Abstract
The development of automatic tools for the three-dimensional reconstruction of the microtubule cytoskeleton is crucial for large-scale analysis of mitotic spindles. Recently, we have published a method for the semiautomatic tracing of microtubules based on 3D template matching (Weber et al., J Struct Biol 178:129–138, 2012). Here, we give step-by-step instructions for the automatic tracing of microtubules emanating from centrosomes in the early mitotic Caenorhabditis elegans embryo. This approach, integrated in the visualization and data analysis software Amira, is applicable to tomographic data sets from other model systems.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wuhr M, Chen Y, Dumont S, Groen AC, Needleman DJ, Salic A, Mitchison TJ (2008) Evidence for an upper limit to mitotic spindle length. Curr Biol 18(16):1256–1261. doi:10.1016/j.cub.2008.07.092
Maddox PS, Bloom KS, Salmon ED (2000) The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae. Nat Cell Biol 2(1):36–41. doi:10.1038/71357
Heath IB (1980) Variant mitoses in lower eukaryotes: indicators of the evolution of mitosis. Int Rev Cytol 64:1–80
O'Toole ET, Winey M, McIntosh JR (1999) High-voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae. Mol Biol Cell 10(6):2017–2031
Winey M, Mamay CL, O'Toole ET, Mastronarde DN, Giddings TH Jr, McDonald KL, McIntosh JR (1995) Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J Cell Biol 129(6): 1601–1615
O’Toole ET, McDonald KL, Mantler J, McIntosh JR, Hyman AA, Muller-Reichert T (2003) Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans. J Cell Biol 163(3):451–456. doi:10.1083/jcb.200304035
Dong Y, Vanden Beldt KJ, Meng X, Khodjakov A, McEwen BF (2007) The outer plate in vertebrate kinetochores is a flexible network with multiple microtubule interactions. Nat Cell Biol 9(5):516–522. doi:10.1038/ncb1576
Mastronarde DN, McDonald KL, Ding R, McIntosh JR (1993) Interpolar spindle microtubules in PTK cells. J Cell Biol 123(6 Pt 1): 1475–1489
McDonald KL, O’Toole ET, Mastronarde DN, McIntosh JR (1992) Kinetochore microtubules in PTK cells. J Cell Biol 118(2):369–383
McDonald KL, Morphew M, Verkade P, Muller-Reichert T (2007) Recent advances in high-pressure freezing: equipment- and specimen-loading methods. Methods Mol Biol 369:143–173
Muller-Reichert T, Srayko M, Hyman A, O’Toole ET, McDonald K (2007) Correlative light and electron microscopy of early Caenorhabditis elegans embryos in mitosis. Methods Cell Biol 79:101–119. doi:10.1016/S0091-679X(06)79004-5
Verkade P (2008) Moving EM: the rapid transfer system as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing. J Microsc 230(Pt 2):317–328. doi:10.1111/j.1365-2818.2008.01989.x
McEwen BF, Marko M (2001) The emergence of electron tomography as an important tool for investigating cellular ultrastructure. J Histochem Cytochem 49(5):553–564
McIntosh R, Nicastro D, Mastronarde D (2005) New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol 15(1):43–51. doi:10.1016/j.tcb.2004.11.009
Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1):71–76. doi:10.1006/jsbi.1996.0013
Weber B, Greenan G, Prohaska S, Baum D, Hege HC, Muller-Reichert T, Hyman AA, Verbavatz JM (2012) Automated tracing of microtubules in electron tomograms of plastic embedded samples of Caenorhabditis elegans embryos. J Struct Biol 178(2):129–138. doi:10.1016/j.jsb.2011.12.004
Stalling D, Westerhoff M, Hege H-C (2005) Amira: a highly interactive system for visual data analysis. In: Charles DH, Chris RJ (eds) Visualization handbook. Butterworth-Heinemann, Burlington, pp 749–767
McDonald K, Muller-Reichert T (2002) Cryomethods for thin section electron microscopy. Methods Enzymol 351:96–123
Muller-Reichert T, Hohenberg H, O’Toole ET, McDonald K (2003) Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure. J Microsc 212(Pt 1): 71–80
Muller-Reichert T, Mantler J, Srayko M, O'Toole E (2008) Electron microscopy of the early Caenorhabditis elegans embryo. J Microsc 230(Pt 2):297–307. doi:10.1111/j.1365-2818.2008.01985.x
Mastronarde DN (1997) Dual-axis tomography: an approach with alignment methods that preserve resolution. J Struct Biol 120(3):343–352. doi:10.1006/jsbi.1997.3919
NVIDIA (2010) NVIDIA CUDA Programming Guide 3.1. http://developer.download.nvidia.com/compute/cuda/3_1/toolkit/docs/NVIDIA_CUDA_C_ProgrammingGuide_31pdf. Accessed 04 Aug 2011
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Redemann, S. et al. (2014). The Segmentation of Microtubules in Electron Tomograms Using Amira. In: Sharp, D. (eds) Mitosis. Methods in Molecular Biology, vol 1136. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0329-0_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0329-0_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0328-3
Online ISBN: 978-1-4939-0329-0
eBook Packages: Springer Protocols