Fernandez-Nicolas A et al. (2022), Vasa nucleates asymmetric translation along the...

ECB-ART-50373

Nat Commun 2022 Apr 20;131:2145. doi: 10.1038/s41467-022-29855-8.

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Vasa nucleates asymmetric translation along the mitotic spindle during unequal cell divisions.

Fernandez-Nicolas A , Uchida A , Poon J , Yajima M .

Abstract
mRNA translation on the spindle is hypothesized to be an essential strategy for the localized production of cell regulators. This mechanism may be important particularly in early embryonic cells, which have a large diffusion volume and that undergo rapid cell divisions. Evidence to test such a hypothesis has been, however, limited. Here, we use an embryo with both symmetric and asymmetric cell divisions and manipulate Vasa protein, an RNA-helicase, on the spindle in live sea urchin embryos. We learned that the spindle serves as a major site of translation and that protein synthesis within a single spindle can be unequal and help drive asymmetric cell divisions during embryogenesis. Recruiting Vasa to the ectopic sub-cellular region induced a new site of translation, disturbed asymmetric translation on the spindle, and changed the cell fate. Based on these observations, we conclude that Vasa functions in localized translation, which provides a spatiotemporal control in protein synthesis and is essential for rapidly developing embryonic cells.

PubMed ID: 35444184
PMC ID: PMC9021227
Article link: Nat Commun
Grant support: [+]

