An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture.
An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture.
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DOI：
10.7554/elife.32418
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发表时间：
2018-01-11
期刊：
影响因子：
7.7
通讯作者：
Salmon ED
中科院分区：
文献类型：
--
作者：
Suzuki A;Long SK;Salmon ED
三维双色荧光共定位具有在纳米尺度上实现精确测量的潜力。在这里，我们优化了一种3D荧光共定位方法，该方法使用色差校正的平均值来产生单个人体运动中心内绿色和红色荧光标记的蛋白质表位之间的平均分离精度约10 nm。准确度关键取决于在荧光质心测定中实现小的标准偏差、整个测量领域的色差和盖片厚度。计算机模拟表明，这些参数的较大标准偏差显著增加了3D测量值，使其偏离真实值。我们的3D结果表明，在中期，内部着丝粒内的CENP-A与外部着丝粒内的Ndc80复合体的微管结合区域之间的蛋白质连接平均为~90 nm。Ndc80复合体在中期完全伸展，在体内表现出与体外结晶学相同的亚基结构。
Two-color fluorescence co-localization in 3D (three-dimension) has the potential to achieve accurate measurements at the nanometer length scale. Here, we optimized a 3D fluorescence co-localization method that uses mean values for chromatic aberration correction to yield the mean separation with ~10 nm accuracy between green and red fluorescently labeled protein epitopes within single human kinetochores. Accuracy depended critically on achieving small standard deviations in fluorescence centroid determination, chromatic aberration across the measurement field, and coverslip thickness. Computer simulations showed that large standard deviations in these parameters significantly increase 3D measurements from their true values. Our 3D results show that at metaphase, the protein linkage between CENP-A within the inner kinetochore and the microtubule-binding domain of the Ndc80 complex within the outer kinetochore is on average ~90 nm. The Ndc80 complex appears fully extended at metaphase and exhibits the same subunit structure in vivo as found in vitro by crystallography.
影响因子：
2
作者：
Fuller CJ;Straight AF
通讯作者：
Straight AF