Year 2021 THESIS TOPIC Subject N° (to be completed by the ED): FUNDING: Requested Acquired Funding origin: Contrat Doctoral Ordinaire Thesis title: Functional analysis of the MAP7/Kinesin-1 complex in vivo keywords: Intracellular Transport, microtubule networks, cell division Unit / team: IGDR-UMR6290 Supervisor’s name: Régis GIET Phone number: 02 23 23 49 98 Email address: regis.giet@univ-rennes1.fr Socio-economic and scientific context (approximately 10 lines): Intracellular transport of vesicles and organelles (cargo) is an essential process that relies on microtubules (MT) These MTs constitute "highways" used by molecular engines for the intracellular transport of cargoes In addition to their function for intracellular transport, these molecular motors and their regulatory proteins are also required for the assembly of complex dynamic MTs-based structures Understanding the role of these motor and regulatory proteins is therefore an important issue in terms of developmental biology and human health, since certain molecular motors, such as Kinesin-1, are mutated in patients suffering from neurodegenerative diseases Our team recently validated the double activation of Kinesin-1 by MAP7, a protein itself associated with MTs Our goal is now to dissect how Kinesin-1 and MAP7 cooperate to regulate the intracellular transport and assembly of MT networks at level of the cell and the whole organism Working hypothesis and aims (approximately lines): Kinesin-1 is a self-inhibited molecule This self-inhibition occurs through intramolecular folding phenomena We have shown that the binding of MAP7 to the self-inhibitory motif of Kinesin-1 prevents its self-inhibition and allows the activation of Kinesin-1 This interaction therefore activates intracellular transport and the assembly of specialised MT structures We have mapped on MAP7 and Kinesin-1 the motifs that allow the interaction between the two proteins, and we have created mutants deficient for the Kinesin-1/MAP7 interaction We propose to replace endogenous MAP7 and Kinesin-1 proteins by their respective mutated variants to measure the impact of MAP7/Kinesin-1 interactions in vivo We propose two hypotheses Firstly, a MAP7 variant that does not bind kinesin-1 should interfere with kinesin-1 dependent processes (transport and organization of microtubule networks) Secondly, a Kinesin-1 that is defective in binding to its MAP7 activator should produce a "hyperactive" molecular motor because the self-inhibition motif is mutated These experiments will enable us to analyse the contribution of the Kinesin-1/MAP7 complex to intracellular transport and the organisation of the dynamic structures of MTs in different cell types (neural stem cells, neurons, oocytes) and at different stages of development Main milestones of the thesis (approximately 12 lines): 1) Replacement of MAP7 and Kinesin-1 by their own interaction-deficient variants labelled by GFP or Dendra (photoactivatable protein) -2) Measurements of intracellular transport and assembly of MT networks in different cell types (neurons, stem cells, oocytes) and at different stages of development (larval and adult stages) -3) Analyses of the localisation dynamics of the different mutants in the cell types by 5D imaging, photo-activation, FRAP Scientific and technical skills required by the candidate (2 lines): Imaging, microscopy, image analysis, biochemistry, preparation of biological samples publications from the team related to the topic (last years): - Mathieu Métivier1, Brigette Y Monroy 3, Emmanuel Gallaud1, Renaud Caous1, Aude Pascal1, Laurent Richard-Parpaillon1, Antoine Guichet2, Kassandra M Ori-McKenney3 and Régis Giet1* Dual control of Kinesin-1 recruitment to microtubules by Ensconsin/MAP7 (2019) Development doi: 10.1242/dev.171579 - Renaud Caous, Aude Pascal, Pierre Romé, Laurent Richard-Parpaillon, Roger Karess, and Régis Giet (2015) Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila Nature Communications 6, Article number: 8879 doi:10.1038/ncomms9879 - Mathieu Métivier1, Emmanuel Gallaud1, Aude Pascal1, Jean-Philippe Gagné2, Guy G Poirier2, Denis Chrétien1, Romain Gibeaux1, Laurent Richard-Parpaillon1, Christelle Benaud1, and Régis Giet1* Drosophila dTBCE recruits tubulin around chromatin to promote mitotic spindle assembly Current Biology 2020 doi: 10.1016/j.cub.2020.11.009 National collaborations: Christian Jaulin Ph D University of Rennes-France Year 2021 R Gibeaux and Denis Chrétien Ph D University of Rennes-France Véronique David Ph D University of Rennes-France Sébastien Huet Ph.D University of Rennes-France Roger Karess, Ph D Institute Jacques Mondod-Paris, France Antoine Guichet, Ph D Institute Jacques Mondod-Paris, France International collaborations : Kassandra Ori-McKenney Ph D University of California-Davis, CA Hiro Ohkura Ph D University of Edinburgh-UK Isabelle Arnal Ph D University of Grenoble-France Mathew Savoian Ph D Institute of Fundamental Sciences Massey NZ Vincent Archambault Ph D IRIC Montreal Canada Guy Poirier Ph.D CHUL Quebec canada ... University of Rennes-France Véronique David Ph D University of Rennes-France Sébastien Huet Ph.D University of Rennes-France Roger Karess, Ph D Institute Jacques Mondod-Paris, France Antoine Guichet,... Guichet, Ph D Institute Jacques Mondod-Paris, France International collaborations : Kassandra Ori-McKenney Ph D University of California-Davis, CA Hiro Ohkura Ph D University of Edinburgh-UK Isabelle... University of Edinburgh-UK Isabelle Arnal Ph D University of Grenoble-France Mathew Savoian Ph D Institute of Fundamental Sciences Massey NZ Vincent Archambault Ph D IRIC Montreal Canada Guy Poirier