition and mobility. 

[Translate to English:] Wissenschaftliche Abbildung Skizze der Wirkung auf fluoreszenzmarkierte künstliche Cluster

The idea behind TOCCSL

The top row presents original data, the bottom row a sketch of the effect on fluorescently labeled artificial clusters. (i) represents the situation where the surface density is too high to resolve single fluorescence-labeled antibody molecules. The analysis region was defined by imaging a round diaphragm onto the object plane. Immediately after bleaching the sample is totally devoid of any fluorescence signal (ii). At the onset of the recovery process, due to Brownian motion single fluorescently labelled biomolecules can be observed as well-separated spots within the analysis region (iii). After a longer recovery time the system reaches the equilibrated state again, similar to the pre-bleach image (iv).

The image of individual diffraction limited signals is described by the point spread function (PSF) of the optical system. The PSF can be well approximated by a two-dimensional Gaussian profile yielding the position (x,y), the brightness B, the local background and the full width at half maximum (FWHM) of single diffraction limited spots. For stoichiometry analysis we make use of the distribution of brightness values ρ(B). It should be a linear combination of the brightness distributions of the various N-mers: ρ(B)=∑N αN ρN(B), with αN specifying the corresponding statistical weights. The theoretical intensity distribution of N co-localized independent emitters, ρN(B), can be calculated from the measured brightness distribution of monomers, ρ1(B), recursively as a series of convolution integrals ρN(B) = ∫ρ1(B) ρN-1(B-B')dB'.

[Translate to English:] Wissenschaftliche Abbildung

© TU Wien

Stoichiometry analysis of a typical TOCCSL experiment

Stoichiometry analysis of a typical TOCCSL experiment, in which we addressed the subunit stoichiometry of the Orai1 ion channel. The left panel shows the brightness distributions of detected signals in the TOCCSL image (grey) and after additional moderate photobleaching to approach the single molecule brightness (black). Original data are plotted as dotted lines, fit results as full lines. We show here the fit results assuming a linear combination of four Gaussian distributions. The weights of the individual Gaussians obtained in the TOCCSL image are displayed in the right panel (black bars), yielding mainly associates of four colocalized protomers. The observed 40% dimers and trimers most likely represent non-fluorescent mGFP; from the Binomial fit, an mGFP maturation degree of 88% was estimated (grey bars)

Our key publications

Monomeric TCRs drive T cell antigen recognition

Brameshuber, M., F. Kellner, B. K. Rossboth, H. Ta, K. Alge, E. Sevcsik, J. Göhring, M. Axmann, F. Baumgart, N. R. J. Gascoigne, S. J. Davis, H. Stockinger, G. J. Schütz, and J. B. Huppa. 2018. Monomeric TCRs drive T cell antigen recognition. Nature Immunology 19(5):487–496, opens an external URL in a new window

We used TOCCSL to revisit the hypothesized oligomeric state of the T cell receptor (TCR). We observed no indication for the presence of TCR dimers or higher structures.

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

Anderluh, A., T. Hofmaier, E. Klotzsch, O. Kudlacek, T. Stockner, H. H. Sitte, and G. J. Schütz. 2017. Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter. Nature Communications 8:14089, opens an external URL in a new window.

We found SERT oligomerization to be dependent on PIP2 levels.

Single molecule analysis reveals coexistence of stable serotonin transporter monomers and oligomers in the live cell plasma membrane

Anderluh, A., E. Klotzsch, A. W. Reismann, M. Brameshuber, O. Kudlacek, A. H. Newman, H. H. Sitte, and G. J. Schütz. 2014. Single molecule analysis reveals coexistence of stable serotonin transporter monomers and oligomers in the live cell plasma membrane. J Biol Chem 289(7):4387-4394, opens an external URL in a new window.

We quantified the stoichiometry of the serotonin transporter (SERT), yielding a broad distribution up to pentamers.

Detection and quantification of biomolecular association in living cells using single-molecule microscopy

Brameshuber, M. and G. J. Schütz. 2012. Detection and quantification of biomolecular association in living cells using single-molecule microscopy. Methods in Enzymology 505: 159-186, opens an external URL in a new window.

We provide a detailed description of the TOCCSL approach, several examples using TOCCSL to address the stoichiometry of membrane proteins and a little “User guide to TOCCSL”.

Two-color single molecule tracking combined with photobleaching for the detection of rare molecular interactions in fluid biomembranes

Ruprecht, V., M. Brameshuber, and G. J. Schütz. 2010. Two-color single molecule tracking combined with photobleaching for the detection of rare molecular interactions in fluid biomembranes. Soft Matter 6(3):568-581, opens an external URL in a new window.

We introduced two-color TOCCSL as an improved tool for single molecule co-localization analysis.

Imaging of Mobile Long-lived Nanoplatforms in the Live Cell Plasma Membrane

Brameshuber, M., J. Weghuber, V. Ruprecht, I. Gombos, I. Horvath, L. Vigh, P. Eckerstorfer, E. Kiss, H. Stockinger, and G. J. Schütz. 2010. Imaging of Mobile Long-lived Nanoplatforms in the Live Cell Plasma Membrane. J Biol Chem 285(53):41765-41771, opens an external URL in a new window.

In this paper we addressed the question, whether the putative marker of lipid rafts, mGFP-GPI, shows cholesterol-dependent association. We found a small but significant fraction of dimers.

Resting state orai1 diffuses as homotetramer in the plasma membrane of live Mammalian cells

Madl, J., J. Weghuber, R. Fritsch, I. Derler, M. Fahrner, I. Frischauf, B. Lackner, C. Romanin, and G. J. Schutz. 2010. Resting state orai1 diffuses as homotetramer in the plasma membrane of live Mammalian cells. J Biol Chem 285(52):41135-41142, opens an external URL in a new window.

We used TOCCSL to study the subunit stochiometry of the ion channel Orai1, yielding essentially tetramers.

Thinning out clusters while conserving stoichiometry of labeling

Moertelmaier, M., M. Brameshuber, M. Linimeier, G. J. Schütz, and H. Stockinger. 2005. Thinning out clusters while conserving stoichiometry of labeling. Appl Phys Lett 87:263903, opens an external URL in a new window.

Proof of principle paper, in which we proposed the TOCCSL method for the first time.

Project Members

Thinning Out Clusters while Conserving the Stoichiometry of Labeling (TOCCSL)

Senior Scientist Dipl.-Ing. Dr.techn.

Mario Brameshuber

Phone: +43 1 58801 134896 Call Mario Brameshuber

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