Les mutations du gène SPG4 codant la spastin sont responsables de la forme la plus fréquente de Paraplégies Spastiques Héréditaires (PSH), des maladies neurologiques caractérisées par une dégénérescence des faisceaux cortico-spinaux. La spastin, ainsi que son homologue p60-katanin sont des enzymes de cassure des microtubules (MSE) essentielles à la croissance des neurones moteurs spinaux (NMS) chez l'embryon de poisson-zèbre mais dont le rôle dans les processus de guidage axonal également dépendant des microtubules (MTs) demeurent énigmatiques. Les principaux objectifs de ma thèse ont consisté à préciser le rôle et le degré de redondance fonctionnelle existant entre ces deux MSE lors de l'établissement des circuits moteurs chez ce téléoste et de clarifier les mécanismes pathogéniques à l¿origine des PSH liées au gène SPG4.J'ai tout d'abord contribué à montrer que la p60-Katanin contrôle la trajectoire des axones des NMS et la mobilité des larves de façon dose-dépendante et non redondante avec la spastin. De plus, notre étude identifie la polyglutamylation des MTs par TTLL6 comme un élément clé de l'activité de la p60-Katanin lors de ce processus. Sur le même modèle, j'ai révélé un rôle différentiel des isoformes majoritaires de la spastin (résultant d¿une traduction alternative, M1 et M61) au cours du développement des NMS en démontrant un rôle coopératif de M1 et d'autres protéines de PSH dans l'inhibition de la voie des BMPs et révélant un rôle pour M61 en aval de la signalisation Neuropilin-1. Ces données suggèrent que l'altération de ces deux grandes voies de signalisation essentielles au développement des NMS pourrait contribuer à la pathogénèse des formes SPG4. / Mutations in SPG4, encoding spastin cause the major form of Hereditary Spastic Paraplegias (HSP), a paralytic disorder characterised by the degeneration of the corticospinal tracts. Spastin and its close homologue p60-katanin are microtubule-severing enzymes (MSE) required for spinal motor neuron (SMN) axon extension during zebrafish development. However, their roles in SMN axon navigation which also rely on microtubules (MTs) remain elusive. My PhD work aimed at refining the functional specificity and redundancy of these MSE during motor circuit wiring and clarifying the physiopathology of SPG4-linked HSP. I have first contributed to show that p60-Katanin controls SMN axon targeting and larval locomotion in a dose-dependent manner. We also demonstrated that Spastin and p60-Katanin play differential roles in SMN navigation and identified TTLL6-mediated MT polyglutamylation as a key event in regulating p60-Katanin activity in this process. Concomitantly, I have conducted a functional analysis of spastin main isoforms (resulting from alternative translation, M1 and M61) during zebrafish development, which reveals their critical and specific involvement in two distinct signalling pathways that are both essential for motor circuit wiring and locomotor behaviours. This study has provided compelling evidences for a concerted role for M1 and other HSP proteins in the down-regulation of the BMP pathway and reveals a specific role for M87 as a downstream effector of Neuropilin-1 signalling. Altogether, our study emphasizes defective BMP signalling as a key pathogenic mechanism in HSP, and shows that dysregulation of the Neuropilin-1 pathway may equally contribute to SPG4-linked HSP.

Plant microtubules play essential roles in cell processes such as cell division, cell elongation, and organelle organization. Microtubules are arranged in highly dynamic and ordered arrays, but unlike animal cells, plant cells lack centrosomes. Therefore, microtubule nucleation and organization are governed by microtubule-associated proteins, including a microtubule-severing protein, katanin. Mutant analysis and in vitro characterization has shown that the highly conserved katanin is needed for the organization of the microtubule arrays in Arabidopsis and rice as well as in a variety of animal models. Katanin is a protein complex that is part of the AAA+ family of ATPases. Katanin is composed of two subunits, katanin-p60, a catalytic subunit and katanin-p80, a regulatory subunit. Spastin is another MT-severing protein that was identified on the basis of its homology to katanin. In animal cells, spastin is also needed for microtubule organization, but its functionality has not yet been investigated in plants. To initiate an exploration of the function of katanin-p60 and spastin in Zea mays, my research goal was to generate tools for the expression and purification of maize katanin-p60 and spastin proteins in vitro. Plasmids that express katanin-p60 and spastin with N-terminal GST tags were designed and constructed via In-Fusion® cloning after traditional cloning methods were not successful. The constructs were expressed in E. coli, then the recombinant proteins were purified. To determine if the GST-tagged proteins are functional, ATPase activity and tubulin polymerization assays were performed. While both GST-katanin-p60 and GST-spastin hydrolyzed ATP indicating that the ATPase domains are functional, the results of the tubulin polymerization assays were less clear and further experimentation is necessary.

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Microtubules (MT) are regulated by multiple categories of proteins, including proteins responsible for severing MTs that are therefore called MT-severing proteins. Studies of katanin, spastin, and fidgetin in animal systems have clarified that these proteins are MT-severing. However, studies in plants have been limited to katanin p60, and little is known about spastin or fidgetin and their function in plants. I looked at plant genomes to identify MT-severing protein homologues to clarify which severing proteins exist in plants. I obtained data from a variety of eukaryotic species to look for MT-severing proteins using homology to human proteins and analyzed these protein sequences to obtain information on the evolution of MT-severing proteins in different species. I focused this analysis on MT-severing proteins in the maize and Arabidopsis thaliana genomes. I created evolutionary phylogenetic trees for katanin-p60, katanin-p80, spastin, and fidgetin using sequences from animal, plant, and fungal genomes. I focused on Arabidopsis spastin and worked to understand its functionality by identifying protein interaction partners. The yeast two-hybrid technique was used to screen an Arabidopsis cDNA library to identify putative spastin interactors. I sought to confirm the putative protein interactions by using molecular tools for protein localization such as the YFP system. Finally, a Biomolecular Fluorescence Complementation (BiFC) assay was initiated as a proof of concept for confirmation of in vivo protein-protein interaction.

No description available.

No description available.