Giardia lamblia, a widespread parasitic protozoan, attaches to the host gastrointestinal epithelium by using the ventral disc, a complex microtubule (MT) organelle. The ‘cup-like’ disc is formed by a spiral MT array that scaffolds numerous disc-associated proteins (DAPs) and higher-order protein complexes. In interphase, the disc is hyperstable and has limited MT dynamics; however, it remains unclear how DAPs confer these properties. To investigate mechanisms of hyperstability, we confirmed the disc-specific localization of over 50 new DAPs identified by using both a disc proteome and an ongoing GFP localization screen. DAPs localize to specific disc regions and many lack similarity to known proteins. By screening 14 CRISPRi-mediated DAP knockdown (KD) strains for defects in hyperstability and MT dynamics, we identified two strains – DAP5188KD and DAP6751KD –with discs that dissociate following high-salt fractionation. Discs in the DAP5188KD strain were also sensitive to treatment with the MT-polymerization inhibitor nocodazole. Thus, we confirm here that at least two of the 87 known DAPs confer hyperstable properties to the disc MTs, and we anticipate that other DAPs contribute to disc MT stability, nucleation and assembly.

贾第鞭毛虫是一种广泛存在的寄生原生动物，通过使用腹盘（一种复杂的微管 (MT) 细胞器）附着在宿主胃肠上皮上。“杯状”圆盘由螺旋 MT 阵列形成，该阵列支撑着众多圆盘相关蛋白 (DAP) 和高级蛋白质复合物。在相间，椎间盘是超稳定的并且具有有限的 MT 动力学；然而，目前尚不清楚 DAP 如何赋予这些特性。为了研究超稳定性的机制，我们通过使用椎间盘蛋白质组和正在进行的 GFP 定位屏幕确认了超过 50 个新 DAP 的椎间盘特异性定位。DAP 定位于特定的椎间盘区域，许多与已知蛋白质缺乏相似性。通过筛选 14 个 CRISPRi 介导的 DAP 敲低 (KD) 菌株的超稳定性和 MT 动力学缺陷，我们鉴定了两种菌株——DAP5188KD 和 DAP6751KD——它们的圆盘在高盐分馏后分离。DAP5188KD 菌株中的椎间盘也对用 MT 聚合抑制剂诺考达唑处理敏感。因此，我们在此确认 87 种已知 DAP 中至少有两种赋予椎间盘 MT 超稳定特性，并且我们预计其他 DAP 有助于椎间盘 MT 的稳定性、成核和组装。