Role of Centrins 2 and 3 in Organelle Segregation and Cytokinesis in <em>Trypanosoma brucei</em>

<div><p>Centrins are calcium binding proteins involved in cell division in eukaryotes. Previously, we have shown that depletion of centrin1 in <em>Trypanosoma brucei</em> (<em>T. brucei</em>) displayed arrested organelle segregation resulting in loss of cytokinesis. In this study we analyzed the role of <em>T. brucei</em> centrin2 (TbCen2) and <em>T. brucei</em> 3 (TbCen3) in the early events of <em>T. brucei</em> procyclic cell cycle. Both the immunofluorescence assay and electron microscopy showed that TbCen2 and 3-deficient cells were enlarged in size with duplicated basal bodies, multinuclei and new flagella that are detached along the length of the cell body. In both TbCen2 and TbCen3 depleted cells segregation of the organelles i.e. basal bodies, kinetoplast and nucleus was disrupted. Further analysis of the cells with defective organelle segregation identified three different sub configurations of organelle mis-segregations (Type 1–3). In addition, in majority of the TbCen2 depleted cells and in nearly half of the TbCen3 depleted cells, the kinetoplasts were enlarged and undivided. The abnormal segregations ultimately led to aborted cytokinesis and hence affected growth in these cells. Therefore, both centrin2 and 3 are involved in organelle segregation similar to centrin1 as was previously observed. In addition, we identified their role in kinetoplast division which may be also linked to overall mis-segregation.</p> </div

Images show direct effect of centrins' depletion on kinetoplast biology and overall organelle segregation in <i>T. brucei</i>.

<p><b>A</b>: Segregation patterns of the basal bodies along with the kinetoplasts during RNAi for TbCen2 and 3. The cells were stained with DAPI for nuclei and kinetoplasts and YL1/2 for basal bodies. Panels 1 and 2 display the typical segregation of basal bodies, kinetoplasts and nucleus in the control cells. Panels 3–5 and 6–8 display the 3 different segregation patterns of the organelles after RNAi of TbCen2 and 3 respectively, other than the typical pattern seen with the control cells (panel 2). Panel 1 is the ‘G’ stage of the cell cycle of the procyclic form with 1K1N, where both the basal bodies and kinetoplast are seen at the posterior region of the cell. Scale bar common for all the images, 5 µm. <b>B</b>: Histogram of cells with 2 basal body pairs displaying each of the 3 new segregation patterns of basal bodies and kinetoplasts in the RNAi cells. For each TbCen2 and 3 RNAi studies, over 100 bi-nucleated cells with abnormal organelle segregation were manually counted and analyzed. Data represent the means ± SD of three independent experiments. The time points at which the cells analyzed after induction was day 3 for TbCen2 RNAi cells and day 2 for TbCen3 RNAi cells. An, anterior end; B, basal body; K, kinetoplast; N, nucleus, Po, posterior end. Scale bars, 5 µm.</p

Sequence of organelle biogenesis and segregation in normal and RNAi cells.

<p><b>A</b>: The control cells sequentially show duplication of DNA content from panel 1 to 2; division into two kinetoplasts from panel 2 to 3; segregation of the separated kinetoplasts to appropriate locations from panel 3 to 4. An, anterior end; K, kinetoplast; N, nucleus, Po, posterior end. Scale bar, 5 µm. <b>B</b>: Histogram showing the percent of TbCen2 and TbCen3 RNAi cells with undivided kinetoplasts in the two nucleated cells at different time points including day 3 for TbCen2 RNAi and day 2 for TbCen3 RNAi. For the TbCen2 and 3 RNAi, average 200 and 170 cells respectively were manually counted and analyzed during each time point. Data represent the means ± SD of three independent experiments.</p

Effect of ablation of centrins on cell shape and organelle number.

<p><b>A</b>: Effects on the duplication and segregation of basal bodies, kinetoplasts and nuclei in TbCen2 and 3-depleted cells. The cells were stained with DAPI for the nuclei and kinetoplasts, YL1/2 for the basal bodies and L8C4 for the flagella. Note the RNAi induced cells are large and pleomorphic in shape with multiple organelles with more than one flagellum and the new flagella are of detached type (middle and lower panels) compared to the control cell with organelles in single number with one attached flagellum (top panel). Scale bar, 5 µm. <b>B</b>: Electron microscopy of centrin-depleted cells. (<b>a</b>) A typical control cell in this particular section shows single flagellum, pair of basal bodies, kinetoplast and nucleus. (<b>b–d</b>) TbCen2-depleted cells with (<b>b</b>) multi basal bodies and the kinetoplast with enlarged size compared to the control in ‘<b>a</b>’, (<b>c</b>) multi nuclei and (<b>d</b>) abnormal kinetoplast. (<b>e–h</b>) TbCen3-depleted cells with (<b>e</b>) multi basal bodies, (<b>f</b>) multi flagella. Inset is the cross-section image of a detached flagellum displaying the normal axoneme (with 9+2 microtubule structure) and the paraflagellar rod, (<b>g</b>) multi nuclei and (<b>h</b>) an abnormal kinetoplast. Scale bars, 500 nm (<b>a</b>, <b>b</b>, <b>d–f</b> and <b>h</b>) and 2 µm (<b>c</b> and <b>g</b>). <b>C & D</b>: Bar graphs showing the percent of <i>T. brucei</i> procyclic cells with duplicated basal bodies (<b>C</b>) and flagella (<b>D</b>). The cells were analyzed on day 3 after induction for TbCen2 RNAi cells and day 2 for TbCen3 RNAi cells. Data represent the means ± SD of three independent experiments. For each TbCen2 and 3 RNAi studies, over 140 cells were manually counted and analyzed. F, flagellum; B, basal body; K, kinetoplast; N, nucleus; P, flagellar pocket, A, axoneme; R, paraflagellar rod; AF, attached flagellum; DF, detached flagellum; An, anterior; Po, posterior.</p

Analysis of cell structure and kinetoplasts and nuclei number in the RNAi cells.

