The role of spatio-temporal nano-organization in regulating the specificity of JAK/STAT signaling – NANOSTAT

We will identify specific differences between the assembly and dynamics of IFNAR and IFNGR in the plasma membrane and in endosomes using single molecule and superresolution imaging techniques . The role of plasma membrane microcompartmentation into actin and lipid-based domains will be explored by using biochemical and genetic modifications. Specifically, we aim to identify how specific features of IFNGR localization into lipid nanodomains, are probably related to specific membrane lipid and extracellular galectin interactions. For a detailed understanding of the molecular interactions involved in the formation of the signaling complex we will identify potential interaction partners by mass spectrometry, which will be validated in living cells by functional surface micropatterning. The role of these interaction partners will be further elucidated by biophysical and functional studies. Based on the detailed mechanistic picture obtained from these studies, we will attempt to systematically swap functional modules between the type I and type II IFN receptors in order to validate their functional relevance in a specific cellular context.

P1 studied the IFNGR2 T168N mutation, which adds a neo-glycan on IFNGR2 subunit and blocks IFN-gamma activity (Nat Genet 2005). P1 showed that IFNGR2 lateral diffusion is confined by sphingolipid/cholesterol nanodomains whereas the IFNGR2 T168N diffusion is confined by actin nanodomains where JAK/STAT activation by IFN-gamma is inhibited. Removing IFNGR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT activation. These experiments establish the physiological role of lipid nanodomains in the control of receptor signaling in vivo (Cell 2016).

P1 next addressed the role of endosomal trafficking in signaling and found that the IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. These data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and JAK/STAT signaling and gene transcription (Nat Commun 2016).

P2 studied the spatiotemporal dynamics of IFNAR and IFNGR in the plasma membrane (PM) by single molecule tracking. Site-specific fluorescence-labeled IFNs were produced. Single molecule imaging revealed similarly low density of signaling complexes for both IFN-Rs as well as random distribution and diffusion in the PM. Dual-color single molecule imaging of ectopically expressed labeled IFN-R subunits showed that both IFNR subunits are not pre-dimerized or spatially pre-organized. While similar diffusion properties were obtained for IFNGR2 wt and T168N in absence of ligand, the mutant was not recruited into the signaling complex. Moreover, P2 established four-color single molecule imaging to simultaneously follow diffusion and interaction of all subunits of both IFNRs.

Suitable fusion proteins for imaging STAT1 and STAT2 recruitment with high resolution were implemented together with fluorescent endosomal markers. Proof-of-concept experiments for imaging receptor trafficking by lattice light sheet microscopy were performed.

We will follow the original program of work with an emphasis on the remaining experiments that require collaboration between the two groups. We will now investigate the role actin-binding proteins and actin connectors to the receptors that have been revealed by mass spectrometry by P1.

P2 will also examine the role of galectins in IFNR diffusion and interaction at the plasma membrane since they have been found associated to IFNGR2 by cell proteomics and to regulate JAK/STAT activation by IFN-??(Cell 2016). P2 will take advantage of the labeling and three-dimensional imaging by lattice light sheet microscopy established in his laboratory in combination with dSTORM to resolve the spatiotemporal receptor organization and dynamics in endosomes.

In another collaborative effort, we will study and validate by advanced fluorescence imaging techniques the potential interactors identified by P1 by proteomics. This validation will be carried out by cell micropatterning recently established by P2 (JCB 2014). By spatial redistribution of bait proteins in the PM by means of micropatterned substrates, interactions with fluorescent-tagged bait proteins can be unambiguously detected quantified in living cells. Assembly of active signaling complexes in micropatterns is currently employed for detecting and quantifying effector recruitment to select most suitable probes for monitoring receptor activation at single molecule level. By means of lattice light sheet microscopy P2 will probe effector interactions at the plasma membrane and in endosomes to compare the efficacies of signal activation in different cellular nanocompartments.

