UC Davis

Cellular morphology plays a key role in cellular function, and cytoskeletal elements are key to defining and maintaining cell shape. Eukaryotic parasites of humans and other animals often possess unique microtubule (MT) organelles. Understanding how these distinctive cytoskeletal features are built and maintained may help us in our battle against these parasites. One source of MT organization is the Striated Fibers (SFs), filamentous structures often found in association with MTs and present across diverse eukaryotic clades. Notably, SFs possess the ability to self-assemble, which extends the possibility that these structures help drive cellular organization. Comprising SFs are a family of proteins called Striated Fiber Assemblins (SFAs).Giardia lamblia is a protist parasite that utilizes a combination of MTs and SFAs to form a unique organelle called the ventral disc. Giardia colonizes the small intestine and causes diarrheal disease worldwide. Motile trophozoites attach to the extracellular surface of intestinal villi with the cup-shaped ventral disc. A current model of attachment proposes that a flexible disc modulates its dome shape to create a seal on the host cell surface. The base of the ventral disc is a highly ordered and complex spiral MT array. The microribbon-crossbridge (MR-CB) complex, a novel protein complex, binds to the disc MTs at regular intervals, almost completely coating all MT protofilaments. The three Giardia SFA homologs localize to the MRs; their role and the functional and structural roles of the MR-CB complex has remained unknown. During interphase, the disc is hyperstable and has limited MT dynamics, and it remains unclear how the Giardia SFAs, or other disc-associated proteins (DAPs) confer these properties. To better understand SFAs through their evolutionary history, we have undertaken a phylogenetic analysis of this protein family, and describe three primary groups which we label Group I, Group II, and Group III. The presence of SFA homologs in the majority of flagellated supergroup lineages implies that SFA homologs were present in the last universal common ancestor and subsequently lost in several linages. SFA structure is highly conserved among excavates, which may indicate that the role of SFAs in this clade is also conserved. To investigate mechanisms of disc MT hyperstability, we screened 14 CRISPRi-mediated DAP knockdown (KD) strains for defects in hyperstability and MT dynamics, and 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 that at least two of the known DAPs confer hyperstable properties to the disc MTs. Additionally, we show that SFAs in the MR-CB complex play a role in maintaining disc spiral structure and stabilizing disc conformation required for parasite attachment. We create stable CRISPR knockouts (KO) of the three MR SFA proteins – beta-giardin, delta-giardin, and SALP1 – and evaluate mutant disc structure and function with light microscopy and biophysical attachment assays. Functional studies of the MR-CB complex have been hampered by the small number of known MR-CB proteins. Therefore, we conducted a co-immunoprecipitation assay on beta-giardin and delta-giardin to identify MR-CB candidate proteins. We localized these candidates using fluorescent tags and targeted them with CRISPRi KD and CRISPR KO. A protein called 15376 is potentially a new MR protein. Understanding the MR-CB complex will shed light on the disc as a whole and guides us towards key insights into how the disc functions during attachment. This work also contributes to our knowledge of how cells construct and maintain complex MT organelles and helps to define the role of SFAs in complex MT structures.