Figure 4. Centrosomal localization of pcnt/PCNT mRNA requires intact polysomes, microtubules, and dynein activity.

(A) HeLa cells were synchronized by a double thymidine block and treated with DMSO vehicle (Control), 208 µM emetine, 3.76 µM harringtonine for 30 min, or 300 µM puromycin for 2 min before anti-PCNT immunostaining and PCNT smFISH. Representative confocal images and quantification of the PCNT mRNA distribution are shown for each condition. The distribution of PCNT mRNA in cells was quantified by measuring the distance between 3D rendered PCNT smFISH signals and the center of the nearest centrosome (labeled by anti-PCNT immunostaining). The fractions of mRNA as a function of distance to the nearest centrosome (binned in 0.5 µm intervals) were then plotted as mean (solid lines) ±95% CI (shading) from three biological replicates. n = 48, 45, 57, and 51 cells for control, emetine, harringtonine, and puromycin conditions, respectively. Note that PCNT mRNA moved away from the centrosome upon the harringtonine or puromycin treatment, but stayed close to the centrosome upon the emetine treatment, similar to the control. (B) Zebrafish embryos were injected with DMSO vehicle or 100 µg/ml nocodazole at the one-cell stage followed by pcnt FISH. (C) HeLa cells were treated with DMSO vehicle or 3 µg/ml nocodazole for 2 hr at 37°C before anti-α-tubulin, anti-PCNT immunostaining, and PCNT smFISH. Note that pcnt/PCNT mRNA in early embryos (B) and in early mitotic cells (C) was no longer enriched at the centrosome after microtubules were depolymerized. (D) HeLa cells were synchronized by RO-3306 and treated with DMSO vehicle or 50 µM ciliobrevin D for 1 hr 25 min before anti-PCNT immunostaining and PCNT smFISH. The distribution of PCNT mRNA in cells was quantified as in (A). n = 63 and 70 cells for control and ciliobrevin D conditions, respectively, from a representative experiment (two technical duplicates per condition). Note that PCNT mRNA was no longer enriched at the centrosome upon the ciliobrevin D treatment. Dashed lines delineate the cell boundaries. Scale bars, 10 µm (A), 100 µm (C), 10 µm (D), and 2 µm (inset in D).

Figure 4—figure supplement 1. Centrosomal localization of zebrafish pcnt mRNA depends on intact polysomes.

RNA in situ hybridization showed that pcnt transcripts were localized to the centrosomes in the buffer-injected embryo (Control) but were diffused throughout the cell in the embryo injected with ~1 nl of 300 µM puromycin at the one-cell stage (Puromycin). Embryos shown are at the two-cell stage. >100 embryos were examined for each condition. Scale bar: 200 µm.

Figure 4—figure supplement 2. More PCNT mRNA was often enriched near the larger centrosome in early mitosis.

In the majority of prophase and prometaphase HeLa cells (~67%), more PCNT mRNA was enriched around the larger centrosome. Data are represented as mean ± SD, ‘n’ indicates the total number of cells analyzed from four biological replicates.

Figure 4—figure supplement 3. Centrosomal localization of human PCNT mRNA during early mitosis is microtubule-dependent.

(A) HeLa cells synchronized to prophase by double thymidine block were treated with 5 µg/ml cytochalasin B for 15 min or 10 µg/ml nocodazole for 30 min at 37°C before fluorescent in situ hybridization with tyramide signal amplification against PCNT mRNA and anti-γ-tubulin immunostaining. Note that nocodazole, but not cytochalasin B, disrupted the centrosomal enrichment of PCNT mRNA. (B) HeLa cells were synchronized to late G2 phase by RO-3306, treated with 3 µg/ml nocodazole for 1 hr 35 min, and then released into mitosis (25 min after RO-3306 washout) in the presence of 3 µg/ml nocodazole. After fixation, anti-PCNT N-terminus immunostaining and PCNT smFISH were performed, followed by quantifications of PCNT mRNA distribution, PCNT protein distribution, and centrosomal PCNT intensities. Representative images of vehicle- and nocodazole-treated cells were shown. Insets are two-fold magnifications of the box regions. Note that upon the nocodazole treatment, PCNT mRNA was no longer enriched at the centrosome and the centrosomal PCNT levels were reduced. Furthermore, in contrast to the vehicle-treated cells in which close to 100% of PCNT proteins was localized to centrosomes, in the nocodazole-treated cells, only ~80% of PCNT proteins was localized to centrosomes, while the rest ~20% of PCNT proteins was dispersed throughout the cytoplasm as small PCNT puncta (see the monochromic ‘PCNT protein’ images and quantification shown as a histogram). n = 49 and 43 cells for control and nocodazole-treated conditions, respectively, from a representative experiment (two technical duplicates per condition). Dashed lines delineate the cell boundaries. a.u., arbitrary unit. Scale bars, 10 µm and 2 µm (insets).