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J Biomed Opt. 2015 Oct;20(10):101206. doi: 10.1117/1.JBO.20.10.101206.

Three-dimensional tracking of plus-tips by lattice light-sheet microscopy permits the quantification of microtubule growth trajectories within the mitotic apparatus.

Author information

1
Center for Advanced Photonics, Image Processing Research Team, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan.
2
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia 20147, United States.
3
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia 20147, United StatescResearch Center for Applied Sciences, Academia Sinica, 128 Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan.
4
Cellular Dynamics Analysis Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan.

Abstract

Mitotic apparatus, which comprises hundreds of microtubules, plays an essential role in cell division, ensuring the correct segregation of chromosomes into each daughter cell. To gain insight into its regulatory mechanisms, it is essential to detect and analyze the behavior of individual microtubule filaments. However, the discrimination of discrete microtubule filaments within the mitotic apparatus is beyond the capabilities of conventional light microscopic technologies. Recently, we detected three-dimensional (3-D) microtubule growth dynamics within the cellular cytoplasmic space using lattice light-sheet microscopy in conjunction with microtubule growth marker protein end-binding 1, a microtubule plus-end-tracking protein, which was fused to green fluorescent protein (EB1-GFP). This technique enables high-resolution 3-D imaging at subsecond intervals. We adapted mathematical computing and geometric representation techniques to analyze spatial variations in microtubule growth dynamics within the mitotic spindle apparatus. Our analytical approach enabled the different dynamic properties of individual microtubules to be determined, including the direction and speed of their growth, and their growth duration within a 3-D spatial map. Our analysis framework provides an important step toward a more comprehensive understanding of the mechanisms driving cellular machinery at the whole-cell level.

PMID:
26527322
DOI:
10.1117/1.JBO.20.10.101206
[Indexed for MEDLINE]

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