<p><b>A</b>: DAPI-stained images of the control and TbCen2 and 3-depleted cells, 4d after RNAi induction. DF, detached flagella; K, kinetoplast; N, nucleus. Scale bar, 5 µm. (<b>B & C</b>) Fluorescence-activated cell sorting analysis for relative DNA content of TbCen2 and 3 depleted cells. <b>B</b>: Histogram plots of cell count by DNA content obtained directly from the flow cytometry analysis are shown. <b>C</b>: The percentage of cells that contain two times the DNA content (2C), four times (4C), and more than 4 times (>4C) were measured at each time point and are shown. Data represent the means ± SD of three independent experiments. <b>D</b>: Bar graph showing increase in the proportion of multinucleated and multikinetoplast cells with time after RNAi knockdown of TbCen2 and 3. Cells after different days of RNAi induction were stained with PI and examined by fluorescence microscope for tabulation of the cells with different number of kinetoplasts and nuclei. The time points at which the cells were analyzed after induction was up to day 5 for TbCen2 RNAi cells and day 4 for TbCen3 RNAi cells. Data represent the means ± SD of three independent experiments. For each TbCen2 and 3 RNAi studies, over 140 cells were manually counted and analyzed.</p

Centrins' RNAi and their effect on parasite growth.

<p><b>A</b>: Northern blots of RNA from induced (I) and uninduced (UN) TbCen2 and 3 cultures. Membranes containing 12 µg total RNA from day 2 uninduced and induced cells were used. Membranes, in addition to hybridization with DNA probes of either specific centrin or α-tubulin (loading control) were reprobed with DNA probes of other centrins (TbCen1-3). <b>B</b>: The effect of TbCen2 and 3 knockdown on the <i>in vitro</i> growth of <i>T. brucei</i> procyclics. The cells were grown with or without tetracycline. The data represent the means of ± SD of the three independent experiments. *p<0.02.</p

<i>T. brucei</i> procyclic cells after TbCen2 or 3 RNAi induction displaying the cell populations that contain undivided kinetoplasts.

<p>DAPI stained cells were used in the analysis. The cells were analyzed on day 3 after induction for TbCen2 RNAi cells and day 2 for TbCen3 RNAi cells.</p>*<p>Cells as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045288#pone-0045288-g004" target="_blank">Figure 4A</a>; panel 3.</p>**<p>Cells as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045288#pone-0045288-g004" target="_blank">Figure 4A</a>; panel 5.</p

Schematic diagram of the organelle segregation and the failure of segregation in the absence of TbCen2 or 3 in <i>T. brucei</i> procyclic form.

<p>Normal cell division and the three different types of abnormal segregation patterns of the organelles during centrin RNAi leading to the failure in cytokinesis and the generation of giant cells with multiple organelles are shown. Color coordination for organelles colored: red, basal body; green, kinetoplast; blue, nucleus.</p

Role Of SMAD5 Over-Expression In ERα Down-Regulation.

<p>(<b>A</b>) Immunoblot analysis showing parental and MCF-7 cells engineered to over-express SMAD5. (<b>B</b>) Immunofluorescence analysis showing that SMAD5 over-expression induces ERα down-regulation in ERα+MCF-7 cells. ERα (Abcam, Cambridge, Massachusetts, USA) was labeled in green, p∼SMAD5 (Cell Signaling Technology, Boston, MA, USA) was labeled in red and nuclei were labeled in blue with DAPI. (<b>C</b>) Graph showing the percentage of cells expressing p∼SMAD5 and ERα in vMCF-7^SMAD5 and parental cells. Experiments were performed in triplicate (+/− s.d.; *p<0.0001 vs. MCF-7; **p<0.0001 vs. MCF-7).</p

Endocrine Resistant Breast Cancer Cells.

<p>(<b>A</b>) Immumohistochemistry staining of low-grade tubular tumors for MCF-7 and high-grade vMCF-7^ΔRaf-1 Primary and Metastatic tumors. Breast cancer xenografts were stained with a polyclonal antibody targeting the ERα (Abcam, Cambridge, Massachusetts, USA). (<b>B</b>) Immunofluorescence analysis showing down-regulation of ERα expression in vMCF-7^ΔRaf-1 1GX cancer cells compared to parental MCF-7 and vMCF-7^ΔRaf-1 cells. (<b>C</b>) Graph showing the percentage of cancer cells harboring an ERα^low/− phenotype from three independent experiments (+/− s.d.; *p<0.0705 vs. MCF-7; **p<0.0001 vs. vMCF-7^ΔRaf-1). (<b>D</b>) Graph showing the percentage of cancer cells in the S phase of the cell cycle during starvation from 17-β estradiol and following treatment with 17-β estradiol (10⁻¹⁰ M) alone or in combination with 4-OH-tamoxifen (10⁻⁷ M) for 48 hours from three independent experiments (+/− s.d.; *p<0.0008 vs. MCF-7; **p<0.0009 vs. vMCF-7^ΔRaf-1).</p