Mitosis is central to life as it guarantees the accurate segregation of replicated chromosomes to daughter cells. This process relies on the spindle, a dynamic and bipolar structure based on microtubules. Spindle morphology varies greatly among species and cells to optimize its function. Size and architecture, in particular, are both essential for accurate chromosome segregation, cell division and cytokinesis. However, it remains unclear how spindle microtubule subpopulations organize into complex assemblies. Especially, how correct spindle morphometrics, at both the size and architectural levels, are established and maintained is poorly understood. We tackle this question at multiple scales, from tubulin and MAPs to microtubules and spindles, following three main complementary objectives.
Understanding the structural basis of microtubule dynamics in vitro
Microtubules organize into complex, highly dynamic and adaptable large-scale assemblies to perform a variety of cellular function, one of the most spectacular assemblies being the spindle. This adaptability relies on the intrinsic property that characterizes microtubules: their dynamic instability. While this property has been discovered almost 40 years ago, the structural basis regulating it is still not understood. In particular, the current models do not describe how tubulin lateral interactions are destabilized by GTP hydrolysis. We therefore analyze at high-resolution microtubules assembled in vitro in the presence of GTP analogues to reveal the structural basis governing these conformational changes.
EB1-gold tracks growing microtubule ends. Background: slice through a cryo-electron tomogram. Color image: High-resolution structure of a microtubule (yellow) fitted into the cryo-electron tomogram density of a microtubule with gold nanoparticles (red) in interaction with its dynamic growing end. The SAXS-based structure of EB1 (blue) has been arbitrarily placed in between the microtubule and gold nanoparticles. Guesdon, Bazile et al. Nat Cell Biol. 2016 sept.
In vivo, microtubule dynamics is finely regulated by MAPs that are able to stabilize or destabilize them. Assembly of large microtubule assemblies such as the metaphase spindle requires a fine regulation between all these actors. Like previous ones on the origins of microtubule dynamics, these studies on MAPs are essential to build realistic models of supra-molecular assemblies such as the spindle. We thus study at high-resolution, on microtubules assembled in vitro, MAPs involved in the architecture of Xenopus egg extract spindles, and understand their binding mode as well as their influence on microtubule structure, dynamics, and supra-molecular architecture.
Related publications
Structural model for differential cap maturation at growing microtubule ends
Estévez-Gallego J*, Josa-Prado F*, Ku S*, Buey RM, Balaguer FA, Prota AE, Lucena-Agell D, Kamma-Lorger C, Yagi T, Iwamoto H, Duchesne L, Barasoain I, Steinmetz MO, Chrétien D, Kamimura S, Díaz JF, Oliva MA
Elife 2020 Mar.
*Co-first authors
Lattice defects induce microtubule self-renewal
Schaedel L, Triclin S, Chrétien D, Abrieu A, Aumeier C, Gaillard J, Blanchoin L, Théry M, John K
Nature Physics, 2019, Jan.
EB1 interacts with outwardly curved and straight regions of the microtubule lattice
Guesdon A*, Bazile F*, Buey RM, Mohan R, Monier S, Rodríguez García R, Angevin M, Heichette C, Wieneke R, Tampé R, Duchesne L, Akhmanova A, Steinmetz MO, Chrétien D
Nat Cell Biol. 2016 Sep.
*Co-first authors
Deciphering the molecular control of spindle architecture in cytoplasmic extracts
Spindles are large assemblies within which different populations of microtubules cooperate to properly build it and regulate its morphology. To simplify the spindle, we study subpopulations independently, while comparing their assembly between two Xenopus species of different spindle sizes and architectures.
Colorful collage of Xenopus laevis (top) and Xenopus tropicalis (bottom) spindles showing spindle size and architecture differences with microtubules labeled with rhodamine tubulin and chromosomes with Hoechst DNA stain. Spindles were assembled in vitro using Xenopus egg extracts. Modified from Gibeaux and Heald, Cold Spring Harb Protoc, 2019 June issue cover Image.
Using the egg extracts of X. laevis and X. tropicalis, we reconstruct microtubule structures assembled from the two major organizing sites that contribute to spindle assembly, (i) the spindle poles and (ii) chromatin. We combine fluorescence microscopy and electron tomography analyses to understand the dynamics and ultrastructural bases of varying size and architecture of these different microtubule populations. We also extract quantitative parameters to combine them into physically realistic simulations using Cytosim to reveal the biophysical basis of the different architectures and scaling properties, and ultimately their implication for the regulation of spindle morphology.
Related publications
Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis.
Kitaoka M, Heald R, Gibeaux R
Cytoskeleton, 2018, Jun.
Subcellular scaling: Does size matter for cell division?
Heald R, Gibeaux R
Curr Opin Cell Biol, 2018, Jun.
Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus.
Gibeaux R, Acker R, Kitaoka M, Georgiou G, van Kruijsbergen I, Ford B, Marcotte EM, Nomura DK, Kwon T, Veenstra GJC, Heald R
Nature. 2018 Jan.
Developing supporting cutting-edge light and electron microscopy tools
To follow and answer the challenges raised by our research projects, we constantly adapt and develop various microscopy methods, including expansion microscopy, correlative protein probes based on our nanoparticle technology, ChromEMT as well as complex analysis pipelines based on our TubuleJ software.
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3D reconstructions of 13 protofilament microtubules assembled in the presence of GDP-BeF3- (left), GTP (middle), and GMPCPP (right), using the TubuleJ software. Taken from Estévez-Gallego, Josa-Prado, Ku et al., eLife, 2020. |
Expansion microscopy of X. laevis (left) and X. tropicalis (right) egg extract spindles, imaged on a conventional microscope with a 40x air objective. Scale bar is 40 µm. |
Related publications
A Fourier-based method for detecting curved microtubule centers: Application to straightening of cryo-electron microscope images.
Blestel S, Kervrann C, Chrétien D
Proceedings of the IEEE International Symposium on Biomedical Imaging: From Nano to Macro., Boston. 2009, June 28-July 1. > pp. 298-301
Robust ligand shells for biological applications of gold nanoparticles.
Duchesne L, Gentili D, Comes-Franchini M, Fernig DG
Langmuir, 2008 Dec.
