The Trypanosoma Brucei Subpellicular Microtubule Array Is Organized Into Functionally Discrete Subdomains Defined By Microtubule Associated Proteins

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.Author summary Many parasitic protists use arrays of microtubules that contact the inner leaflet of the plasma membrane, typically known as subpellicular microtubule arrays, to shape their cells into forms that allow them to efficiently infect their hosts. While subpellicular arrays are found in a wide range of parasites, very little is known about how they are assembled and maintained. Trypanosoma brucei, which is the causative agent of human African trypanosomiasis, has an elaborate subpellicular array that produces the helical shape of the parasite, which is essential for its ability to move within crowded and viscous solutions. We have identified a series of proteins that have a range of localization patterns within the array, which suggests that the array is regulated by subdomains of array-associated proteins that likely tune the local properties of the microtubules to suit the stresses found at different parts of the cell body. These results establish a foundation to understand how trypanosomatid subpellicular arrays are built, shaped, and maintained, which has not previously been considered.