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First published online 8 November 2005
doi: 10.1242/jcs.02659

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The Parkin co-regulated gene product, PACRG, is an evolutionarily conserved axonemal protein that functions in outer-doublet microtubule morphogenesis

Helen R. Dawe^*, Helen Farr^*, Neil Portman, Michael K. Shaw and Keith Gull

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK

View larger version (76K): [in a new window]   Fig. 1. Two PACRG paralogues in T. brucei. Protein sequence alignment of two T. brucei PACRG-like proteins (Tb03.4808.210 and Tb09.210.1470) with gene products from M. musculus (XP_128418), C. reinhardtii (C_20334), D. melanogaster (CG179349 and CG15120) and C. elegans (NP_495496). Orthology was determined by examination of candidate sequences including reciprocal BLAST. Identical residues are shown in dark blue; similar residues are shown in light blue.

View larger version (31K): [in a new window]   Fig. 2. Ablation of PACRG AB produces a slow-growth phenotype. Q-PCR data (A,C,E) and representative growth curves (B,D,F) of cells undergoing RNAi of TbPACRG A (A,B), TbPACRG B (C,D) or TbPACRG AB (E,F). A reduction of mRNA signal of TbPACRG A (black bars) was seen in RNAi TbPACRG A-treated cells (A). However, there was no alteration in growth kinetics in TbPACRG A RNAi-treated cells (B, triangles) compared to non-induced controls (B, squares). A reduction of mRNA signal of TbPACRG B (grey bars) was observed in RNAi TbPACRG B-induced cells (C), but again, no alteration in growth kinetics was observed compared to the control (D). A reduction of mRNA signal of TbPACRG A (black bars) and TbPACRG B (grey bars) was observed in TbPACRG AB RNAi-treated cells (E) with the representative growth curve (F) showing a growth defect starting 72 hours after induction (triangles) compared to non-induced controls (squares). For the growth curves (B,D,F), cells were maintained in log phase by diluting the culture every 48 hours. Q-PCR values are percentages (mean±s.e.m.) of the control cell signal (A,C,E).

View larger version (140K): [in a new window]   Fig. 3. Induced cells build a flagellum, however daughter kinetoplast position is impaired. Phase-contrast images of control cells (A,B) and TbPACRG AB RNAi-induced cells at 72 hours (C-F) with nuclear and kinetoplast DNA overlaid in blue. (A) A non-induced cell with one kinetoplast and one nucleus (1K1N). (B) A non-induced cell with duplicated nucleus and kinetoplast (2K2N cell) showing correct positioning of both kinetoplasts. (C) A TbPACRG AB RNAi-induced cell with correct positioning of the kinetoplast. (D) A TbPACRG AB RNAi-induced cell undergoing division where the distance between the daughter kinetoplast (arrow) and the daughter nucleus is reduced compared to that of the parent cell. Note the formation of the new flagellum (NF). (E) A TbPACRG AB RNAi-induced 1K1N cell where the kinetoplast is mislocalised. (F) A TbPACRG AB RNAi-induced cell undergoing cytokinesis where the daughter kinetoplast (arrow) is mispositioned, anterior to the daughter nucleus. Bar, 5 µm.

View larger version (46K): [in a new window]   Fig. 4. PACRG AB RNAi induction causes loss of outer-doublet microtubules. (A) Transmission electron microscopy images of TbPACRG AB non-induced cells (a) and RNAi-induced cells 72 hours after induction (b-d). Note the loss of variable numbers of outer-doublet microtubules in b-d. (B) Drawing of relevant structures in a T. brucei flagellum. Numbers refer to the convention of numbering outer doublets according to their position relative to the central pair and PFR. (C) Graph of frequency of loss of specific outer-doublet microtubules. Bar, 200 nm.

View larger version (38K): [in a new window]   Fig. 5. Multiple outer-doublet loss occurs more frequently at the anterior of the cell. (A-E) The number of outer doublets was quantified at various positions along the axoneme. Graphs show the number of outer doublets present plotted against the frequency of occurrence in flagella profiles observed. Inset images show representative micrographs of flagella profiles at each position. The arrow in D indicates the break between two outer doublets that is observed prior to doublet loss. (F) Drawing of a T. brucei cell indicating the positions along the flagellum quantified in A-E.

View larger version (49K): [in a new window]   Fig. 6. The A and B tubules terminate simultaneously. Serial electron micrographs (left to right) showing loss of an outer doublet along a single axoneme. The black arrows indicate the position of the doublet that will be lost. Note the electron-dense lumen in the A tubule (red arrow) immediately prior to doublet loss. Bar, 200 nm.

View larger version (121K): [in a new window]   Fig. 7. GFP-TbPACRG fusion proteins localise to the axoneme. Phase-contrast microscopy (A,C,E,G) and fluorescence (B,D,F,H) images of detergent-extracted trypanosome cytoskeletons. DNA is labelled with DAPI (blue), and GFP is identified by anti-GFP polyclonal antibody (green). (A,B) Cells expressing GFP-TbPACRG A. (C,D) Cells expressing GFP-TbPACRG B. (E,F) Cells expressing GFP-TbPACRG A where the PFR is identified by the L8C4, anti-PFR, monoclonal antibody (red). Note the GFP signal extends proximal to the PFR staining (F, arrow). (G,H) Cells expressing GFP-TbPACRG A where the distal end of the basal body is identified by the BBA4 monoclonal antibody (red). Bar, 5 µm.