The distance that kinesin-1 holds its cargo from the microtubule surface measured by fluorescence interference contrast microscopy
The distance that kinesin-1 holds its cargo from the microtubule surface measured by fluorescence interference contrast microscopy
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DOI：
10.1073/pnas.0510400103
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发表时间：
2006-10-24
影响因子：
11.1
通讯作者：
Diez, Stefan
中科院分区：
文献类型：
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作者：
Kerssemakers, Jacob;Howard, Jonathon;Diez, Stefan
Kinesin-1 是一种运动蛋白，可携带细胞货物，例如沿着微管 (MT) 的膜结合细胞器。同二聚体运动分子包含两个 N 端运动结构域（运动“头”）、一个长卷曲螺旋结构域（“杆”或“茎”）和两个小球状“尾”结构域。关于驱动蛋白的头部如何沿着 MT 行走以及尾部如何参与货物结合和自抑制，人们已经了解了很多。然而，人们对这根棒的作用知之甚少。在这里，我们通过测量 MT 在 ATIP 存在的情况下在驱动蛋白涂层表面上滑动的高度来研究主动运输过程中杆的延伸。为了以纳米精度进行高度测量，我们使用了荧光干涉对比显微镜，该显微镜基于反射表面附近物体的荧光自干涉。使用原位校准方法，我们确定驱动蛋白-1 分子将滑动 MT 提升至表面上方 17 +/- 2 nm（平均值 +/- SEM）。当改变周围核苷酸的组成或通过枯草杆菌蛋白酶消化去除MT带负电荷的-COOH末端时，我们发现测量的距离没有显着变化。尽管这个距离明显短于运动分子的轮廓长度（大约 60 nm），但它可能足以防止蛋白质与 MT 结合或防止细胞器干扰运输。
Kinesin-1 is a motor protein that carries cellular cargo such as membrane-bounded organelles along microtubules (MTs). The homodimeric motor molecule contains two N-terminal motor domains (the motor "heads"), a long coiled-coil domain (the "rod" or "stalk"), and two small globular "tail" domains. Much has been learned about how kinesin's heads step along a MT and how the tail is involved in cargo binding and autoinhibition. However, little is known about the role of the rod. Here, we investigate the extension of the rod during active transport by measuring the height at which MTs glide over a kinesin-coated surface in the presence of ATIP. To perform height measurements with nanometer precision, we used fluorescence interference contrast microscopy, which is based on the self-interference of fluorescent light from objects near a reflecting surface. Using an in situ calibrating method, we determined that kinesin-1 molecules elevate gliding MTs 17 +/- 2 nm (mean +/- SEM) above the surface. When varying the composition of the surrounding nucleotides or removing the negatively charged -COOH termini of the MTs by subtilisin digestion, we found no significant changes in the measured distance. Even though this distance is significantly shorter than the contour length of the motor molecule (approximate to 60 nm), it may be sufficient to prevent proteins bound to the MTs or prevent the organelles from interfering with transport.