Department of Physics and Astronomy, College of Science, Clemson University, Clemson, South Carolina, United States of America.
Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, South Carolina, United States of America.
PLoS Pathog. 2022 Sep 26;18(9):e1009984. doi: 10.1371/journal.ppat.1009984. eCollection 2022 Sep.
Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids. Trypanosoma brucei flagella beat with a bending wave that propagates from the flagellum's tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein-associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex-type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi in both wild-type and FLAM3, a flagellar attachment zone protein, knockdown cells and quantified TbLC2's effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to-tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids' unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally.
鞭毛运动对于致病锥虫的细胞形态、活力和毒力至关重要。布氏锥虫的鞭毛以弯曲波的形式运动，这种波从鞭毛的尖端传播到底部，而不是像其他真核生物那样从底部到尖端。数以千计的动力蛋白马达蛋白协调其活动，以驱动纤毛弯曲波的传播。动力蛋白相关的轻链和中间链调节轴丝动力蛋白的生物物理机制。Tctex 型外臂动力蛋白轻链 2（LC2）调节莱茵衣藻鞭毛弯曲波的传播方向、幅度和频率。然而，Tctex 型轻链在调节 T. brucei 运动中的作用尚不清楚。在这里，我们使用生物信息学、原位分子标记和免疫荧光显微镜相结合的方法，在布氏锥虫的前鞭毛体（procyclic form）中鉴定出一种 Tctex 型轻链（TbLC2）。我们使用 RNAi 敲低野生型和鞭毛附着区蛋白 FLAM3 敲低细胞中的 TbLC2 表达，并量化 TbLC2 对锥虫细胞生物学和生物物理学的影响。我们发现，TbLC2 敲低降低了锥虫细胞游动的定向持久性，诱导了不对称的纤毛弯曲波形，调节了基底到尖端和尖端到基底拍打模式之间的偏向，并增加了拍打频率。总的来说，我们的发现与 TbLC2 作为轴丝动力蛋白活性的下调因子的模型一致，该模型稳定了锥虫细胞特有的从尖端到底部的纤毛拍打波形。我们的工作揭示了轴丝动力蛋白调节机制，这些机制有助于致病锥虫独特的从尖端到底部的纤毛拍打性质，以及这些机制如何更普遍地影响动力蛋白驱动的纤毛运动。
