Cytoplasmic preassembly of the flagellar outer dynein arm complex in Trypanosoma brucei

Abstract

The outer dynein arm (ODA) is a large, multimeric protein complex essential for ciliary motility. The composition and assembly of ODA are best characterized in the green algae Chlamydomonas reinhardtii, where individual ODA subunits are synthesized and preassembled into a mature complex in the cytosol prior to ciliary import. The single-cellular parasite Trypanosoma brucei contains a motile flagellum essential for cell locomotion and pathogenesis. Similar to human motile cilia, T. brucei flagellum contains a two-headed ODA complex arranged at 24 nm intervals along the axonemal microtubule doublets. The subunit composition and the preassembly of the ODA complex in T. brucei, however, have not been investigated. In this study, we affinity-purified the ODA complex from T. brucei cytoplasmic extract. Proteomic analyses revealed the presence of two heavy chains (ODAα and ODAβ), two intermediate chains (IC1and IC2) and several light chains. We showed that both heavy chains and both intermediate chains are indispensable for flagellar ODA assembly. Our study also provided biochemical evidence supporting the presence of a cytoplasmic, preassembly pathway for T. brucei ODA.

FIGURE 1:

Depletion of ODAα reduces flagellar ODA. (A–C) Cells with stable expression of Ty:mNG:Ty tagged IC1, IC2, or ODAβ were induced for ODAα-RNAi for 48 h. Flagellar ODA components were visualized in RNAi and control (uninduced) cells by immunofluorescence using an antibody directed against the Ty tag. (D–F) Quantitation of flagellar IC1, IC2, and ODAβ intensity, reported by anti-Ty. Mean flagellum intensity was measured along a 1-µm segment (spline size = 5) at the free-hanging, distal end of the trypanosome flagellum (arrowhead). As control, PFR1 intensity along the flagellum was also measured. The results were presented as scatter diagrams with the bar representing the median. In D, N = 94 for control and N = 100 for ODAα-RNAi cells. In E, N = 100 for control, and N = 93 for ODAα-RNAi cells. In F, N = 93 for control, and N = 86 for ODAα-RNAi cells.

FIGURE 2:

Depletion of ODAα leads to accumulation of IC1 and IC2 in the detergent-soluble fraction. Indicated T. brucei cells were induced for RNAi for 48 h, followed by extraction with PEME buffer containing 1% NP-40. Whole cell lysate (w), detergent-insoluble pellet (p), and detergent-soluble supernatant (s) were fractionated by SDS–PAGE. The distribution of specified ODA components in RNAi and control cells was examined by anti-Ty. The flagellar cytoskeletal protein PFR1 was also labeled as a loading and extraction control.

FIGURE 3:

IC1 is present in different complexes in T. brucei cytosol. (A) Cytoplasmic extracts from T. brucei cells coexpressing Ty:mNG:Ty:ODAα and Ty:mNG:Ty:IC1 (top panels), or cells expressing Ty:mNG:Ty:ODAβ only (bottom panels) were fractionated by 5–25% sucrose gradient ultracentrifugation. The fractions were analyzed by immunoblots using anti-Ty. (B) Cells with stable expression of Ty:mNG:Ty:IC1 were induced for ODAα-RNAi for 48 h. Cytoplasmic extracts from both control and RNAi cells were analyzed by sucrose gradients and immunoblots using anti-Ty. Other than the indicated ODA components, many weaker bands were detected in some sucrose fractions shown in A and B. These may represent degradation products of the tagged ODA subunits. (C) Cytoplasmic extracts from cells with stable endogenous expression of Ty:mNG:Ty:IC1 and YFP:IC2 were fractionated by 5–25% sucrose gradient ultracentrifugation. Fractions 7–8 were pooled and incubated with GFP-Trap or empty magnetic beads. Input (10% total), proteins bound to the beads (50%), and unbound fractions (10% total) were analyzed by immunoblots. * marks a nonspecific band detected by anti-GFP (Abcam) in cell extracts but did not bind to GFP-Trap. ** marks a band not detectable in cell extracts but enriched on GFP-Trap.