1.Chmiest, D., Sharma, N., Zanin, N., Viaris de Lesegno, C., Shafaq-Zadah, M., Sibut, V., Dingli, F., Hupé, P., Wilmes, S., Piehler, J., Loew, D., Johannes, L., Schreiber., G and C. Lamaze. 2016. Spatiotemporal Control of Interferon-induced JAK/STAT Signaling and Gene Transcription by the Retromer Complex. Nat Commun 7:13476

2. Blouin, C.M., Hamon Y., Gonnord, P., Boularan, C., Kagan, J., Viaris de Lesegno, C., Ruez, R., Mailfert, S., Bertaux, N., Loew, D., Wunder, C., Johannes, L., Vogt, G., Contreras, F-X., Marguet, D., Casanova, J-L., Galés, C., He, H-T. and C. Lamaze. 2016. Glycosylation-Dependent IFN-?R Partitioning in Lipid and Actin Nanodomains is Critical for JAK Activation. Cell 166: 920-34. doi: 10.1016/j.cell.2016.07.003

3. Lamaze, C., and C.M. Blouin. 2017. Receptor Lipid nanodomain Partitioning and Signaling. Cell Cycle 16(3):237-238

Ligand-induced signal propagation through specific receptors in the plasma membrane is a key process for the cell in order to fulfill its complex role within multicellular organisms. While principal paradigms of how this process is mediated by different receptor types have been established, the molecular and biophysical parameters, which control the specificity of signal activation by the ligand, are far from being clear. A key challenge is to understand the so-called paradox of signaling specificity where a limited set of intracellular signaling effectors can control myriads of signaling outputs. This paradox is particularly well illustrated by the type I interferon receptors (IFNRs) where as many as 13 different IFNs can bind and activate the same IFN? receptor. Complex lateral interactions between integral membrane proteins in the lipid bilayer are believed to play an important role for receptor activation by its ligands. In addition to these interactions at the plasma membrane, increasing evidence shows that the dynamics of endomembranes can play a key role in the selective activation of signaling receptors.

The overall aim of this interdisciplinary and collaborative project is to uncover how the specificity of JAK/STAT signaling specificity is controlled by the nano-compartmentalization of the activated IFNR complex at the plasma membrane and in endosomes. IFNRs represent one of the best paradigms to investigate the complexity of these mechanisms since both type I IFNAR (IFN?lpha/?eta? and type II IFNGR (IFN?amma) activate a common STAT1 molecule but display a distinct transcriptional output. P1 has recently shown that IFNR endocytic trafficking and lipid-based nano-organization at the plasma membrane play a key role in the selectivity of JAK/STAT activation by type I and type II IFNs, respectively. P2 has established advanced imaging techniques for monitoring IFNR assembly and spatiotemporal dynamics using single molecule localization imaging techniques. The role of plasma membrane nano-compartmentalization into actin and lipid-based domains will be explored by using biochemical and genetic modifications. Specifically, we aim to identify how specific features of IFNGR localization into lipid rafts, are probably related to specific lipid and galectin interactions. We will identify new interaction partners by mass spectrometry (P1), which will be validated in living cells by functional surface micropatterning (P2). The role of these interaction partners will be further elucidated by biophysical (P2) and functional studies (P1). Based on the detailed mechanistic picture obtained from these studies, we will attempt to systematically swap functional modules between the type I and type II IFN receptors in order to validate their functional relevance in a specific cellular context (P1).

The combination of cell biology assays (P1) with biophysics and advanced biophotonics approaches (P2) will give us the unprecedented opportunity to test the provocative hypothesis that the localization and clustering of the activated IFNR complex into distinct membrane nano-compartments (plasma membrane for IFNGR and the early endosome for IFNAR) together with the different spatiotemporal regulation of endosomal IFNR trafficking selectively condition the mechanistics of JAK/STAT activation.

The philosophy of our project lies in the emergence of new concepts based on the quantification of biological observables at a single molecule level through the development of innovating experimental and analytical tools. Ultimately, we expect to unravel the molecular basis that conditions the reactivity and the dynamics of the biological macromolecules (IFNRs) and their complexes (JAK/STAT cytosolic partners) when cells are activated by IFNs. Thus, there is an intrinsic link that brings together three different research fields, namely cell biology, in vitro membrane model systems and photonics, at the conceptual and experimental levels.