Antibodies: LOC548620 Ab2 phospho-rps6 Ab1 tubb1 Ab9
Morpholinos: ddx4 MO1

Article Images: [+] show captions

	Fig. 1. Vasa dynamics during asymmetric cell division.a Vasa distribution in early embryogenesis. b Time-lapse images of the vegetal half of the Vasa-GFP (green) embryo during micromere formation (arrows) at the 8â16-cell stage presented as maximum 2D-projection. 2xmCherry-EMTB (magenta)20 was used as counter-staining to visualize microtubules. These experiments were performed at least three independent times. Scale barsâ=â10âÎ¼m. c Kaede-Vasa enrichment model. Unphotoconverted Kaede-Vasa (green) on the spindle around the chromosomes is photoconverted and emits red fluorescence. Dynamics of photoconverted Kaede-Vasa were monitored over time to determine if Vasa enrichment in the micromeres was accomplished through differential degradation, translocation, or upregulation of translation on the micromere side of the spindle. If decreasing levels of photoconverted Kaede-Vasa on both sides of the spindle is complemented by an increase in unphotoconverted Kaede-Vasa on the micromere side of the spindle during anaphase, it suggests that upregulated translation is responsible for the asymmetric enrichment of Vasa in micromeres.
	Fig. 2. Photoconverted and unphotoconverted Kaede-Vasa dynamics during symmetric (a) or asymmetric (d) cell division.a, d A whole Z-section of the cell (0.44âÎ¼m per slice; ~90 slices in total) was imaged and analyzed every 1-min and presented as maximum 2D-projection. The upper leftmost column shows Kaede-Vasa expression prior to photoconversion (green) while the remaining columns are magnified images of the region squared by a white dashed line. Images were taken at varying minute intervals as indicated in the corner, following photoconversion (magenta) during cell division. The area marked by a red circle is a photoconverted area (activation ROI). Kaede-Vasa levels were compared between the vegetal side (V) and the animal side (A) of the spindle during asymmetric cell division (B) or both sides of animal blastomeres (A1 and A2) during symmetric cell division (A). Scale barsâ=â10âÎ¼m. b, e A cartoon diagram depicting a measurement area and protocol for comparing fluorescent signal intensity across the mitotic spindles in either the animal (b) or vegetal blastomeres (e). Photoconversion was performed at an 8-cell stage metaphase. The major photoconverted area (magenta) typically appears in a trapezoid by following the shape of the spindle, which is labeled as A, V, A1, or A2 and indicates the area of the intensity measured (Detection ROI), respectively. The relative intensity of A1 over A2 for symmetric cell division and V over A for asymmetric cell division was compared and shown in graphs (c) and (f). c, f Kaede-Vasa intensity level during symmetric (c) and asymmetric (f) cell division was measured for detection ROI and normalized to its initial value. The green Kaede-Vasa was maximum ~2.29 folds higher in the vegetal side (V) compared to the animal side (A) of the spindle during asymmetric cell division, whereas nearly equal in both sides of the spindles (A1 and A2) during symmetric cell division (p-value, 0.0094). All experiments were performed at least three independent times and the representative analyses are shown (nâ=â3 each). Unpaired t-test was used for all statistical analyses in this figure. **pâ<â0.01. Columns represent meansâÂ±âSD or SEM. Source data are provided as a Source Data file.
	Fig. 3. Kaede-Vasa does not translocate within a spindle or from the cytoplasm.a Kaede-Vasa was photoconverted on one side of the spindle at the 8-cell stage metaphase during asymmetric cell division (A or V) or during symmetric cell division (A1 or A2). These experiments were performed at least three independent times and a representative image series of the embryo is shown. The cell was photoconverted only for the animal side (A) of the spindle during asymmetric cell division. Scale barsâ=â10âÎ¼m. b Diagrams indicating where photoconversion was performed (magenta) and detection ROIs were measured. c Photoconverted Kaede-Vasa (red line) showed a significant increase immediately after photoconversion yet decreased at a similar rate with that on the other side of the spindle, suggesting Kaede-Vasa did not translocate to the other side of the spindle yet rather remained at the original site during M-phase. Each experiment was repeated at least three times and a representative slope ratio out of three individual embryos is shown in column graphs on the right. Each slope ratio was nearly equal on both sides of the spindle. d A majority of Kaede-Vasa in the cytoplasmic region was photoconverted during M-phase. A time series of the vegetal blastomere images undergoing asymmetric cell division is shown. e A diagram indicating where photoconversion was performed (magenta) and detection ROIs (S1, C2) were measured. Scale barsâ=â10âÎ¼m. f Photoconverted Kaede-Vasa remained in the cytoplasm (C2) during M-phase, yet the signal was increased in the spindle region (S1) after S-phase entry. These experiments were performed at least three independent times and the measurement results of two other embryos are shown in Supplementary Fig. S4f, g. g A summary model of Vasa dynamics during cell cycle progression. Increased Vasa protein synthesis occurs on the vegetal side of the spindle during asymmetric cell division. Vasa is released from the sub-cellular structures (e.g., microtubules) upon S-phase entry and partly translocates back to the spindle at the beginning of the next M-phase. Source data are provided as a Source Data file.
