IL-15 Trans-Presentation Is an Autonomous, Antigen-Independent Process.

IL-15 plays a pivotal role in the long-term survival of T cells and immunological memory. Its receptor consists of three subunits (IL-15Rα, IL-2/15Rß, and γc). IL-15 functions mainly via trans-presentation (TP), during which an APC expressing IL-15 bound to IL-15Rα presents the ligand to the ßγc receptor-heterodimer on a neighboring T/NK cell. To date, no direct biophysical evidence for the intercellular assembly of the IL-15R heterotrimer exists. Ag presentation (AP), the initial step of T cell activation, is also based on APC-T cell interaction. We were compelled to ask whether AP has any effect on IL-15 TP or whether they are independent processes. In our human Raji B cell-Jurkat T cell model system, we monitored inter-/intracellular protein interactions upon formation of IL-15 TP and AP receptor complexes by Förster resonance energy transfer measurements. We detected enrichment of IL-15Rα and IL-2/15Rß at the synapse and positive Förster resonance energy transfer efficiency if Raji cells were pretreated with IL-15, giving direct biophysical evidence for IL-15 TP. IL-15Rα and MHC class II interacted and translocated jointly to the immunological synapse when either ligand was present, whereas IL-2/15Rß and CD3 moved independently of each other. IL-15 TP initiated STAT5 phosphorylation in Jurkat cells, which was not further enhanced by AP. Conversely, IL-15 treatment slightly attenuated Ag-induced phosphorylation of the CD3ζ chain. Our studies prove that in our model system, IL-15 TP and AP can occur independently, and although AP enhances IL-15R assembly, it has no significant effect on IL-15 signaling during TP. Thus, IL-15 TP can be considered an autonomous, Ag-independent process.

Biophysical characterization of histone H3.3 K27M point mutation.

Lysine 27 to methionine (K27 M) mutation of the histone variant H3.3 drives the formation of an aggressive glioblastoma multiforme tumor in infants. Here we analyzed how the methionine substitution alters the stability of H3.3 nucleosomes in vitro and modifies its kinetic properties in live cells. We also determined whether the presence of mutant nucleosomes perturbed the mobility of the PRC2 subunit Ezh2 (enhancer-of-zeste homolog 2). We found that K27 M nucleosomes maintained the wild-type molecular architecture both at the level of bulk histones and single nucleosomes and followed similar diffusion kinetics to wild-type histones in live cells. Nevertheless, we observed a remarkable differential recovery of Ezh2 in response to transcriptional stress that was accompanied by a faster diffusion rate of the mobile fraction of Ezh2 and a significantly increased immobile fraction, suggesting tighter chromatin binding of Ezh2 upon transcription inhibition. The differential recovery of Ezh2 was dependent on transcription, however, it was independent from K27 M mutation status. These biophysical characteristics shed more light on the mechanism of histone H3.3 K27M in glioma genesis in relation to the kinetic properties of Ezh2.

High throughput FRET analysis of protein-protein interactions by slide-based imaging laser scanning cytometry.

Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site.

Conformation of the c-Fos/c-Jun complex in vivo: a combined FRET, FCCS, and MD-modeling study.

The activator protein-1 transcription factor is a heterodimer containing one of each of the Fos and Jun subfamilies of basic-region leucine-zipper proteins. We have previously shown by fluorescence cross-correlation spectroscopy (FCCS) that the fluorescent fusion proteins Fos-EGFP and Jun-mRFP1, cotransfected in HeLa cells, formed stable complexes in situ. Here we studied the relative position of the C-terminal domains via fluorescence resonance energy transfer (FRET) measured by flow cytometry and confocal microscopy. To get a more detailed insight into the conformation of the C-terminal domains of the complex we constructed C-terminal labeled full-length and truncated forms of Fos. We developed a novel iterative evaluation method to determine accurate FRET efficiencies regardless of relative protein expression levels, using a spectral- or intensity-based approach. The full-length C-terminal-labeled Jun and Fos proteins displayed a FRET-measured average distance of 8 +/- 1 nm. Deletion of the last 164 amino acids at the C-terminus of Fos resulted in a distance of 6.1 +/- 1 nm between the labels. FCCS shows that Jun-mRFP1 and the truncated Fos-EGFP also interact stably in the nucleus, although they bind to nuclear components with lower affinity. Thus, the C-terminal end of Fos may play a role in the stabilization of the interaction between activator protein-1 and DNA. Molecular dynamics simulations predict a dye-to-dye distance of 6.7 +/- 0.1 nm for the dimer between Jun-mRFP1 and the truncated Fos-EGFP, in good agreement with our FRET data. A wide variety of models could be developed for the full-length dimer, with possible dye-to-dye distances varying largely between 6 and 20 nm. However, from our FRET results we can conclude that more than half of the occurring dye-to-dye distances are between 6 and 10 nm.

A biophysical approach to IL-2 and IL-15 receptor function: localization, conformation and interactions.

Interleukin-2 and interleukin-15 (IL-2, IL-15) are key participants in T and NK cell activation and function. Sharing the beta and gamma receptor subunits results in several common functions: e.g. the promotion of T cell proliferation. On the other hand, due to their distinct alpha receptor subunits, they also play opposing roles in immune processes such as activation induced cell death and immunological memory. Divergence of signaling pathways must ensue already at the plasma membrane where the cytokines interact with their receptors. Therefore understanding molecular details of receptor organization and mapping interactions with other membrane proteins that might influence receptor conformation and function, are of key importance. Biophysical/advanced microscopic methods (fluorescence resonance energy transfer (FRET), fluorescence crosscorrelation spectroscopy (FCCS), near-field scanning optical microscopy (NSOM), X-ray crystallography, surface plasmon resonance, NMR spectroscopy) have been instrumental in clarifying the details of receptor structure and organization from the atomic level to the assembly and dynamics of supramolecular clusters. In this short review some important contributions shaping our current view of IL-2 and IL-15 receptors are presented.

Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling.

The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 µM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.

FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells.

The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor.