Figure 6. De novo centriole biogenesis... | Rockefeller University Press

Figure 6.

$De novo centriole biogenesis is partially impaired in PCM-depleted Drosophila cells. (A) DMEL-cultured cells were treated with RNAi against Plk4 over the course of 12 d to deplete their centrioles. mCherry (mCh) RNAi was used as negative control. Cells treated with RNAi against Plk4 gradually lost centrioles during proliferation. On day 10, samples were obtained for fixation and staining, and centriole-depleted cells were treated with RNAi against individual PCM components. On day 12, Plk4 translation was allowed to recover. On day 16, cells were fixed and stained by immunofluorescence (IF). We targeted for individual PCM components—Cnn, Asl, D-Plp, Spd2, or γ-tubulin 23C—or combinations of these molecules previously shown to be essential for PCM assembly in cycling cells—Cnn + Spd2, Cnn + D-Plp, Spd2 + D-Plp, or Cnn + Spd2 + D-Plp (Gomez-Ferreria et al., 2007; Conduit et al., 2014; Lerit et al., 2015; Feng et al., 2017; Citron et al., 2018). Additionally, we depleted all four PCM components—Cnn + Asl + D-Plp + Spd2 (referred to as All PCM)—required for PCM maintenance (Pimenta-Marques et al., 2016). (B) Maximum-intensity z projections of fluorescence from DMEL cells after 10 d treatment with RNAi against Plk4 (top row) or mCherry (mCh, bottom row). Cells were stained for centriolar markers Sas4 (magenta) and Cp110 (green), in addition to DAPI against DNA (blue). Inset squares in each fluorescence channel are shown at higher magnification on the right (scale bar, 1 µm). Knock-down of Plk4 (bottom row) caused loss of centrioles, as reported by the absence of spot signals in the green and magenta channels. Note that it is common for a small fraction of untreated DMEL cultured cells to have either too many (more than four) or too few (less than two) centrioles (Bettencourt-Dias et al., 2005). This is found in most cell lines from D. melanogaster as they are permissive to those changes. In contrast to vertebrate cells, a p53-dependent cell cycle arrest does not occur in the presence of numerical centrosome abnormalities in these insect cells. (C) Maximum-intensity z projections of fluorescence from centriole-depleted DMEL cells after 6 d treatment with RNAi against mCherry (top row), allowing recovery of normal centriole number, or against γ-tubulin 23C (bottom row) leading to little or no recovery of centrioles. Staining, color code, and insets are as in B. (D) Quantification of cells with centrioles after 10 and 16 d of RNAi treatment. Centriole number was scored in >300 cells per treatment, per experiment. The bars represent the average of proportions obtained in two or four independent experiments (gray squares) for the conditions listed. Superscripts denote statistical significance in treatments. ns (not significant), P ≥ 0.05; *, P < 0.05; ***, P < 0.001 (Pearson’s χ2 test, and two-proportions z test on pooled data). The top dashed arch denotes statistical difference between the mCh (control) and every other condition. γ-tub, γ-tubulin.$

De novo centriole biogenesis is partially impaired in PCM-depleted Drosophila cells. (A) DMEL-cultured cells were treated with RNAi against Plk4 over the course of 12 d to deplete their centrioles. mCherry (mCh) RNAi was used as negative control. Cells treated with RNAi against Plk4 gradually lost centrioles during proliferation. On day 10, samples were obtained for fixation and staining, and centriole-depleted cells were treated with RNAi against individual PCM components. On day 12, Plk4 translation was allowed to recover. On day 16, cells were fixed and stained by immunofluorescence (IF). We targeted for individual PCM components—Cnn, Asl, D-Plp, Spd2, or γ-tubulin 23C—or combinations of these molecules previously shown to be essential for PCM assembly in cycling cells—Cnn + Spd2, Cnn + D-Plp, Spd2 + D-Plp, or Cnn + Spd2 + D-Plp (Gomez-Ferreria et al., 2007; Conduit et al., 2014; Lerit et al., 2015; Feng et al., 2017; Citron et al., 2018). Additionally, we depleted all four PCM components—Cnn + Asl + D-Plp + Spd2 (referred to as All PCM)—required for PCM maintenance (Pimenta-Marques et al., 2016). (B) Maximum-intensity z projections of fluorescence from DMEL cells after 10 d treatment with RNAi against Plk4 (top row) or mCherry (mCh, bottom row). Cells were stained for centriolar markers Sas4 (magenta) and Cp110 (green), in addition to DAPI against DNA (blue). Inset squares in each fluorescence channel are shown at higher magnification on the right (scale bar, 1 µm). Knock-down of Plk4 (bottom row) caused loss of centrioles, as reported by the absence of spot signals in the green and magenta channels. Note that it is common for a small fraction of untreated DMEL cultured cells to have either too many (more than four) or too few (less than two) centrioles (Bettencourt-Dias et al., 2005). This is found in most cell lines from D. melanogaster as they are permissive to those changes. In contrast to vertebrate cells, a p53-dependent cell cycle arrest does not occur in the presence of numerical centrosome abnormalities in these insect cells. (C) Maximum-intensity z projections of fluorescence from centriole-depleted DMEL cells after 6 d treatment with RNAi against mCherry (top row), allowing recovery of normal centriole number, or against γ-tubulin 23C (bottom row) leading to little or no recovery of centrioles. Staining, color code, and insets are as in B. (D) Quantification of cells with centrioles after 10 and 16 d of RNAi treatment. Centriole number was scored in >300 cells per treatment, per experiment. The bars represent the average of proportions obtained in two or four independent experiments (gray squares) for the conditions listed. Superscripts denote statistical significance in treatments. ns (not significant), P ≥ 0.05; *, P < 0.05; ***, P < 0.001 (Pearson’s χ² test, and two-proportions z test on pooled data). The top dashed arch denotes statistical difference between the mCh (control) and every other condition. γ-tub, γ-tubulin.