Control of assembly of extra-axonemal structures: the paraflagellar rod of trypanosomes

Abstract

statementEukaryotic flagella are complex organelles. In many organisms there are extra-axonemal structures including the trypanosome paraflagellar rod. We have discovered two cytoplasmic proteins that are required for paraflagellar rod formation. AbstractEukaryotic flagella are complex microtubule based organelles and in many organisms there are extraaxonemal structures present, including the outer dense fibres of mammalian sperm and the paraflagellar rod (PFR) of trypanosomes. Flagellum assembly is a complex process occurring across three main compartments, the cytoplasm, the transition fibre-transition zone, and the flagellum. It begins with translation of protein components, followed by their sorting and trafficking into the flagellum, transport to the assembly site and then incorporation. Flagella are formed from over 500 proteins; the principles governing axonemal component assembly are relatively clear. However, the coordination and sites of extra-axonemal structure assembly processes are less clear.We have discovered two cytoplasmic proteins in T. brucei that are required for PFR formation, PFR assembly factors 1 and 2. Deletion of either PFR-AF1 or PFR-AF2 dramatically disrupted PFR formation and caused a reduction in the amount of major PFR proteins. The presence of cytoplasmic factors required for PFR formation aligns with the concept of processes occurring across multiple compartments to facilitate axoneme assembly and this is likely a common theme for extra-axonemal structure assembly.

Monoallelic antigen expression in trypanosomes requires a stage-specific transcription activator

“…These cell lines were generated using pJ1339, an expression construct which carries homology arms for integration in the tubulin locus. We have previously described the TREU927 PCF 1339 cell line 41 . To generate the Lister 427 BSF 1339 cell line, pJ1339 was linearised by restriction digest with Hind III and transfection into BSFs (see Electroporation and drug selection).…”

Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

“…We used a similar approach by C-terminally tagging one allele of the paraflagellar rod (PFR) protein PFR2 with the GFP variant mClover3 (mClv3) in SmOx 427 cells [31]. The PFR is a paracrystalline structure closely associated with the flagellum that is thought to provide additional rigidity to the axoneme to facilitate T. brucei motility [32]. Nearcomplete depletion of PFR2 causes severe motility defects in procyclic cells, but by targeting only the tagged allele with RNAi directed against mClv3, it should be possible to remove the PFR2-mClv3 from the cells selectively while leaving the wild-type (WT) pool of the protein intact [33,34].…”

Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

“…We used a similar approach by Cterminally tagging one allele of the paraflagellar rod (PFR) protein PFR2 with the GFP variant mClover3 (mClv3) in SmOx 427 cells (29). The PFR is a paracrystalline structure closely associated with the flagellum that is thought to provide additional rigidity to the axoneme to facilitate T. brucei motility (30). Near-complete depletion of PFR2 causes severe motility defects in procyclic cells, but by targeting only the tagged allele with RNAi directed against mClv3, it should be possible to remove the PFR2-mClv3 from the cells selectively while leaving the wild-type (WT) pool of the protein intact (31,32).…”

“…Cells were imaged over 24 h, imaged every 10 min with 4% LED power,30 ms exposure, and 2×2 pixel binning. A.…”

“…125 μL of cell mixture were plated as previously described. Cells were imaged over 24 h, using 40×/1.3 NA oil objective, imaging every 10 min with simultaneous split-DIC/Fluorescent imaging at 4% LED power, with 30 TbPLKas and mNeonGreen expressing 427 sentinels were incubated with 2.5 μM 3MB-PPi for 3 h, followed by plating of 1×10 6 cells from each condition in a total of 125 μL. Cells were imaged over 24 h, imaged every 10 min with 4% LED power, 30 ms exposure, and 2×2 pixel binning.…”