Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis

paper

mitosis

microscopy

joint first author

Author

Nixon FM, Honnor TR, Clarke NI, Starling GP, Beckett AJ, Johansen AM, Brettschneider JA, Prior IA, Royle SJ.

Doi

https://doi.org/10.1242/jcs.203877

Citation

Nixon, F.M., Honnor, T.R., Clarke, N.I., Starling, G.P., Beckett, A.J., Johansen, A.M., Brettschneider, J.A., Prior, I.A., and Royle, S.J. (2017). Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis. J Cell Sci 130, 1845–1855.

Abstract

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; and fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of microtubules within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle.