Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates

Abstract

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

Keywords: cell biology; centrosome; human; mRNA localization; translation; zebrafish.

Figure 1.. Pericentrin (pcnt) mRNA is localized to centrosomes in early zebrafish embryos.

(A) RNA in situ hybridization of transcripts of different PCM components in four-cell stage zebrafish embryos. Note that while the mRNA of cep152, cep192, and cep215 displayed a pan-cellular distribution, pcnt mRNA was concentrated at two distinct foci in each cell. (B) RNA in situ hybridization showed similar dot-like patterns of pcnt transcripts with two non-overlapping antisense probes. The signals were lost in two maternal-zygotic (MZ) pcnt mutants. (C) Fluorescent RNA in situ hybridization and anti-γ-tubulin co-staining demonstrated the centrosomal localization of pcnt mRNA. n > 300 (pcnt-001 probe), n > 100 (pcnt-002 probe), n > 50 (cep152, cep192, or cep215 probe); all the embryos showed the same RNA distribution patterns as shown in the representative images. More than 100 MZpcnt^tup2 or MZpcnt^tup5 embryos were examined; none of them showed visible pcnt RNA in situ signals. Embryos were examined between 2- and 16-cell stages with representative four-cell stage embryos shown. Dashed lines delineate the cell boundaries. Scale bars: 200 µm or 25 µm (inset in C).

Figure 1—figure supplement 1.. Sequences of two Cas9-induced frameshift mutations (alleles pcnt^tup2 and pcnt^tup5) in the zebrafish pcnt gene.

The wild-type reference sequence is on the top. The guide RNA targets the exon 2 of the pcnt transcript (encoded by ENSDARG00000033012). The target site is underlined and the protospacer-adjacent motif (PAM) is in orange (on the reverse strand). Insertions and deletions (indels) are indicated by blue lowercase letters and dashes, respectively. The net change of each indel mutation is noted at the right of each sequence (+, insertion; −, deletion).

Figure 2.. Human PCNT mRNA is localized to centrosomes during early mitosis.

(A) Synchronized HeLa cells were subjected to fluorescent in situ hybridization with tyramide signal amplification against PCNT mRNA and anti-γ-tubulin immunostaining. Note that PCNT mRNA was localized to centrosomes predominantly during prophase (pro) and prometaphase (prometa). (B) Quantification of PCNT mRNA localization at centrosomes during different cell cycle stages. Data are represented as mean with standard deviation (SD) from three biological replicates, with the total number of cells analyzed indicated. Dashed lines delineate the cell boundaries. Scale bars: 10 µm and 2 µm (inset).

Figure 2—figure supplement 1.. Non-overlapping antisense probes and two independent in situ methods confirm centrosomal localization of PCNT mRNA during early mitosis.

HeLa cells were subjected to fluorescent in situ hybridization (FISH) with tyramide signal amplification (TSA) against PCNT mRNA with a 1000-nt probe (A) or the Stellaris single-molecule FISH (smFISH) with a set of 48 20-nt fluorescent probes (B). Note that both methods (probing two distinct regions in each method) showed similar distributions of PCNT mRNA at centrosomes during early mitosis, with the Stellaris smFISH showing mRNA at near single-molecule resolution. Scale bars: 10 µm and 2 µm (inset).

Figure 2—figure supplement 2.. Zebrafish pcnt mRNA is localized to centrosomes of mitotic retinal neuroepithelial cells in vivo.

Retinal neuroepithelial cells of 1-day-old zebrafish were subjected to pcnt FISH, anti-γ-tubulin, and anti-phospho-Histone H3 (PH3) immunostaining. Note that pcnt mRNA was localized to the centrosome of the mitotic (Cell 1, PH3+), but not of the non-mitotic cell (Cell 2). Retinas from >40 embryos were examined. Scale bars: 10 µm and 2 µm (inset).

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).

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).

Figure 5.. Centrosomal localization of PCNT mRNA/polysomes contributes to PCNT incorporation into mitotic centrosomes.

(A) Centrin-GFP RPE-1 cells—at either late G2 or early M phase—were subjected to anti-PCNT immunostaining. Representative confocal images are shown for each condition. A ‘fire’ lookup table (LUT) was used to show PCNT signal intensities. The sum intensity of anti-PCNT signals from both centrosomes of each cell was measured and plotted. (B) Numbers of PCNT mRNA at different cell cycle stages of Centrin-GFP RPE-1 cells were determined by PCNT smFISH. S phase/early G2 cells were identified by EdU labeling for 30 min. (C) HeLa cells were treated with vehicle control or 300 µM puromycin for 2 min before anti-PCNT immunostaining. The sum intensity of anti-PCNT signals from both centrosomes of each prophase or prometaphase cell was measured and plotted. Data are represented as mean ±95% CI. ‘n’ indicates the total number of cells analyzed from two (A), three (B), and two (C) biological replicates. p-values were obtained with Student’s t-test (two-tailed). a.u., arbitrary unit. Scale bar: 10 µm.

Figure 6.. ASPM mRNA is enriched at centrosomes in a translation-dependent manner during mitosis.

Prometaphase/metaphase RPE-1 cells were treated with vehicle (Control) or 300 µM puromycin for 2 min at 37°C (Puromycin) before fixation, followed by anti-PCNT immunostaining and ASPM smFISH. Representative confocal images and quantification of the ASPM mRNA distribution are shown for each condition. The distribution of ASPM mRNA in cells was quantified by measuring the distance between 3D rendered ASPM 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 two biological replicates. n = 76 and 81 cells for control and puromycin conditions, respectively. Note that ASPM mRNA was enriched at the centrosomes/spindle poles of the metaphase cell, but became dispersed throughout the cell upon a short puromycin treatment. Scale bars: 10 µm.

Figure 6—figure supplement 1.. ASPM mRNA is enriched at centrosomes in a translation-dependent manner during mitosis.

Prometaphase HeLa cells were treated with vehicle (Control) or 300 µM puromycin for 2 min at 37°C (Puromycin) before fixation, followed by anti-PCNT immunostaining and ASPM smFISH. Representative confocal images and quantification of the ASPM mRNA distribution are shown for each condition. The distribution of ASPM mRNA in cells was quantified as in Figure 6. Data are from a representative experiment (two technical duplicates per condition). n = 17 and 27 cells for control and puromycin conditions, respectively. Note that ASPM mRNA was enriched at the centrosomes/spindle poles of the prometaphase cell, but became dispersed throughout the cell upon a short puromycin treatment. Scale bars: 10 µm.

Figure 7.. A model of co-translational targeting of PCNT polysomes toward the centrosome during centrosome maturation.

During the late G2/M transition, translation of PCNT mRNA is upregulated by an as yet unknown mechanism. The partially translated PCNT nascent polypeptide starts to interact with the dynein motor complex once the dynein light intermediate chain 1 (LIC1)-interacting domain in the N-terminal half of PCNT is synthesized and folded. It will be interesting to test if PLK1 phosphorylation of S1235 and S1241 within the LIC1-interacting domain initiates this PCNT-dynein interaction. Subsequently, this nascent polypeptide-dynein interaction allows the entire polysome, which is still actively translating PCNT mRNA, to be transported along the microtubule toward the centrosome. This co-translational targeting mechanism may maximize efficiency of PCNT production and delivery to the centrosome, prevent ectopic accumulation of PCNT outside of centrosomes, and/or facilitate integration of PCNT into the expanding PCM during early mitosis. It remains to be determined if other PCM components (e.g. CEP215) interact with PCNT co- and/or post-translationally.