The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement

Abstract

AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalizes with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re-positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non-equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasize differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite.

Fig 1

tyGFP:TbAIR9 is a cytoskeletal-associated protein but is not present in the flagellum. A. Western blot of lysates of procyclic (PCF) and bloodstream (BSF) T. brucei Lister 427 wild-type (−) and 427 pHG172 (tyGFP:TbAIR9; expected size = 139 kDa) (+) cells, probed with anti-GFP antibody (upper panels). The sizes of the molecular weight markers are indicated. Lower panels: same samples probed with anti-OPB antiserum as a loading control. B and C. Fluorescence microscopy images of tyGFP:TbAIR9-expressing procyclic (B) or bloodstream (C) stage cells. Panels from left to right: DIC images, DAPI staining (blue), tyGFP:TbAIR9 (green). The number of nuclei (N) and kinetoplasts (K) per cell are indicated, and arrowheads point to the kinetoplasts. D. Cytoskeletal preparations of procyclic (PCF, upper panels) and bloodstream (BSF, lower panels) T. brucei. Image panels as for (B and C). E. (Immuno)fluorescence analysis of procyclic whole cells. From left to right: DIC image; tyGFP:AIR9 (direct fluorescence; green)/DAPI (blue); anti-β-tubulin (KMX; red)/DAPI (blue); GFP/KMX/DAPI merge. Arrows indicate flagellum with a β-tubulin signal but not tyGFP:AIR9 fluorescence. Scale bars (B–E): 5 µm. F. Subcellular fractionation of procyclic cells expressing tyGFP:TbAIR9. Western blots of fractions [whole cell lysate (WCL), supernatants (S1-3) and pellets (P1-3), see Experimental procedures for full details] were probed with anti-TY, anti-PFR1/2, anti-β-tubulin and anti-EF1α antibodies, as indicated. A total of 10⁶ cell equivalents were loaded per lane.

Fig 2

Depletion of TbAIR9 following induction of RNAi in procyclic trypanosomes occurs preferentially from the posterior end and reduces growth rate. A. Cumulative growth curves for two independent TbAIR9 RNAi cell lines in the presence (+ tet) or absence (− tet) of tetracycline. B. Western blot of lysates of TbAIR9 RNAi cell lines expressing tyGFP:TbAIR9 from one endogenous allele, probed with anti-GFP antibody or with anti-OPB antiserum as a loading control. Upper panels: clone 1; lower panels: clone 2. Two tyGFP:TbAIR9 blots of different exposures are presented for clone 1 to show residual tyGFP:TbAIR9 at 48 h post induction. Samples of cells grown in the presence or absence of tetracycline were taken every 24 h for 72 h, as indicated. C. Fluorescence microscopy images of TbAIR9 RNAi cell lines expressing tyGFP:TbAIR9 from one endogenous allele. Cells were prepared for imaging at the time points (in hours) indicated. Panels from left to right: DIC images, DAPI staining (blue), tyGFP:TbAIR9 (green), GFP/DAPI merge. Arrowheads point to kinetoplasts. Scale bars: 5 µm.

Fig 3

Depletion of TbAIR9 in procyclic T. brucei results in aberrant cell division. A. DAPI staining of TbAIR9 RNAi cells following induction, at the time points indicated. The number of nuclei (N) and kinetoplasts (K) in > 200 cells/time point are indicated. ‘Other’ comprise mainly multinucleate cells. B. Images of DAPI-stained cells (left panels: DAPI; right panels: DAPI/DIC merge) with abnormal N/K configurations, as indicated. C. DNA content (flow cytometry) analysis of TbAIR9 RNAi cells following induction, at the time points indicated. The ploidies of the peaks are indicated. D. DAPI stained images [as in (B)] of aberrantly dividing 2N2K cells. Arrowheads point to kinetoplasts. Scale bars: 10 µm. Data presented are for clone 1.

