Figure 3. Centrosomally localized PCNT mRNA undergoes active translation.

(A) A strategy of using smFISH and double immunofluorescence (IF) to distinguish between newly synthesized and full-length PCNT proteins (see text for details). The location and size of the epitopes for anti-PCNT N- and C-terminus antibodies, proportionally scaled to the full-length human PCNT protein, are indicated. (B) Prometaphase HeLa cells were subjected to PCNT smFISH and anti-PCNT immunostaining against the N- and C-terminus of PCNT protein (PCNT N-term and PCNT C-term). Note that the putative active translation sites were labeled by PCNT N-term IF and PCNT smFISH, but not by PCNT C-term IF (top panel). However, upon the puromycin treatment (300 µM for 2 min at 37°C, bottom panel), PCNT N-term IF signals were no longer colocalized with PCNT smFISH signals, indicating that those PCNT N-term IF signals on RNA represent nascent PCNT polypeptides. Orange boxes show higher contrast of selected areas (dashed orange boxes) for better visualization. The low-magnification images corresponding to the magnified insets are shown in monochrome (individual channels) and color (merged channels). (C) PCNT smFISH signals between 1 and 3 µm radius from the centrosome center were quantified for the presence of anti-PCNT N-term IF signals with or without a short puromycin treatment. Data are represented as mean ±95% CI (confidence intervals) from three biological replicates, with the total number of cells analyzed indicated. p-value was obtained with Student’s t-test (two-tailed). Scale bars: 5 µm and 0.5 µm (inset).

Figure 3—figure supplement 1. Visualization of active translation in live cells using the SunTag/PP7 system.

(A) SunTag/PP7 system overview, adapted from Wang et al. (2016). (B) HeLa cells stably expressing scFv-GFP and tdPCP-tdTomato transfected with SunTag-ODC-PP7 reporter. Individual polysomes (GFP⁺) and mRNA (tdTomato⁺) were shown. (C) Translation foci in the same field before and after adding 300 µM puromycin for 1 min. Scale bars: 10 µm.

Figure 3—figure supplement 2. Mean radius of mitotic centrosomes of HeLa cells.

Data are represented as mean ±95% CI from three biological replicates.

Figure 3—figure supplement 3. Colocalization of anti-PCNT N-terminus, anti-ribosomal protein S6, and PCNT smFISH signals near the centrosome during early mitosis.

HeLa (A) or RPE-1 (B) cells at prophase were subjected to anti-PCNT N-terminus, anti-ribosomal protein S6 double immunostaining, and PCNT smFISH. Note that when colocalization of these three signals was assessed within thin optical sections (e.g. 1 to 3 confocal z-sections, 0.3 µm per z section), many PCNT mRNA molecules positive for anti-PCNT N-terminus signals near the centrosome were also positive for anti-ribosomal protein S6 (RPS6) signals (arrows), suggesting that these PCNT mRNA molecules were undergoing active translation. Scale bars: 10 µm and 2 µm (inset).