Microscopy, Fluorescence; Signal Transduction
Adaptive character of responses to signals from the environment is a fundamental property of all living organisms. At the cellular level, it is brought about by a highly integrated process of transmembrane and intracellular signal transduction. Although many signaling molecules have been identified, how exactly the dynamics of their interactions ensure the ultimate specificity and adaptive character of cellular responses remains poorly understood. How changes in flux of substrates via an enzyme affects signalling specificity? How intracellular localization of a protein is coupled to its signaling role? What is the function of a given protein in network regulation?
To address these questions, we employ a combination of biochemical and advanced imaging techniques. We are developing tools to visualize the functional state of signaling molecules in live cells to determine how spatial distribution, regulation of specific activity and the corresponding changes in the flux of substrates via individual enzymes determine their signaling function. Furthermore, to examine the physiological relevance of individual enzymes in signaling, we will be developing new tools to selectively manipulate their localization and activity in live cells.
Techniques, methods & infrastructure
Imaging techniques: laser scanning confocal (LSCM), spinning disk, structured illumination microscopy (SIM), Förster resonant energy transfer (FRET), fluorescence lifeime imaging microscopy (FLIM), fluorescence correlation spectroscopy (FCS, FCCS), immunofluorescence/immunocytochemistry (IF/ICC).
Biochemical techniques: chemical- and light-induced dimerization, reversible dimerization, mass spectrometry, protein expression and purification, size exclusion chromatography (SEC), affinity chromatography.
Computation techniques: unsupervised image analysis (MatLab), stochastic modeling using cellular automata (MatLab).
Model systems: primary and cultured mammalian cells (including primary and cultured human T cells).
- Yudushkin, I.A. & Vale, R.D., 2010. Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3'
- Yudushkin, I.A. et al., 2007. Live-Cell Imaging of Enzyme-Substrate Interaction Reveals Spatial Regulation of PTP1B. Science, 315(5808), pp.115-119. Available at: http://dx.doi.org/10.1126/science.1134966.
- Ebner, M. et al., 2017. PI(3,4,5)P3 Engagement Restricts Akt Activity to Cellular Membranes. Molecular Cell, 65(3), pp.416-431.e6. Available at: http://dx.doi.org/10.1016/j.molcel.2016.12.028.
- Ebner, M. et al., 2017. Localization of mTORC2 activity inside cells. The Journal of Cell Biology, 216(2), pp.343-353. Available at: http://dx.doi.org/10.1083/jcb.201610060.