Exploring microtubule-based motor circuit wiring via the analysis of the Fidgetin-like 1 interactome

Lors du développement du système nerveux, la formation de circuits fonctionnels dépend de la capacité des axones à traduire les signaux de guidage perçus dans l’environnement en un remodelage morphologique du cône de croissance, afin qu’il atteigne les bonnes cibles synaptiques. Différentes machineries cellulaires sous-tendent ce remodelage, tels que le trafic membranaire et le cytosquelette. Mon équipe de thèse a identifié l’ATPase Fidgetin-like 1 (Fignl1) comme un acteur phare de la navigation axonale des neurones moteurs spinaux de poisson zèbre, par sa régulation de la dynamique des microtubules. Ma thèse a consisté à préciser les mécanismes cellulaires et moléculaires par lesquels la Fignl1 régule la navigation axonale, en initiant l’analyse de l’interactome de cette ATPase. Une approche gêne candidat, basée sur Rad51 – le seul partenaire d’interaction publié de Fignl1 –, a ainsi pu révéler le rôle de cette recombinase dans la navigation axonale des neurones moteurs spinaux du téléoste, et son association potentielle avec Fignl1 lors de ce processus. D’autre part, l’analyse d’un crible double hybride m’a conduite à identifier un nouveau mécanisme impliquant la Fignl1 en tant que régulateur clé du trafic vésiculaire rétrograde au sein d’axones en développement. Enfin, en me concentrant sur la voie de signalisation netrin 1/DCC, j’ai initié la caractérisation des signalisations de guidage régulant le comportement giratoire du cône de croissance par le biais de la Fignl1. Mes travaux de thèse établissent ainsi le rôle central de la Fignl1 dans la navigation axonale, par ses fonctions multiples dans le remodelage du cytosquelette et la régulation du trafic membranaire.During nervous system development, the wiring of functional circuits requires developing axons to accurately sense and translate environmental guidance signals into morphological changes of the growth cone, enabling it to reach the right synaptic targets. This growth cone remodelling is mediated by the concerted action of different intracellular machineries, such as membrane trafficking and the cytoskeleton. My PhD team has recently identified the Fidgetin-like 1 ATPase (Fignl1) as a critical player of zebrafish spinal motor axon navigation, via its regulation of microtubule dynamics. The aim of my PhD was to further unravel the cellular and molecular mechanisms by which Fignl1 regulates axon navigation, via the analysis of the Fignl1 interactome. A candidate gene approach, focused on the sole published Fignl1 binding partner – Rad51 – first revealed a role for this recombinase in zebrafish motor axon pathfinding, and its potential association with Fignl1 in this process. Additionally, a global approach – based on a yeast two-hybrid screen – led to the identification of a new mechanism involving Fignl1 as a key regulator of retrograde vesicular trafficking in navigating axons. Finally, using the netrin-1/DCC candidate pathway, I have initiated the characterisation of upstream signalling cascades converging onto Fignl1 to regulate axon navigation. Taken together, my PhD results highlight the multifaceted role of Fignl1 in axon pathfinding, via its multiple functions in the regulation of cytoskeletal dynamics and membrane trafficking

Microtubule remodelling as a driving force of axon guidance and pruning

International audienceThe establishment of neuronal connectivity relies on the microtubule (MT) cytoskeleton, which provides mechanical support, roads for axonal transport and mediates signalling events. Fine-tuned spatiotemporal regulation of MT functions by tubulin post-translational modifications and MT-associated proteins is critical for the coarse wiring and subsequent refinement of neuronal connectivity. The defective regulation of these processes causes a wide range of neurodevelopmental disorders associated with connectivity defects. This review focuses on recent studies unravelling how MT composition, post-translational modifications and associated proteins influence MT functions in axon guidance and/or pruning to build functional neuronal circuits. We here summarise experimental evidence supporting the key role of this network as a driving force for growth cone steering and branch-specific axon elimination. We further provide a global overview of the MT-interactors that tune developing axon behaviours, with a special emphasis on their emerging versatility in the regulation of MT dynamics/structure. Recent studies establishing the key and highly selective role of the tubulin code in the regulation of MT functions in axon pathfinding are also reported. Finally, our review highlights the emerging molecular links between these MT regulation processes and guidance signals that wire the nervous system

