Antony Team

Previous and current research

The installation in 2008 of a new Tecnai F30 tomography microscope has allowed us and other EMBL groups to develop projects aiming at resolving the 3D organisation of cells in various contexts. The focus of interest in the team is the organisation of microtubular cytoskeleton arrays in both fission yeast and budding yeast as well as in the Xenopus mitotic spindle. For this purpose we use electron tomography, which not only allows the reconstruction, modelling and quantification of subcellular elements, but also enables the visualisation of a number of fine structural features which would not be detectable by conventional EM.

Fission yeast microtubule dynamics: In fission yeast we have studied microtubule regrowth after depolymerisation of the whole microtubular array using the carbendazim microtubule depolymerising drug (MBC). Upon drug wash-out we captured the early stages of microtubule bundle reassembly and observed detailed features of the growing microtubules. This analysis was carried out by Johanna Höög in collaboration with the Brunner group (both formerly EMBL).

Microtubule bundling factors in fission yeast: In parallel we also investigated microtubule bundling factors in fission yeast. To understand inter-microtubule bonds, we reconstructed microtubule arrays in strains with deletions affecting the bundling function. Ase1 (encoding a non-motor homodimer protein) deletion strain shows a loss of parallel organisation of the bundles despite some overlapping area where microtubules remain associated but with a reduced inter-microtubule spacing. We observed that cells lacking ase1p and klp2p can still bundle microtubules but with an altered intermicrotubule spacing.We further identified Dis1p (XMAP215 homologue) as an alternative microtubule bundling factor which accumulates at microtubule overlap areas in cells that are deleted for both ase1 and klp2. Dis1p appears to be a critical factor for the maintenance of interphase microtubules. The project was carried out by Helio Roque in collaboration with the Brunner group (both formerly EMBL).

Spatial organisation of microtubules in the budding yeastmating pathway: A new project has been started by Romain Gibeaux (PhD student) in collaboration with the Knop group (page 13) concerning the morphological and molecular analysis of the karyogamy process in the budding yeast mating pathway. This process involves dynamic events with microtubule connections between the two nuclei and associated SPBs which are instrumental in bringing the two nuclei in close apposition until nuclear fusion. The morphological 3D analysis of these events are currently carried out by electron tomography performed on shmooing cells and fused cells in the process of nuclear congression. The molecular basis of the process and the role played by key molecules, either SPB or microtubule-associated, is studied in collaboration with the Knop group.

Future projects and goals

In conjunction with in-house and external research groups (collaboration with Nédélec group and Rebecca Heald (UCB)) we are now starting a major project aiming to reconstruct the Xenopus laevis mitotic spindle from egg extracts. Cryofixation and preparation of samples for tomography acquisition are the first steps. The large-scale reconstruction of such a huge structure, or parts of it, will be performed using extensive montaging and joining of tomograms. In the course of this project we intend to elucidate the spindle microtubule architecture at high resolution, and in doing so derive information aboutmicrotubule polarity, in particular in themidzone of the spindle. XavierHeiligenstein (PhD student) and Erin Tranfield (postdoc) are working on this project.