	Fig. 4. In vivo or real-time detection of localized translation.a The OPP (red) and Vasa (green) staining at the 16-cell stage. i and e arrowheads indicate the micromere and macromere sides of the spindle areas, respectively, analyzed in (c). A whole Z-section of the cell (1âÎ¼m per slice; ~35 slices in total) presented as a maximum 2D-projection. b (Left) % of the OPP signal on the spindle area with/without emetine (0.5âÎ¼M). (Right) % of the HPG signal on the spindle. nâ=â10 for all groups. Adjusted p-value <0.0001. c The OPP level comparison between the micromere side (i) and the macromere side (a) of the spindle at telophase. nâ=â18, 14, 32, 12, 16, 9 from the left-right columns. dâf Vasa (red; e) and HPG (green; f) signals enriched on the micromere side (i) of the spindle compared to the macromere side (a). nâ=â50, 33, 70, 31, 38, 24 from the left-right columns of graph (e). nâ=â19, 11, 23, 9, 13, 9 from the left-right columns of graph (f). Adjusted p-value <0.0001. A whole Z-section of the cell (1âÎ¼m per slice; ~35 slices in total) presented as a maximum 2D-projection. g TC-tag imaging procedure. h Live imaging of TC-tagged GFP (green) during micromere formation in 3âÎ¼M ReAsH-EDT2 (red): Vasa-GFP with no TC-tag (negative control), TC-Vasa-GFP (wild type), and TC-Vasa-ÎC-term-GFP (non-functional mutant). Time codes are the time post ReAsH-EDT2 exposure. A sub-Z-section (~5âÎ¼m) of the embryo around the spindle area was imaged every 15-s. All experiments were repeated at least three independent times. () in all graphs indicate the total number of embryos analyzed. The box plot defined by two box lines indicates the 25th and 75th percentile, respectively, with a centerline at the 50th percentile and the minima and maxima whiskers. *pâ<â0.001, **pâ<â0.0001. Columns represent meansâÂ±âSD or SEM. Two-way ANOVA was used for all statistical analyses except for the Spindle HPG (b, right) that used paired t-test in this figure. Source data are provided as a Source Data file. Scale barsâ=â10âÎ¼m.
	Fig. 5. Vasa C-terminus is responsible for localized translation on the spindle.a A schematic diagram of Vasa deletion or mutation constructs tested in this study. VasaC1-3 series are the constructs mutated for the Vasa-C-terminal regions (see also Supplementary Fig. S7 for detail). b Live imaging of the 8â16-cell embryonic cell injected with each of the Vasa mutants fused to GFP. Vasaâs spindle localization was lost in Vasa-C-terminal mutants (e.g., Vasa-ÎC-term, Vasa-C1-3, and Vasa-C3), yet not in other mutants except for Vasa-7F that lacks all of the conserved motifs important for DEAD-box helicase activitiesÂ (SupplementaryÂ Fig. S7). c Localized translation on the spindle was measured by the OPP signal level in each embryo injected with each of the Vasa mutants along with Vasa morpholino antisense oligo to knock down endogenous Vasa activity. The OPP signal appears on the spindle when the introduced Vasa mutants are functional. A whole Z-section of the cell of interest (1âÎ¼m per slice; ~35 slices in total) was imaged, analyzed, and presented as a maximum 2D-projection. The whole embryo images are presented in Supplementary Fig. S7. d Vasa C-terminus deletion or mutation resulted in the reduced signal of localized translation on the spindle. The relative OPP intensity level on the spindle versus in the cytoplasm was obtained as follows: The average signal level of three detection ROIs was obtained per embryo. A total of ~27 embryos were analyzed in this manner per sample group and the average value was presented in the graph. () indicates the total number of ROIs measured for each sample group. All experiments were performed at least three independent times. Adjusted p-value <0.0001. nâ=â81, 45, 36, 48, 48, 36, 27, 45, 33, 27, 18, 33 from the left-right columns of graph (d). The box plot defined by two box lines indicates the 25th and 75th percentile, respectively, with a centerline at the 50th percentile and minima and maxima whiskers. Two-way ANOVA was used for all statistical analyses in this figure. ****pâ<â0.0001. Columns represent meansâÂ±âSD or SEM. All scale barsâ=â10âÎ¼m. Source data are provided as a Source Data file.
