Over the last years we have developed a method, which allows to arrange specific membrane proteins according to micro- or nanopatterns of adjustable size directly in the live cell plasma membrane. Thereby we can generate areas enriched or depleted in the protein of interest within the very same cell membrane. Currently, we apply this method to i) measure biomolecular interactions in the live cell plasma membrane, and to ii) quantify the hydrodynamic size of membrane proteins.
To generate the micropatterns, we first fabricate streptavidin patterns of adjustable geometry on glass slides via microcontact printing. To foster cell adhesion and to passivate the non-coated parts of the surface, the interspaces are filled with fibronectin. Finally, biotinylated antibody specific to the expressed GFP-tagged bait is bound to streptavidin. By this approach, antibody patterns with sizes of typically 3µm down to 80nm can be generated. Cells expressing the GFP-tagged bait will be grown on such surfaces, leading to specific immobilization of the bait.

Steps of micropattering printing and cell attachment

© TU Wien

Micropatterning

Micropatterning of membrane proteins in the live cell plasma membrane. PDMS stamps are inked with streptavidin solution (i), washed (ii), and transferred onto glass slides (iii). After backfilling with fibronectin (iv), slides are incubated with biotinylated capture antibody against the protein of interest (GFP-tagged bait).

Measuring biomolecular interactions in the live cell plasma membrane

Characterization, especially quantification, of protein interactions in live cells is usually not an easy endeavor. We have developed a straightforward method to identify and quantify the interaction of a membrane protein (“bait”) and a fluorescently labeled interaction partner (“prey”) (membrane-bound or cytosolic) in live cells using Total Internal Reflection Fluorescence (TIRF) microscopy. The bait protein is immobilized within patterns in the plasma membrane (e.g., via an antibody); the bait–prey interaction strength can be quantified by determining the prey bulk fluorescence intensity with respect to the bait patterns. This method is particularly suitable for the analysis of weak, transient interactions that are not easily accessible with other methods.

Scientific illustration of the principle of protein microstructuring in the plasma membrane

Principle of protein microstructuring in the plasma membrane

(A) Sketch and (B) TIRF image of a cell grown on a micro-patterned substrate. Bait antibody is arranged in a regular pattern of 3 μm sized dots with 3 μm interspaces. The bait protein (unlabeled) reorganizes according to the antibody patterns, but the fluorescently labeled prey protein is distributed homogeneously in the plasma membrane, indicating no interaction between bait and prey protein. Scale bar is 7 μm. (C, D) As in (A, B), but here the prey protein interacts strongly with the bait protein and localizes according to the bait patterns. The cell outline is indicated by a dashed white contour line.

Quantifying the hydrodynamic size of membrane proteins

We are also using the micropatterning approach for studying the size of plasma membrane proteins: one interaction partner (obstacle) is immobilized, and the influence on the diffusional motion of the other partner (tracer) is assessed. Using microcontact printing we can routinely generate densities of immobilized obstacles up to 10,000 molecules per µm², which corresponds to average nearest neighbor distances of 10nm. Using GPI-anchored proteins both as obstacle and tracer, we observed up to 50% reduction of tracer-mobility merely due to the presence of the immobilized obstacles. Much higher reductions in diffusional motion down to complete tracer arrest could be achievable, if larger protein complexes were used as tracers. Tracer and obstacle sizes can be quantified from the dependence of the mobility ratio DON⁄DOFF  on obstacle density.

Scientific illustration shows protein microstructuring and analysis

Protein microstructuring and analysis

(A) shows a fluorescence image of the obstacle distribution – here mGFP-GPI – in a living T24 cell. (B) For analysis, diffusion constants of the tracer are determined by single molecule tracking, both in the obstacle-enriched (“ON”) and -depleted (“OFF”) regions of the plasma membrane.

Our key publications

A Fast and Simple Contact Printing Approach to Generate 2D Protein Nanopatterns

Lindner, M., A. Tresztenyak, G. Fülöp, W. Jahr, A. Prinz, I. Prinz, J. G. Danzl, G. J. Schütz, and E. Sevcsik. 2019. A Fast and Simple Contact Printing Approach to Generate 2D Protein Nanopatterns. Front. Chem. 6(655), opens an external URL in a new window.

Using a special composite stamp, we could reduce the achievable pattern size down to 80nm. 

