Disc-associated proteins (DAPs) mediate the unusual hyperstability of Giardia's ventral disc

Giardia lamblia, a widespread parasitic protozoan, attaches to the host gastrointestinal epithelium 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 might confer these properties. To investigate mechanisms of hyperstability, we confirmed the disc-specific localization of over 50 new DAPs identified using both a disc proteome and an ongoing GFP localization screen. DAPs localize to specific disc regions, and many lack homology 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 drug nocodazole. Thus, while we confirm that at least two of 87 known DAPs confer hyperstable properties to the disc MTs, we anticipate that other DAPs contribute to disc MT stability, nucleation, and assembly.