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

We present a method to identify and characterize interactions between a fluorophore-labeled protein ('prey') and a membrane protein ('bait') in live mammalian cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait extracellular domain. Bait-prey interactions are assayed through the redistribution of the fluorescent prey. We used the method to characterize the interaction between human CD4, the major co-receptor in T-cell activation, and human Lck, the protein tyrosine kinase essential for early T-cell signaling. We measured equilibrium associations by quantifying Lck redistribution to CD4 micropatterns and studied interaction dynamics by photobleaching experiments and single-molecule imaging. In addition to the known zinc clasp structure, the Lck membrane anchor in particular had a major impact on the Lck-CD4 interaction, mediating direct binding and further stabilizing the interaction of other Lck domains. In total, membrane anchorage increased the interaction lifetime by two orders of magnitude.

Identification of immobile single molecules using polarization-modulated asynchronous time delay and integration-mode scanning.

We report the development of a data acquisition method for identifying single molecules on large surfaces with simultaneous characterization of their absorption dipole. The method is based on a previously described device for microarray readout at single molecule sensitivity (Hesse, J.; Sonnleitner, M.; Sonnleitner, A.; Freudenthaler, G.; Jacak, J.; Höglinger, O.; Schindler, H.; Schütz, G. J. Anal. Chem. 2004, 76, 5960-5964). Here, we introduced asynchronous time delay and integration- (TDI-) mode imaging to record also the time course of fluorescence signals: the images thus contain both spatial and temporal information. We demonstrate the principle by modulating the signals via rotating excitation polarization, which allows for discriminating static absorption dipoles against multiple or freely rotating single absorption dipoles. Experiments on BSA carrying different numbers of fluorophores demonstrate the feasibility of the method. Protein species with an average labeling degree of 0.55 and 2.89 fluorophores per protein can be readily distinguished.

Implementation of alternating excitation schemes in a biochip-reader for quasi-simultaneous multi-color single-molecule detection.

We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects, e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer, the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera readout yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to approximately 10 nm was shown by scanning immobilized 100 nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two-color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody-receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application.

in-vivo detection of protein-protein interactions on micro-patterned surfaces.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

Detection of protein-protein interactions in the live cell plasma membrane by quantifying prey redistribution upon bait micropatterning.

Our understanding of complex biological systems is based on high-quality proteomics tools for the parallelized detection and quantification of protein interactions. Current screening platforms, however, rely on measuring protein interactions in rather artificial systems, rendering the results difficult to confer on the in vivo situation. We describe here a detailed protocol for the design and the construction of a system to detect and quantify interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in living cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput, making it applicable as a screening tool. The proof-of-concept is demonstrated for the interaction between CD4, a major coreceptor in T-cell signaling, and Lck, a protein tyrosine kinase essential for early T-cell signaling.

Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation.

STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca(2+) entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca(2+) store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca(2+) inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.