Antony Team
Cellular electron tomography of cells
Nuclear congression in the budding yeast. 3D reconstruction of mating yeast cells after plasma (blue-green) fusion has occurred. The two nuclei are brought together by forces exerted by microtubules emanating from the two SPBs of opposite cells. (Picture by Romain Gibeaux; collaboration with Michael Knop’s group. Bar, 300 nm).
Previous and current research
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 (ET), 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. We are equipped with a Tecnai F30 tomography microscope (FEI), which is mostly devoted to plastic electron tomography projects.
Our work has focussed on key areas, such as resolving the 3D organisation of microtubule arrays 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 analysed the morphology of the microtubule ends by ET so as to determine the type of ends of the growing microtubules. This analysis was carried out by Johanna Höög in collaboration with Damian Brunner’s group (both formerly EMBL). We also investigated microtubule bundling factors in fission yeast, Ase1 (a non-motor homodimer protein) and further identified Dis1p (XMAP215 homologue) as an alternative microtubule bundling factor that 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. This work was done by Helio Roque in collaboration with Damian Brunner’s group.
Another project is to investigate the role and organisation of microtubules in the budding yeast mating path- way, led by Romain Gibeaux (PhD student) in collaboration with Michael Knop’s group (EMBL). The project concerns the morphological and molecular analysis of the karyogamy process in the budding yeast mating pathway. We recently focused on the nuclear congression by resolving the detailed microtubule organisation and microtubule connection with cells’ organelles. These experiments are carried out by electron tomography. In parallel we are searching for molecular players associated with microtubules that are also involved in force generation by designing a set of mutant analysis coupled with live-cell fluorescence microscopy. This work is currently ongoing.
Future projects and goals
In collaboration with in-house and external research groups (Nédélec group and Rebecca Heald, UCB) we run a major project aiming to reconstruct, at EM resolution, the Xenopus laevis mitotic spindle assembled from egg extracts. Samples are cryofixed by high pressure freezing and prepared for tomography acquisition. The large-scale reconstruction of such a huge structure, or parts of it, is being 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 about microtubule polarity. In particular, we are interested in understanding the structural organisation of microtubules in the midzone of the spindle and at the poles. Xavier Heiligenstein (PhD student) and Erin Tranfield (postdoc) are currently working on this project.