A micropatterning platform for quantifying interaction kinetics between the T cell receptor and an intracellular binding protein

Motsch, V., M. Brameshuber, F. Baumgart, G. J. Schütz, and E. Sevcsik. 2019. A micropatterning platform for quantifying interaction kinetics between the T cell receptor and an intracellular binding protein. Scientific Reports 9(1):3288, opens an external URL in a new window.

We used the micropatterning approach to measure the kinetics of ZAP-70 exchange on the TCR complex.

Protein Micropatterning Assay: Quantitative Analysis of Protein–Protein Interactions

Schütz G.J., Weghuber J., Lanzerstorfer P., Sevcsik E. 2017. Protein Micropatterning Assay: Quantitative Analysis of Protein–Protein Interactions. Methods in Molecular Biology 1550: 261-270

Here, we give guidelines on how to conduct a protein micropatterning experiment and describe potential pitfalls and propose solutions.

Monte Carlo simulations of protein micropatterning in biomembranes: effects of immobile sticky obstacles

Arnold, A. M., E. Sevcsik, and G. J. Schütz. 2016. Monte Carlo simulations of protein micropatterning in biomembranes: effects of immobile sticky obstacles. Journal of Physics D: Applied Physics 49(36):364002, opens an external URL in a new window.

In this theoretical paper, we quantitatively characterized the influence of immobile sticky obstacles on the mobility ratio DON/DOFF , as it would be measured in a micropatterning experiment. We use this paper as basis of our analysis of micropatterning experiments.

GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

Sevcsik, E., M. Brameshuber, M. Fölser, J. Weghuber, A. Honigmann, and G. J. Schütz. 2015. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane. Nat Commun 6:6969, opens an external URL in a new window.

Here, we arranged a putative lipid raft marker in micropatterns, and quantified the influence on diffusional properties of other membrane constituents. Contrary to previous believes, we did not observe the presence of a liquid ordered phase

Detection of Protein-protein Interactions on Micro-patterned Surface

Julian Weghuber, Mario Brameshuber, Stefan Sunzenauer,Manuela Lehner, Christian Paar, Thomas Haselgrübler, Michaela Schwarzenbacher, Martin Kaltenbrunner, Clemens Hesch, Wolfgang Paster, Bettina Heise, Alois Sonnleitner, Hannes Stockinger, and Gerhard J. Schütz. 2010. Detection of Protein–Protein Interactions in the Live Cell Plasma Membrane by Quantifying Prey Redistribution upon Bait Micropatterning. Methods in Enzymology 472: 133-151, opens an external URL in a new window.
and
Julian Weghuber, Stefan Sunzenauer, Mario Brameshuber, Birgit Plochberger, Clemens Hesch, Gerhard J. Schütz. 2010. In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces. Journal of Visualized Experiments 37.

We describe here a detailed protocol for the design and the construction of the micropatterning system to detect and quantify interactions between a fluorophore-labeled protein (‘‘prey’’) and a membrane protein (‘‘bait’’) in living cells.

Micropatterning for quantitative analysis of protein-protein interactions in living cells

Schwarzenbacher, M., M. Kaltenbrunner, M. Brameshuber, C. Hesch, W. Paster, J. Weghuber, B. Heise, A. Sonnleitner, H. Stockinger, and G. J. Schütz. 2008. Micropatterning for quantitative analysis of protein-protein interactions in living cells. Nat Methods 5(12):1053-1060, opens an external URL in a new window.

Our first micropatterning paper. We introduced the approach by studying the interaction between CD4 (coreceptor in T cell signaling) and Lck (key kinase in early T cell signaling).

Further Reading

Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering

Cai, H., J. Muller, D. Depoil, V. Mayya, M. P. Sheetz, M. L. Dustin, and S. J. Wind. 2018. Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering. Nature Nanotechnology 13:610–617, opens an external URL in a new window.

The TCR was arranged in different nanopatterns, using a nanofabricated single-molecule array platform. Clear differences in T cell signaling were observed, which were depending on the lateral spacing between the ligands.

Micropatterning of costimulatory ligands enhances CD4+ T cell function

Shen, K., V. K. Thomas, M. L. Dustin, and L. C. Kam. 2008. Micropatterning of costimulatory ligands enhances CD4+ T cell function. Proc Natl Acad Sci U S A 105(22):7791-7796, opens an external URL in a new window.

In this paper, two proteins relevant for T cell signaling were simultaneously micropatterned in different arrangements, yielding first indications for spatial requirements in the presentation of stimulus and costimulus.