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation

International audienceDevelopment of SpiCee, a genetically encoded calcium scavenger d SpiCee inhibits calcium-dependent downstream pathways d SpiCee enables cell-specific manipulation of calcium-dependent processes in vivo d Subcellular targeting confers cell compartment specificity to SpiCee In Brief Ros et al. develop SpiCee, a genetically encoded calcium chelator that enables the manipulation of this second messenger in single cells with subcellular specificity. SpiCee alters the migration of developing cortical neurons in vivo. Targeting of lipid rafts prevents axonal repulsion, whereas exclusion from this subcellular compartment does not

FIGNL1 associates with KIF1Bβ and BICD1 to restrict dynein transport velocity during axon navigation

International audienceNeuronal connectivity relies on molecular motor-based axonal transport of diverse cargoes. Yet the precise players and regulatory mechanisms orchestrating such trafficking events remain largely unknown. We here report the ATPase Fignl1 as a novel regulator of bidirectional transport during axon navigation. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we showed that Fignl1 binds the kinesin Kif1bβ and the dynein/dynactin adaptor Bicaudal D-1 (Bicd1) in a molecular complex including the dynactin subunit dynactin 1. Fignl1 colocalized with Kif1bβ and showed bidirectional mobility in zebrafish axons. Notably, Kif1bβ and Fignl1 loss of function similarly altered zebrafish motor axon pathfinding and increased dynein-based transport velocity of Rab3 vesicles in these navigating axons, pinpointing Fignl1/Kif1bβ as a dynein speed limiter complex. Accordingly, disrupting dynein/dynactin activity or Bicd1/Fignl1 interaction induced motor axon pathfinding defects characteristic of Fignl1 gain or loss of function, respectively. Finally, pharmacological inhibition of dynein activity partially rescued the axon pathfinding defects of Fignl1-depleted larvae. Together, our results identify Fignl1 as a key dynein regulator required for motor circuit wiring

Preliminary results of centralized HER2 testing in ductal carcinoma in situ (DCIS): NSABP B-43

NSABP B-43 is the first prospective, randomized phase III multi-institution clinical trial targeting high-risk, HER2-positive DCIS. It compares whole breast irradiation alone with WBI given concurrently with trastuzumab in women with HER2-positive DCIS treated by lumpectomy. The primary aim is to determine if trastuzumab plus radiation will reduce in-breast tumor recurrence. HER2-positive DCIS was previously estimated at >50 %, occurring primarily in ER-negative, comedo-type DCIS of high nuclear grade. There has been no documented centralized multi-institutional HER2 analysis of DCIS. NSABP B-43 provides a unique opportunity to evaluate this in a large cohort of DCIS patients. Patients undergoing lumpectomy for DCIS without evidence of an invasive component are eligible. A central review of each patient’s pure DCIS lesion is carried out by immunohistochemistry analysis. If the lesion is 2+, FISH analysis is performed. Patients whose tumors are HER2 3+ or FISH-positive are randomly assigned to receive two doses of trastuzumab during WBI or WBI alone. NSABP B-43 opened 11/9/08. As of 7/31/2013, 5,861 patients have had specimens received centrally, and 5,645 of those had analyzable blocks; 1,969 (34.9 %) were HER2 positive. A total of 1,428 patients have been accrued, 1,137 (79.6 %) of whom have follow-up information. The average follow-up time for the 1,137 patients is 23.3 months. No grade 4 or 5 toxicity has been observed. In NSABP B-43 the HER2-positive rate for pure DCIS among patients undergoing breast-preserving surgery is 34.9 %, lower than the previously reported rate. No trastuzumab-related safety signals have been observed. Interest in this trial has been robust