Benda, A. | Search bioRxiv

bioRxiv2020

Clustering of CD3ζ is sufficient to initiate T cell receptor signaling

T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of CD3{zeta} chains in a light controlled manner. We showed that spatial clustering of the CD3{zeta} intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of CD3{zeta}, Zap70, PLC{gamma}, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate CD3{zeta} phosphorylation upon CD3{zeta} clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed CD3{zeta} clusters at the plasma membrane. Taken together, our data suggest that clustering of the TCR can initialize proximal TCR signaling and thus constitute a biophysical mechanism of TCR triggering.

immunology↗

bioRxiv2020

Single-color Fluorescence Lifetime Cross-Correlation Spectroscopy in vivo

The ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence based methods like two-color fluorescence cross-correlation spectroscopy can provide this information but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross-talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photo-stability, bleaching and fluorescence brightness can have an impact. Here we take advantage of lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross-talk. To establish sc-FLCCS we first engineered and tested multiple GFP-like fluorescent proteins for their suitability. This identified a novel GFP variant termed slmGFP (short lifetime monomeric GFP) with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and slmGFP were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color(dc)-FCCS measurements. STATEMENT OF SIGNIFICANCEThe quantification of protein concentrations and protein-protein interactions in vivo is a crucial information needed for the understanding of complex processes inside cells. Determination of such information is unfortunately still challenging. Fluorescence-based method like fluorescence cross-correlation spectroscopy (FCCS) is the only method which provides this information in vivo and almost in the real time, however it suffers from limitations caused by experimental setup and biological origin of fluorescent proteins. We present single-color fluorescence lifetime cross-correlation spectroscopy as an alternative to FCCS, which uses the information of fluorescence lifetime to overcome some of these limitations. We challenged the method and determined its advantages and limitations and demonstrated the applicability of the method on the proteins of yeast proteasome.

cell biology↗