The microtubule cytoskeleton plays crucial roles in regulating a vast range of biological processes in different cell types, in particular in neurons that strongly depend on intracellular transport for cargo delivery over long distances. Posttranslational modifications (PTMs) of tubulin recently emerged as modulators of microtubule properties and functions, which is expected to dynamically adapt them to specific cellular functions. Neuronal microtubules are strongly enriched in the PTM polyglutamylation, which was shown to affect the transport of different cargos. Strikingly, perturbed polyglutamylation causes neurodegeneration in mice and humans. How the impact of polyglutamylation on neuronal microtubules translates into such drastic physiological effects is, however, not understood. In my project I aim at determining how polyglutamylation affects physiological functions that are driven by microtubule-based transport in neurons. I will use primary neurons with different levels and patterns of tubulin polyglutamylation and a novel CRISPR/Cas9 technology to endogenously label cargos. Super-resolution microscopy techniques will be applied to precisely determine how this PTM temporally and spatially regulates cargo distribution to their target sites, and how this contributes to neuronal homeostasis. The proposed work will provide a mechanistic understanding of how tubulin PTMs control the physiological roles of the microtubule cytoskeleton at cellular level. This will expand our knowledge of the role of the tubulin code in regulating organism homeostasis, which might provide a mechanistic explanation for its role in neurodegeneration.

The BREAST CANCER TYPE 2 SUSCEPTIBILITY (BRCA2) is an evolutionarily conserved central regulator of homologous recombination (HR) in most eukaryotes. During HR, BRCA2 delivers the recombinases – RADiation sensitive 51 (RAD51) and Disrupted Meiotic cDNA 1 (DMC1) – at the DNA repair site. Its functions rely on a conserved canonical DNA binding domain (DBD). Surprisingly, BRCA2 homologs exist without a canonical DBD and are still essential for HR. We named here BRCA2 homologs with DBD as “canonical BRCA2” and without DBD as “non-canonical BRCA2”. Contrary to the intensive research performed on canonical BRCA2, the non-canonical BRCA2 homologs are understudied. How non-canonical BRCA2 lacking DBD participates in HR is unknown, and the comparative mechanistic understanding of canonical BRCA2 and non-canonical BRCA2 functions is also lacking. Further, human BRCA2 self-associates in a DBD-dependent manner and undergoes conformational changes upon interaction with the RAD51 during HR repair. The underlying structural mechanisms of BRCA2 oligomerization and conformational reorganization are currently elusive. The FLASHMOB project builds on a newly identified yet unpublished BRCA2 homolog in Physcomitrium patens, named PpBRCA2. Our preliminary genetic and biochemical data show that PpBRCA2 is a new non-canonical BRCA2. As Physcomitrium patens is a model plant for plant genetics and genomics analysis, PpBRCA2 is a unique tool to examine the functional mechanisms of a non-canonical BRCA2, and compare them with those of the canonical BRCA2 from Arabidopsis thaliana (AtBRCA2). The objective of the FLASHMOB project is to address three important and original questions: 1) What are the mechanistic differences/similarities between canonical (with DBD) and non-canonical (without DBD) BRCA2? 2) What is the structural mechanism of BRCA2 oligomerization? 3) How does the RAD51/DMC1 association impart conformational changes in BRCA2? The FLASHMOB project is organized in four tasks. 1) The cellular roles of PpBRCA2 are unknown. Task 1 seeks to characterize the in vivo functions of the newly identified PpBRCA2 in somatic and meiotic DNA repair by using classical genetic and cytological approaches. 2) The mechanistic understanding of how non-canonical BRCA2 copes with the lack of DBD is currently unknown. Task 2 focuses on assessing the mechanistic divergence or similarities between non-canonical PpBRCA2 and canonical AtBRCA2 through genetics and proteomics approaches. These tasks will identify new mechanisms involving BRCA2 proteins in plants, which are currently largely unknown. 3) Contrary to the large-sized human BRCA2 protein which is difficult to purify, PpBRCA2 has a small size and can be purified: it is a tool of choice for the structural analysis of BRCA2 mechanisms and is amenable to high-resolution structural studies. Task 3 aims at decoding the structural mechanism of PpBRCA2 self-association and reorganization upon RAD51 interaction by a combination of structural biology techniques. This should lead to novel molecular insights into BRCA2 functions. 4) Biochemical properties of a non-canonical BRCA2 are so far undefined. Task 4 undertakes the in vitro characterization of the biochemical properties of PpBRCA2 such as DNA binding activity and role in RAD51 presynaptic filament assembly. The expected outcome is the functional insight into how non-canonical BRCA2 promotes HR. The FLASHMOB project brings together a consortium of teams with highly complementary expertise, know-how and facilities in genetics, biochemistry, biophysics, structural biology and cytology. Organized in four independent but complementary tasks, the FLASHMOB project will discover new mechanisms involving BRCA2, and establish a structural and functional comparison between canonical and non-canonical BRCA2, which will have a direct impact on our understanding of BRCA2 functions in genomic stability in plants and other eukaryotes.

Membrane receptors control fundamental physiological processes in cells, and are major targets of medical drugs. The goal of this project is to investigate the nanoscale motion of the membrane receptor Fas and its functional role in maintaining immune surveillance. Fas is ubiquitously expressed in human body and has significant roles in disease progressions. A type I single pass transmembrane protein, Fas is known for its ‘dual character’ in triggering signaling pathways leading to both cell survival and cell death. In the presence of its ligand, the receptor undergoes higher-order clustering to form a death-inducing signaling complex (DISC) in the intracellular region. Immune cells use Fas-mediated DISC formation as a mechanism to ‘kill’ virus infected or malignant cells. The Fas ligand, which is a type II transmembrane protein, can be cleaved which results in its soluble variant. Unlike the membrane anchored Fas ligand, the cleaved variant is known to induce an alternative motility inducing signaling complex (MISC) in Fas receptor that results in cell migration. Although the functions of the Fas receptor (and the notion of duality) are well established, how it selects for non-apoptotic or apoptotic pathways is an open question. It has been postulated that the membrane bound and cleaved variants of the Fas ligand induce different structural orientation and conformations in the intracellular domains of the receptors to control DISC/MISC formation. However, due to the immediate higher order aggregations upon ligand-binding and the presence of other modulating proteins during in-vivo experiments, it has been a great challenge to test this hypothesis. This project will investigate the biophysical mechanism behind the duality in full-length Fas receptors by exploiting single-molecule Förster resonance energy transfer (smFRET) and membrane nanodisc platform. Mechanistic understanding of Fas transmembrane signaling has both scientific and pharmaceutical significance.