Molecular mechanisms and functions of tubulin heterogeneity – HetTub

In order to reach the goal of the present project, we will develop the tools required to study tubulin isoforms and modifications in vitro. This will be done using a highly ambitious protein engineering approach in order to obtain recombinant tubulin that can be modified in a systematic and controllable manner. A set of classical biochemical and structural biology approaches will be used to characterize the modifying enzymes. The data obtained will feed into ongoing functional work carried out in the lab on a cellular and organism level in order to understand the molecular mechanisms in their functional context.

The first 6 months of the project have resulted in a large number of reagents allowing to pursue the proposed work. In brief, a careful bioinformatic analysis has allowed to create synthetic genes for various tubulin isoforms as well as prokaryotic/eukaryotic tubulin chimera. A large effort has been put into designing and cloning expression vectors for bacterial, insect and mammalian expression of tubulin isoforms/chimera, as well as modifying enzymes and domains thereof. Stable cell lines have been created and expression tests are ongoing. A technical platform for rapid protein production has been established.

The results obtained will be used to address specific questions in a cellular context in order to understand the biological implications of these modifications. The latter part of the project will be fully integrated into a host lab context that applies an interdisciplinary approach ranging from in vitro systems over cell biology to mouse models in order to study these microtubule modifications.
In summary, this project will address how precise microtubule identity marks are generated by tubulin posttranslational modifications, and how these marks are translated into specialized microtubule functions. This work might have a fundamental impact on the understanding of microtubule functions in cells and organisms.

All results obtained so far are preliminary and have not given rise to publications or patents

Microtubules are key components of the eukaryotic cytoskeleton involved in a multitude of essential cellular functions. In the past decades, thorough studies have elucidated the biophysical properties of microtubules, their interactions with multiple microtubule-associated proteins and molecular motors, as well as many specialized cellular functions of the microtubule network. While many of the basic functions and properties of microtubules have been studied in great detail, microtubules have generally been considered as homogeneous macromolecular assemblies. This is contrasted by the obvious existence of microtubule identities in cells, which allow microtubules to acquire specific properties for functional specialisation. The proposed project addresses two aspects of microtubule identity, isoform distribution and posttranslational modifications. The latter have been recognized as emerging regulatory mechanism in generating distinct microtubule identities.
To study how spatially and temporally controlled modification patterns determine the properties and the functional fate of microtubules, this project will focus on two specific tubulin modifications, polyglutamylation and polyglycylation. The enzymes that catalyse these two modifications have been identified and characterized previously by the Janke lab and one of the main goals of this project is to investigate the molecular mechanisms that determine the specificity of these modifying enzymes and control the generation of modification patterns on selected microtubules. In order to reach this goal, we will also develop the tools required to study tubulin isoforms and modifications in vitro. This will be done using a highly ambitious protein engineering approach in order to obtain recombinant tubulin that can be modified in a systematic and controllable manner. The results obtained will then be used to address specific questions in a cellular context in order to understand the biological implications of these modifications. The latter part of the project will be fully integrated into a host lab context that applies an interdisciplinary approach ranging from in vitro systems over cell biology to mouse models in order to study these microtubule modifications.
In summary, this project will address how precise microtubule identity marks are generated by tubulin posttranslational modifications, and how these marks are translated into specialized microtubule functions. This work might have a fundamental impact on the understanding of microtubule functions in cells and organisms.