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, 2014, 893272

Tracking the Biogenesis and Inheritance of Subpellicular Microtubule in Trypanosoma Brucei With Inducible YFP-α-tubulin

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Tracking the Biogenesis and Inheritance of Subpellicular Microtubule in Trypanosoma Brucei With Inducible YFP-α-tubulin

Omar Sheriff et al. Biomed Res Int.

Abstract

The microtubule cytoskeleton forms the most prominent structural system in Trypanosoma brucei, undergoing extensive modifications during the cell cycle. Visualization of tyrosinated microtubules leads to a semiconservative mode of inheritance, whereas recent studies employing microtubule plus end tracking proteins have hinted at an asymmetric pattern of cytoskeletal inheritance. To further the knowledge of microtubule synthesis and inheritance during T. brucei cell cycle, the dynamics of the microtubule cytoskeleton was visualized by inducible YFP-α-tubulin expression. During new flagellum/flagellum attachment zone (FAZ) biogenesis and cell growth, YFP-α-tubulin was incorporated mainly between the old and new flagellum/FAZ complexes. Cytoskeletal modifications at the posterior end of the cells were observed with EB1, a microtubule plus end binding protein, particularly during mitosis. Additionally, the newly formed microtubules segregated asymmetrically, with the daughter cell inheriting the new flagellum/FAZ complex retaining most of the new microtubules. Together, our results suggest an intimate connection between new microtubule formation and new FAZ assembly, consequently leading to asymmetric microtubule inheritance and cell division.

Figures

Figure 1
Figure 1
Inducible expression of YFP-α-tubulin in T. brucei. Cells stably transfected with pLew-YFP-α-tubulin were cultivated in the absence or presence of tetracycline to induce YFP-α-tubulin expression. Samples were taken at various time points for immunoblots (a), growth curve analyses (b), and cell fractionation studies (c). YFP-α-tubulin was detected as early as 2 hours after induction. Continuous induction led to slightly increased YFP-α-tubulin level and had little effect on parasite proliferation. Immunoblots of detergent extracted YFP-α-tubulin cells indicated that only a small amount of YFP-α-tubulin was incorporated into the detergent insoluble cytoskeleton (P). T: total cells; S: detergent soluble fraction.
Figure 2
Figure 2
Incorporation of inducible YFP-α-tubulin in early cell cycle stages. pLew-YFP-α-tubulin cells were induced for 8 hours, extracted with 1% NP-40, and fixed for staining with anti-GFP (for YFP-α-tubulin), α-tubulin, YL1/2, FAZ and DAPI. In the early cell cycle stage, neither the kinetoplast (small blue dot) nor the nucleus (large blue dot) had duplicated. Basal bodies duplication is one of the earliest events of the cell cycle. * marks the posterior tip of the parasite cell; arrowheads: basal bodies; white lines: new FAZ; arrow: flagellum.
Figure 3
Figure 3
YFP-α-tubulin is incorporated primarily in the region between the old and new FAZ in duplicating cells. In the duplicating cells, kinetoplast has duplicated and segregated before the nucleus. Mitotic cells containing an intranuclear spindle can also be observed. Samples were processed as in Figure 2. Arrowheads: basal bodies; white lines: new FAZ; arrow: intranuclear spindle.
Figure 4
Figure 4
Asymmetric inheritance of newly formed subpellicular microtubules in T. brucei cell division. In these postmitotic cells, both kinetoplasts and nuclei have been duplicated and segregated. The partitioning of intracellular organelles and the cytoskeleton network into the daughter cells become evident. Samples were processed as in Figure 2. Arrowheads: basal bodies; white lines: new FAZ.
Figure 5
Figure 5
Subpellicular microtubule plus end dynamics revealed by EB1. Cells stably expressing YFP-EB1 (a) or Ty1-EB1 (b) were fixed with cold methanol and labeled with DAPI for DNA. YFP-EB1 cells were also immunolabeled with anti-α-tubulin which revealed the total microtubule profile in a parasite cell (c). A polyclonal anti-EB1 was used to label microtubule plus ends throughout the cell cycle (d). Cells double labeled for anti-EB1 and FAZ revealed a possible nonspecific labelling of anti-EB1 along the FAZ region (e). Arrows, EB1 staining at the posterior tip of the cell; double headed arrow: elongated EB1 pattern during mitosis; white lines: possible nonspecific EB1 labelling near FAZ.
Figure 6
Figure 6
GCP2-RNAi affects new FAZ extension. Cells with a stably integrated GCP2-RNAi construct were grown with tetracycline to induce RNAi or without as control. To monitor the efficiency of RNAi, GCP2-RNAi cells were transfected to allow transient transfection of YFP-GCP2 (a). Samples were then taken every 24 hours after induction for growth assay ((a); results shown as mean ± SD, n = 3) and immunoblotting with anti-YFP and anti-BiP (inset). For quantitation of cell cycle effects (b), 400 cells were scored for their DNA contents in each of 3 independent experiments and the results shown as mean ± SD. For motility assays ((c), (d)), uninduced control and cells induced for GCP2-RNAi for 48 hours were diluted in fresh medium, imaged at 2 frames/second for 1 minute, and the movement of individual cells tracked (c) and velocity calculated (d). The 2D-tracks of ~60 cells from three independent experiments were generated by in silico tracking on movies. The velocity results are shown as mean velocity ± SEM of 3 independent experiments with 20–25 cells per experiment. The effect of GCP2 depletion on the new FAZ and flagella elongation was monitored in >100 biflagellated cells in control or cells induced for GCP-RNAi for 48 hours ((e), (f)). The length of new FAZ was plotted against corresponding new flagellum length for each cell measured (e). Alternatively, cells were grouped based on new flagellum length range and FAZ length (shown as mean length ± SEM) was plotted against the flagellum length range (f).

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