Fig 4

Depletion of TbAIR9 in procyclic trypanosomes results in aberrant positioning of the nucleus and kinetoplast. A. Prevalence of nucleus/kinetoplast (N/K) positioning defects following induction of TbAIR9 RNAi. Data presented in Fig. 3A are reclassified according to whether cells displayed normal (standard font) or abnormal (bold italic font) N/K positioning. Data presented are for clone 1. B–D. DAPI stained images of 1N1K, 1N2K and 2N2K cell types, respectively, illustrating the range of N/K positioning phenotypes observed. Left panels: DAPI; right panels: DAPI/DIC merge. For reference, examples of cells displaying normal N/K positions (KN, KKN and KNKN, light blue font) for each cell type are presented in the top left of each figure panel. Organelles are listed in order from posterior to anterior. K^∧: kinetoplast juxta-positioned to nucleus. Arrowheads point to kinetoplasts. Scale bars: 10 µm. Asterisk in (B) indicates posterior end of cell.

Fig 5

Morphometric analysis of procyclic TbAIR9 RNAi cells. Cells were stained with anti-β-tubulin antibody (green) to outline the cytoskeleton and flagellum and co-stained with DAPI to visualize the nucleus (N) and kinetoplast (K). A. The following dimensions of the cell were measured in 1N1K cells: length of the cell (L); middle of the kinetoplast to posterior tip (K-P); centre of the nucleus to posterior tip (N-P), calculated as indicated. The kinetoplast to anterior tip (K-A) and nucleus to anterior tip (N-A) dimensions were calculated by subtracting K-P and N-P dimensions, respectively, from L. Measurements were binned as indicated and frequency distributions plotted for length (B), K-P (D), K-A (E), N-P (F) and N-A (G). Black bars: uninduced cells; grey bars: cells induced with tetracycline (tet) for 72 h. Data presented are for clone 1 [n = 100 (− tet) and 128 (+ tet)]. C. YL1/2 staining of procyclic TbAIR9 RNAi cells, 72 h post induction with tetracycline (tet). Left panels: DIC images; middle panels: YL1/2 staining (red); right panels: YL1/2/DAPI merge. Staining of the posterior end and the basal bodies is visible. Scale bars: 3 µm.

Fig 6

Depletion of TbAIR9 in bloodstream T. brucei arrests growth. A. Cumulative growth curve of two independent TbAIR9 RNAi cell lines expressing tyGFP:TbAIR9 from one endogenous allele in the presence (+ tet) or absence (− tet) of tetracycline. B. Western blot of lysates of TbAIR9 RNAi cell lines expressing tyGFP:TbAIR9 from one endogenous allele, probed with anti-GFP antibody or with anti-OPB antiserum as a loading control. Upper blots: clone 1; lower blots: clone 2. Samples of cells grown in the presence or absence of tetracycline were taken at the time points indicated. C. Fluorescence microscopy images of TbAIR9 RNAi cell lines expressing tyGFP:TbAIR9 from one endogenous allele. Cells were prepared for imaging at the time points (in hours) indicated. At 24 h post induction, the majority of cells had only a very faint tyGFP:TbAIR9 signal (see left cell in first panel set) – a few had significant staining (right cell in first panel set) and a few had no signal at all (second panel set). Top left: DIC images; top right: DAPI staining (blue); bottom left: tyGFP:TbAIR9 (green); bottom right: GFP/DAPI merge. Arrowheads point to kinetoplasts. Scale bars: 5 µm.

Fig 7

Depletion of TbAIR9 from bloodstream parasites impairs cytokinesis. A. DAPI staining of TbAIR9 RNAi cells at the time points indicated, following induction. The number of nuclei (N) and kinetoplasts (K) in > 200 cells/time point are indicated. ‘Other’ comprise mainly multinucleate cells. ‘Dividing’ cells were those with a visible cleavage furrow, or those in abscission; ‘non-dividing’ cells had no visible furrow and were not in abscission. B. DNA content (flow cytometry) analysis of TbAIR9 RNAi cells at the time points indicated, following induction. The ploidies of the peaks are indicated. C. Image of DAPI-stained 2N2K cell, undergoing cytokinesis to result in 0N1K and 2N1K progeny (left: DAPI; right: DAPI/DIC merge). Arrowheads point to kinetoplasts. D. DAPI stained images [as for (C)] of a dividing multinucleate cell (left) and a rounded multinucleate cell (right). Scale bars: 10 µm. E. Prevalence of nuclei and kinetoplast positioning and cytokinesis defects in 2N2K cells. Data presented are for clone 2.