Yamashita Norio, Morita Masahiko, Legant Wesley R, Chen Bi-Chang, Betzig Eric, Yokota Hideo, Mimori-Kiyosue Yuko
Center for Advanced Photonics, Image Processing Research Team, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan.
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia 20147, United States.
J Biomed Opt. 2015 Oct;20(10):101206. doi: 10.1117/1.JBO.20.10.101206.
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.
有丝分裂装置由数百根微管组成，在细胞分裂中起着至关重要的作用，确保染色体正确分离到每个子细胞中。为深入了解其调控机制，检测和分析单个微管丝的行为至关重要。然而，区分有丝分裂装置内离散的微管丝超出了传统光学显微镜技术的能力范围。最近，我们结合微管生长标记蛋白末端结合蛋白1（一种微管正端追踪蛋白，与绿色荧光蛋白融合，即EB1-GFP），利用晶格光片显微镜检测了细胞质空间内的三维（3-D）微管生长动态。该技术能够以亚秒级间隔进行高分辨率三维成像。我们采用数学计算和几何表示技术来分析有丝分裂纺锤体装置内微管生长动态的空间变化。我们的分析方法能够确定单个微管的不同动态特性，包括其生长方向和速度以及在三维空间图中的生长持续时间。我们的分析框架朝着在全细胞水平上更全面地理解驱动细胞机制的机制迈出了重要一步。
