The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins

Abstract

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.

Genome-wide subcellular protein localisation in the flagellate parasite Trypanosoma brucei

“…T. brucei has a number of life cycle developmental forms with characteristic morphologies and substantial remodelling of the cortical cytoskeleton is required for their generation. We identified 10 subdomains of the cortical cytoskeleton (Figure S4D,E), greatly extending the previous discovery of a posterior-ventral domain marked by PAVE1 (Tb927.8.2030) 67,68 , with particular complexity seen in the anterior and posterior tip subdomains. Like the flagellum tip, the cortical cytoskeleton posterior tip maintained its protein cohort over the cell cycle.…”

“…brucei flagellar-driven motility is critical for virulence in the mammalian host and development in the tsetse fly 64,65 . The flagellum and other cytoskeleton-based structures have important functions associated with specific subdomains 25,46,47,[66][67][68] and we identified numerous proteins associated with axoneme, cortical cytoskeleton and FAZ subdomains (Figure S4). In particularly, we showed that the flagellum tip was a complex structure (Figure S4A).…”

Endocytosis inTrypanosoma cruziDepends on Proper Recruitment and Regulation of Functionally Redundant Myosin Motors

“…In order to localize MyAP with greater precision within the SPC itself, we conducted expansion microscopy on T. cruzi epimastigotes combined with an IFA against the Ty-epitope using our MyAP-Ty complemented line. Utilizing the TAT-1 anti-α-tubulin antibody (26, 27) to highlight parasite microtubules, we were able to demonstrate that MyAP-Ty specifically targets to the microtubule rootlets rather than the membrane tubule of the SPC ( Figure 3E left panel). In addition, we examined the localization of both MyoF-Ty and MyoC-Ty using this same methodology and found that these motors also associated with the SPC microtubules ( Figure 3E middle and right panels respectively) thus validating previous observations of MyoF using immuno-electron microscopy (28).…”

Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi

“…The reduction of cell size was an unexpected result, though the shape of trypanosomatids depends on subpellicular microtubules (SM) (Sinclair et al, 2021), and T. brucei cell motility seems to shape SM (Sun et al, 2018). Sun et al showed that flagellar detachment caused by RNAi of FLA1BP leads to abnormal SM handedness compared with parasites with a normal flagellar wave.…”