Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, TX 77030, USA.
Department of Biological Sciences, National University of Singapore, 117543, Singapore.
J Cell Sci. 2015 Jul 1;128(13):2361-72. doi: 10.1242/jcs.168377. Epub 2015 May 13.
Adhesion of motile flagella to the cell body in Trypanosoma brucei requires a filamentous cytoskeletal structure termed the flagellum attachment zone (FAZ). Despite its essentiality, the complete molecular composition of the FAZ filament and its roles in FAZ filament assembly remain poorly understood. By localization-based screening, we here identified a new FAZ protein, which we called FAZ2. Knockdown of FAZ2 disrupted the FAZ filament, destabilized multiple FAZ filament proteins and caused a cytokinesis defect. We also showed that FAZ2 depletion destabilized another new FAZ filament protein and several flagellum and cytoskeleton proteins. Furthermore, we identified CC2D and KMP11 as FAZ2 partners through affinity purification, and showed that they are each required for maintaining a stable complex. Finally, we demonstrated that FAZ filament proteins are incorporated into the FAZ filament from the proximal region, in contrast to the flagellum components, which are incorporated from the distal tip. In summary, we identified three new FAZ filament proteins and a FAZ filament protein complex, and our results suggest that assembly of the FAZ filament occurs at the proximal region and is essential to maintain the stability of FAZ filament proteins.
在布氏锥虫中，游动鞭毛与细胞体的黏附需要一种称为鞭毛附着区（FAZ）的丝状细胞骨架结构。尽管 FAZ 至关重要，但 FAZ 丝的完整分子组成及其在 FAZ 丝组装中的作用仍知之甚少。通过基于定位的筛选，我们在这里鉴定了一种新的 FAZ 蛋白，我们称之为 FAZ2。FAZ2 的敲低破坏了 FAZ 丝，使多种 FAZ 丝蛋白不稳定，并导致胞质分裂缺陷。我们还表明，FAZ2 耗竭使另一种新的 FAZ 丝蛋白和几种鞭毛和细胞骨架蛋白不稳定。此外，我们通过亲和纯化鉴定了 CC2D 和 KMP11 为 FAZ2 伴侣，并表明它们各自对于维持稳定的复合物都是必需的。最后，我们证明 FAZ 丝蛋白从近端区域掺入 FAZ 丝，而与鞭毛成分相反，鞭毛成分从远端尖端掺入。总之，我们鉴定了三种新的 FAZ 丝蛋白和一个 FAZ 丝蛋白复合物，我们的结果表明 FAZ 丝的组装发生在近端区域，对于维持 FAZ 丝蛋白的稳定性是必需的。
