An efficient cumate-inducible system for procyclic and bloodstream form Trypanosoma brucei.

In Trypanosoma brucei, the tetracycline-inducible system enables tightly-regulated, highly-efficient expression of recombinant proteins or double-stranded RNA in both procyclic and bloodstream form cells, providing useful molecular genetic tools to study gene functions. An alternative, vanillic acid-inducible system is recently described for procyclic T. brucei, providing ∼18-fold increase in GFP reporter expression upon induction (Sunter JD. Mol. Biochem. Parasitol. 2016, 207:45-48). Here we describe a cumate-inducible system that allows efficient, tunable gene expression showing >300-fold increase in GFP expression upon induction. The cumate-inducible system can be used alone or together with the tetracycline-inducible system, in both procyclic and bloodstream form T. brucei. Efficient cumate-inducible expression is also achieved in T. brucei-infected mice.

The bi-lobe-associated LRRP1 regulates Ran activity in Trypanosoma brucei.

Cilia and flagella are conserved eukaryotic organelles important for motility and sensory. The RanGTPase, best known for nucleocytoplasmic transport functions, may also play a role in protein trafficking into the specialized flagellar/ciliary compartments, although the regulatory mechanisms controlling Ran activity at the flagellum remain unclear. The unicellular parasite Trypanosoma brucei contains a single flagellum necessary for cell movement, division and morphogenesis. Correct flagellum functions require flagellar attachment to the cell body, which is mediated by a specialized flagellum attachment zone (FAZ) complex that is assembled together with the flagellum during the cell cycle. We have previously identified the leucine-rich-repeat protein 1 LRRP1 on a bi-lobe structure at the proximal base of flagellum and FAZ. LRRP1 is essential for bi-lobe and FAZ biogenesis, consequently affecting flagellum-driven cell motility and division. Here, we show that LRRP1 forms a complex with Ran and a Ran-binding protein, and regulates Ran-GTP hydrolysis in T. brucei. In addition to mitotic inhibition, depletion of Ran inhibits FAZ assembly in T. brucei, supporting the presence of a conserved mechanism that involves Ran in the regulation of flagellum functions in an early divergent eukaryote.

Trypanosoma brucei FKBP12 differentially controls motility and cytokinesis in procyclic and bloodstream forms.

FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.

Biochemical characterization of the bi-lobe reveals a continuous structural network linking the bi-lobe to other single-copied organelles in Trypanosoma brucei.

Trypanosoma brucei, a unicellular parasite, contains several single-copied organelles that duplicate and segregate in a highly coordinated fashion during the cell cycle. In the procyclic stage, a bi-lobed structure is found adjacent to the single ER exit site and Golgi apparatus, forming both stable and dynamic association with other cytoskeletal components including the basal bodies that seed the flagellum and the flagellar pocket collar that is critical for flagellar pocket biogenesis. To further understand the bi-lobe and its association with adjacent organelles, we performed proteomic analyses on the immunoisolated bi-lobe complex. Candidate proteins were localized to the flagellar pocket, the basal bodies, a tripartite attachment complex linking the basal bodies to the kinetoplast, and a segment of microtubule quartet linking the flagellar pocket collar and bi-lobe to the basal bodies. These results supported an extensive connection among the single-copied organelles in T. brucei, a strategy employed by the parasite for orderly organelle assembly and inheritance during the cell cycle.