La régulation des propriétés et des interactions des microtubules par le 'tubulin code'

par Jijumon A.s.

Projet de thèse en Sciences de la vie et de la santé

Sous la direction de Carsten Janke et de Thomas THOMAS MüLLER-REICHERT.

Thèses en préparation à Paris Saclay , dans le cadre de Structure et Dynamique des Systèmes Vivants , en partenariat avec Stress génotoxiques et Cancer (laboratoire) et de Université Paris-Sud (établissement de préparation de la thèse) depuis le 03-10-2016 .


  • Résumé

    In this project, we will develop novel techniques allowing us to reproducibly measuring alterations in microtubule dynamic instability, as well as variations in the interactions with microtubule-associated proteins and molecular motors in vitro. We will combine the generation of recombinant tubulin with controlled posttranslational modifications with in vitro reconstructions of microtubule assemblies, force and motility measurements, and micro-fabrication. Tubulin is subject to a large range of variations referred to as the ‘tubulin code'. Tubulin in mammals and many other organisms is expressed from different genes, giving rise to tubulin isotypes, and is then modified by a panoply of posttranslational modifications. The variety of tubulin genes and different types of posttranslational modifications is believed to alter the basic microtubule properties, e.g. their dynamic instability, as well as their interactions with specific associated proteins including molecular motors (reviewed in Janke et al., 2014). There are several indications that the tubulin code plays an important role for microtubule functions. For instance, we have shown that altering tubulin posttranslational modifications can affect cilia and flagella (Rogowski et al., 2009, Bosch Grau et al., 2013), can lead to neurodegeneration (Rogowski et al., 2010), and can deregulate cell division (Barisic et al., 2015). Mutations of specific tubulin isotypes, on the other hand, have been demonstrated to generate a whole range of human pathologies, in particular neurodevelopmental and neurodegenerative disorder (reviewed in Chakraborti et al., 2016). These first functional data demonstrate the key importance of the tubulin code to regulate microtubule functions, however the molecular mechanisms of these regulatory events have remained largely unexplored. Based on the recent technological advances in our team, we can now tackle the in vitro reconstruction of microtubule assemblies with controlled tubulin-code status to directly measure the impact of this code on microtubule properties and functions. The key methodology of this project is the generation and purification of tubulin with controlled tubulin-code signatures. This has so far not been possible, and most in vitro reconstructions performed so far used tubulin that was purified from brain. Our team has now set up a novel approach to purify tubulin of high purity from cell lines (Barisic et al., 2015, Nirschl et al., 2016), which allows us to vary the tubulin code by either expressing different tubulin isotypes, as well as mutated tubulin variants, or/and by generating posttranslational modifications ‘à la carte' with the different modifying enzymes we have available (Janke et al., 2005, van Dijk et al., 2007, Rogowski et al., 2009, 2010). After having purified a set of tubulin variants, we will establish basic in vitro experiments that allow us to measure (i) the dynamic instability, (ii) the association of a set of microtubule-associated proteins with microtubules, and (iii) the association and motility parameters of several molecular motors. We will use established experimental setting and techniques for these analyses. In the second part, we will develop microfluidic chambers to assemble mosaic microtubules from different tubulin building blocks and test their behavior in the same set of in vitro experiments, this time focusing on the transition zones. In summary, we will develop an in vitro setting that allows us for the first time to test the molecular role of the tubulin code in vitro.

  • Titre traduit

    Control of microtubule interactions and properties by the tubulin code.


  • Résumé

    In this project, we will develop novel techniques allowing us to reproducibly measuring alterations in microtubule dynamic instability, as well as variations in the interactions with microtubule-associated proteins and molecular motors in vitro. We will combine the generation of recombinant tubulin with controlled posttranslational modifications with in vitro reconstructions of microtubule assemblies, force and motility measurements, and micro-fabrication. Tubulin is subject to a large range of variations referred to as the ‘tubulin code'. Tubulin in mammals and many other organisms is expressed from different genes, giving rise to tubulin isotypes, and is then modified by a panoply of posttranslational modifications. The variety of tubulin genes and different types of posttranslational modifications is believed to alter the basic microtubule properties, e.g. their dynamic instability, as well as their interactions with specific associated proteins including molecular motors (reviewed in Janke et al., 2014). There are several indications that the tubulin code plays an important role for microtubule functions. For instance, we have shown that altering tubulin posttranslational modifications can affect cilia and flagella (Rogowski et al., 2009, Bosch Grau et al., 2013), can lead to neurodegeneration (Rogowski et al., 2010), and can deregulate cell division (Barisic et al., 2015). Mutations of specific tubulin isotypes, on the other hand, have been demonstrated to generate a whole range of human pathologies, in particular neurodevelopmental and neurodegenerative disorder (reviewed in Chakraborti et al., 2016). These first functional data demonstrate the key importance of the tubulin code to regulate microtubule functions, however the molecular mechanisms of these regulatory events have remained largely unexplored. Based on the recent technological advances in our team, we can now tackle the in vitro reconstruction of microtubule assemblies with controlled tubulin-code status to directly measure the impact of this code on microtubule properties and functions. The key methodology of this project is the generation and purification of tubulin with controlled tubulin-code signatures. This has so far not been possible, and most in vitro reconstructions performed so far used tubulin that was purified from brain. Our team has now set up a novel approach to purify tubulin of high purity from cell lines (Barisic et al., 2015, Nirschl et al., 2016), which allows us to vary the tubulin code by either expressing different tubulin isotypes, as well as mutated tubulin variants, or/and by generating posttranslational modifications ‘à la carte' with the different modifying enzymes we have available (Janke et al., 2005, van Dijk et al., 2007, Rogowski et al., 2009, 2010). After having purified a set of tubulin variants, we will establish basic in vitro experiments that allow us to measure (i) the dynamic instability, (ii) the association of a set of microtubule-associated proteins with microtubules, and (iii) the association and motility parameters of several molecular motors. We will use established experimental setting and techniques for these analyses. In the second part, we will develop microfluidic chambers to assemble mosaic microtubules from different tubulin building blocks and test their behavior in the same set of in vitro experiments, this time focusing on the transition zones. In summary, we will develop an in vitro setting that allows us for the first time to test the molecular role of the tubulin code in vitro.