Yale University, New Haven, Connecticut.
Yale University, New Haven, Connecticut.
Biophys J. 2019 Aug 20;117(4):679-687. doi: 10.1016/j.bpj.2019.07.004. Epub 2019 Jul 9.
Cilia and flagella are long, slender organelles found in many eukaryotic cells, where they have sensory, developmental, and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. These motor proteins drive motility by using energy derived from the hydrolysis of ATP to generate a bending wave, which travels down the axoneme. As a first step toward visualizing the ATPase activity of the axonemal dyneins during bending, we have investigated the kinetics of nucleotide binding to axonemes. Using a specially built ultraviolet total internal reflection fluorescence microscope, we found that the fluorescent ATP analog methylanthraniloyl ATP (mantATP), which has been shown to support axonemal motility, binds all along isolated, immobilized axonemes. By studying the recovery of fluorescence after photobleaching, we found that there are three mantATP binding sites: one that bleaches rapidly (time constant ≈ 1.7 s) and recovers slowly (time constant ≈ 44 s), one that bleaches with the same time constant but does not recover, and one that does not bleach. By reducing the dynein content in the axoneme using mutants and salt extraction, we provide evidence that the slow-recovering component, but not the other components, corresponds to axonemal dyneins. The recovery rate of this component, however, is too slow to be consistent with the activation of beating observed at higher mantATP concentrations; this indicates that the dyneins may be inhibited due to their immobilization at the surface. The development of this method is a first step toward direct observation of the traveling wave of dynein activity.
纤毛和鞭毛是长而细的细胞器，存在于许多真核细胞中，具有感觉、发育和运动功能。所有的纤毛和鞭毛都包含一个称为轴丝的基于微管的结构。在运动的纤毛和鞭毛中，轴丝在其大部分长度上都含有来自轴丝动力蛋白家族的动力蛋白。这些动力蛋白通过利用来自 ATP 水解的能量产生弯曲波来驱动运动，该弯曲波沿着轴丝传播。作为可视化轴丝动力蛋白 ATP 酶活性在弯曲过程中的第一步，我们研究了核苷酸结合到轴丝的动力学。使用专门构建的紫外线全内反射荧光显微镜，我们发现荧光 ATP 类似物甲氨酰基-ATP（mantATP），已被证明支持轴丝运动，可结合到分离的固定轴丝上。通过研究光漂白后荧光的恢复，我们发现有三个 mantATP 结合位点：一个快速漂白（时间常数约为 1.7 秒）且恢复缓慢（时间常数约为 44 秒），一个以相同的时间常数漂白但不恢复，一个不漂白。通过使用突变体和盐提取减少轴丝中的动力蛋白含量，我们提供的证据表明，慢速恢复的组分（而不是其他组分）对应于轴丝动力蛋白。然而，该组分的恢复速率太慢，与在较高 mantATP 浓度下观察到的拍打激活不一致；这表明由于它们在表面的固定，动力蛋白可能被抑制。该方法的发展是直接观察动力蛋白活性传播波的第一步。
