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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Mitchison T, Kirschner M (1984) Dynamic instability of microtubule growth. Nature 312:237–242
Walker RA, O’Brien ET, Pryer NK et al (1988) Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. J Cell Biol 107:1437–1448
Shelden E, Wadsworth P (1993) Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific. J Cell Biol 120:935–945
Zwetsloot AJ, Tut G, Straube A (2018) Measuring microtubule dynamics. Essays Biochem 62:725–735. https://doi.org/10.1042/EBC20180035
Verde F, Dogterom M, Stelzer E et al (1992) Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. J Cell Biol 118:1097–1108
Minden JS, Agard DA, Sedat JW, Alberts BM (1989) Direct cell lineage analysis in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos. J Cell Biol 109:505–516. https://doi.org/10.1083/jcb.109.2.505
Kellogg DR, Mitchison TJ, Alberts BM (1988) Behaviour of microtubules and actin filaments in living Drosophila embryos. Development 103:675–686. https://doi.org/10.1242/dev.103.4.675
Wadsworth P, Sloboda RD (1983) Microinjection of fluorescent tubulin into dividing sea urchin cells. J Cell Biol 97:1249–1254. https://doi.org/10.1083/jcb.97.4.1249
Hamaguchi Y, Toriyama M, Sakai H, Hiramoto Y (1985) Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage. J Cell Biol 100:1262–1272. https://doi.org/10.1083/jcb.100.4.1262
Salmon ED, Leslie RJ, Saxton WM et al (1984) Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. J Cell Biol 99:2165–2174. https://doi.org/10.1083/jcb.99.6.2165
Castoldi M, Popov AV (2003) Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr Purif 32:83–88. https://doi.org/10.1016/S1046-5928(03)00218-3
Hyman AA (1991) Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence. J Cell Sci Suppl 14:125–127
Prodon F, Chenevert J, Hébras C et al (2010) Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors. Development 137:2011–2021. https://doi.org/10.1242/dev.047845
Lacroix B, Letort G, Pitayu L et al (2018) Microtubule dynamics scale with cell size to set spindle length and assembly timing. Dev Cell 45:496–511 e6. https://doi.org/10.1016/j.devcel.2018.04.022
Yasuo H, McDougall A (2018) Practical guide for ascidian microinjection: phallusia mammillata. Adv Exp Med Biol 1029:15–24. https://doi.org/10.1007/978-981-10-7545-2_3
Lacroix B, Bourdages KG, Dorn JF et al (2014) In situ imaging in C. elegans reveals developmental regulation of microtubule dynamics. Dev Cell 29:203–216. https://doi.org/10.1016/j.devcel.2014.03.007
Lacroix B, Maddox AS (2014) Microtubule dynamics followed through cell differentiation and tissue biogenesis in C. elegans. Worm 3:e967611. https://doi.org/10.4161/21624046.2014.967611
Jaffe LA, Terasaki M (2004) Quantitative microinjection of oocytes, eggs, and embryos. Methods Cell Biol 74:219–242
Belmont LD, Hyman AA, Sawin KE, Mitchison TJ (1990) Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62:579–589
Verde F, Labbe JC, Doree M, Karsenti E (1990) Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature 343:233–238. https://doi.org/10.1038/343233a0
Mitchison TJ, Ishihara K, Nguyen P, Wuhr M (2015) Size scaling of microtubule assemblies in early xenopus embryos. Cold Spring Harb Perspect Biol 7:a019182. https://doi.org/10.1101/cshperspect.a019182
Rieckhoff EM, Berndt F, Elsner M et al (2020) Spindle scaling is governed by cell boundary regulation of microtubule nucleation. Curr Biol 30:4973–4983.e10. https://doi.org/10.1016/j.cub.2020.10.093
Srayko M, Kaya A, Stamford J, Hyman AA (2005) Identification and characterization of factors required for microtubule growth and nucleation in the early C. elegans embryo. Dev Cell 9:223–236. https://doi.org/10.1016/j.devcel.2005.07.003
Li G, Moore JK (2020) Microtubule dynamics at low temperature: evidence that tubulin recycling limits assembly. Mol Biol Cell 31:1154–1166. https://doi.org/10.1091/mbc.E19-11-0634
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3557-5_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3556-8
Online ISBN: 978-1-0716-3557-5
eBook Packages: Springer Protocols