Abstract
During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms.
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Acknowledgments
We thank all members of Alex McDougall’s and Remi Dumollard’s lab and Stefania Castagnetti’s lab at LBDV, IMEV in Villefranche-sur-mer, France, Julien Dumont’s lab in Institut Jacques Monod in Paris and Anna Castro’s and Thierry Lorca’s lab at CRBM in Montpellier, France. We are grateful to the Clytia team especially Evelyn Houliston and Tsuyoshi Momose at IMEV for their assistance and for providing Clytia hemisphaerica gametes. We thank Carsten Janke’s lab (Institut Curie, Orsay, France) for providing help with tubulin purification and Lydia Besnardeau (LBDV, IMEV) for cloning the pET11-mouseH2B-RFP-6His plasmid. We are also grateful to Christian Sardet for fruitful discussions and his inspiring comments. This work benefited from access to the Institut de la Mer de Villefranche including the Imaging Platform PIM (member of MICA microscopy platform), an EMBRC-France and EMBRC-ERIC Site. Financial support was provided by ANR-10-INBS-02. The project was initiated thanks to EMBRC FR – AAP2019 – n° 3238 (B. Lacroix). The work was funded by ANR MTDiSco ANR-20-CE13-0033 (B. Lacroix) and the European Research Council ERC-CoG ChromoSOMe 819179 (J. Dumont).
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Chenevert, J. et al. (2024). Measuring Mitotic Spindle and Microtubule Dynamics in Marine Embryos and Non-model Organisms. In: Castro, A., Lacroix, B. (eds) Cell Cycle Control. Methods in Molecular Biology, vol 2740. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3557-5_12
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DOI: https://doi.org/10.1007/978-1-0716-3557-5_12
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