Nanoscale regulation of cell-surface receptor signaling

Cell surface receptors survey and relay information to ensure the development and survival of multicellular organisms. In plants, receptor kinases and receptor proteins are the main ligand-binding cell-surface receptors perceiving self, non-self, and modified-self molecules. While over the past decades tremendous efforts led to a detailed understanding of the genetic- and structural-bases underlying ligand-induced formation of receptors complexes for archetypical signaling pathways, their spatial and temporal regulation remains obscure. Taking perception of bacterial flagellin by the immune receptor kinase FLS2 and it’s co-receptor BAK1 as model system we investigate how is cell surface signaling orchestrated. We show that the co-receptor BAK1 is recruited “on demand” into pre-formed FLS2 membrane nanodomains to form signaling active nano-platforms. We unveil that formation of FLS2-BAK1 nanoscale assemblies is regulated by a tri-partite receptor module for endogenous secreted peptides. Our data suggest that plant and mammals evolved distinct spatio-temporal logic to orchestrate cell surface signaling. Finally, we propose a conceptual framework for the formation of nanoscale assemblies regulating cell surface signaling.

Surface plasmon resonance to study anti-HLA antibodies: transfer of a basic science method to the bedside of organ transplant recipients and beyond

Human leukocyte antigen (HLA) donor-specific antibodies are key serum biomarkers for assessing the outcome of solid organ transplanted patients. The current clinical tools, which are fluorescence-based methods, are semi-quantitative and then lack in-depth characterization of anti-HLA antibodies. We hypothesized that measuring anti-HLA antibodies active concentration, i.e. the fraction that really interacts with donor HLA, and their affinity could help deciphering their pathogenicity. Surface plasmon resonance (SPR) is recognized as the gold-standard for measuring binding kinetics of antibodies but also their active concentration, without calibration curves. Through tight collaboration with the INSERM-CNRS ARNA unit and the IECB’s SPR platform we pushed forward the strategies of immobilization/regeneration to determine the active concentration and binding kinetics of mouse and human monoclonal anti-HLA antibodies. We then correlate binding kinetics of these monoclonal antibodies to the structure of their cognate class I HLA epitopes. We thereafter established a patented method to correct non-specific binding when using SPR with complex samples, in our case patients’ serum. Clinical studies are ongoing to determine whether active concentration of anti-HLA antibodies is a useful biomarker in lung and kidney transplantation. Preliminary results indicate that this SPR-based assay may help to better understand anti-HLA antibodies pathogenicity and to improve solid organ recipients’ management. Beyond anti-HLA antibodies, this assay could be of interest in various fields, especially infectious diseases and vaccines. Our work is another example of how basic science methods can be transferred from the bench to bedside.

Non-coding small RNAs as versatile regulators of germline gene expression programs

The RNA-guided targeting of nucleic acids is an ancient and conserved mechanism of cellular immunity that has been evolutionary adapted and diversified to regulate eukaryotic gene expression. In animal germ cells, PIWI-interacting small RNAs (piRNAs) have been extensively characterized as a defense mechanism targeting transposable elements (TEs) to promote fertility and genome integrity. In a nutshell: loaded into PIWI effector proteins, piRNA sequences provide mRNA targeting specificity by antisense-complementarity, promoting gene silencing through a variety of mechanisms. Yet, piRNA sequences do not necessarily match TEs, pointing to extended possibilities in gene regulation.

Studying piRNA pathway functions in the context of the developing C. elegans germline we show that spermatogenic genes are susceptible to piRNA-mediated transcriptional silencing, and this function is required to ensure proper germline gene expression patterning and germ cell differentiation. In addition, our work revealed an intriguing aspect of small RNA biology: piRNA pathway components localize into diverse and distinct phase separated condensates present in the nuclear periphery. These condensates, also known as germ granules, are enriched in RNA-binding proteins and RNAs and are suspected to regulate post-transcriptional processes important for germ cell fate specification and function. Our results show that the organization of germ granules changes dynamically during development, and only a particular configuration enable nuclear piRNA silencing at a specific time and location in the germline tissue. This suggests that changes in germ granule composition directly influence nuclear processes through the modulation of small RNA related activities.

Overall the results of this work show that the function of piRNAs can be repurposed to regulate endogenous transcriptional programs during development and might contribute to expand the notion that piRNAs do not only function as a cellular immune system but also act as extremely versatile regulators of gene expression in animals.