	Fig. 6. Vasa nucleates ectopic translation on the membrane.a membrane-mCherry-Vasa (magenta, upper panels) was recruited to the membrane (outlined by a white circle) and away from the spindle (arrow) (nâ=â10), while Vasa-mCherry (magenta; control; lower panels) was enriched specifically on the spindle (nâ=â10). EMTB-3xGFP (green) was used as a microtubule marker. b Vasa-GFP (green) mRNA was coinjected with membrane-mCherry-Vasa or Vasa-C3 (red) mRNA. The overlapping signal ofÂ GFP and mCherry is shown in orange. membrane-mCherry-Vasa, nâ=â33; membrane-mCherry-Vasa-C3 (nâ=â29). c The graph represents the overlapping mean fluorescence on the cortex (green/ magenta) of controls versus experimental conditions. Average values from 3 detection ROIs per embryo were obtained for each group, nâ=â30 for membrane-mCherry-Vasa-C3 (a total of 90 ROIs) and nâ=â18 for membrane-mCherry-Vasa (a total of 54 ROIs). Adjusted p-value, 0.022. d New protein synthesis (HPG signal, green) was detected on the plasma membrane with membrane-mCherry-Vasa (arrows, nâ=â18), but little with membrane-mCherry-Vasa-C3 (nâ=â30) in 16-cell stage embryos. A whole Z-section of the cell of interest (1âÎ¼m per slice; ~35 slices in total) presented as a maximum 2D projection. e The graph represents the overlapping mean fluorescence on the cortex (green/ magenta) of the control versus experimental condition. Average values from 3 detection ROIs per embryo were obtained for each group, nâ=â10 for membrane-mCherry-Vasa-C3 (a total of 30 ROIs) and nâ=â45 for membrane-mCherry-Vasa (a total of 135 ROIs). Adjusted p-value, 0.009. f Membrane-mCherry-Vasa injected embryos failed in gastrulation as well as Vasa localization in the germline (arrows). Membrane-mCherry (magenta), Vasa-GFP (green). A whole Z-section of the cell of interest (1âum per slice; ~35 slices in total) presented as a maximum 2D projection. All experiments were performed at least three independent times. All scale bars except for (f)â=â10âÎ¼m; Scale bar of (f)â=â20âÎ¼m. Multiple t-tests were used for all statistical analyses in this figure. pâ<â0.05, *pâ<â0.01. Columns represent meansâÂ±âSD or SEM. Source data are provided as a Source Data file.
	Fig. 7. Optogenetic disruption of Vasa localization on the spindle during asymmetric cell division altered localized translation and cell fate of the micromeres.aâd Upon blue light irradiation, Vasa-mCherry-LOV was recruited to actin, resulting in Vasa-less micromeres at the 16-cell stage in the experimental group. A level of translation was detected by FIAsH-EDT2 at the 16-cell stage and was increased in the micromere-side of the spindle in the control group (a and b) whereas decreased in that of the experimental group (c and d). A whole Z-section of the cell of interest (0.44âÎ¼m per slice; ~90 slices in total) was imaged and analyzed every 1-min and presented as a maximum 2D-projection. A bright green spot (*) is a non-specific debris that tends to fluoresce under the microscope. Each experiment was repeated at least three times and the measurement results of two other embryos are shown in Supplementary Fig. S10f. e, f Localization of Î²-catenin-GFP was tracked by time-lapse imaging of every 15âmin for ~5âh after activation (approximately, ~12 hpf). In controls, GFP-Î²-catenin enriched into the vegetal most cells (e, arrows), whereas its localization became uniform or random in the experimental groups (f, arrows). Arrowheads indicate ectopic localization of GFP-Î²-catenin. A whole Z-section of the embryo of interest (1âÎ¼m per slice; ~50 slices in total) was imaged and is presented as a maximum 2D-projection. The embryos were lightly squashed to completely immobilize during imaging. Therefore, the embryos that successfully formed micromeres under this condition were selected for the analysis and the embryos in a similar angle were compared between control and experimental groups in this study. These experiments were performed at least three independent times. All Scale barsâ=â10âÎ¼m. Source data are provided as a Source Data file.
	Fig. 8. Models for Vasa enrichment and new protein synthesis on the mitotic spindle.a A model for asymmetric translation on the spindle during micromere formation. Metaphase: Vasa (purple) is equally abundant on both sides of the spindle, so as are levels of active translation. Anaphase: Increased active translation on the micromere side of the spindle facilitates increased Vasa accumulation in the same region. Increased Vasa also increases translation activity, and new protein synthesis occurs at a higher level on the micromere side relative to the macromere side. Telophase: the micromere has a higher level of protein synthesis, resulting in the micromere-specific fate differentiation. b A model for ectopic translation nucleated by membrane-targeted Vasa. Membrane-targeted Vasa recruits Vasa protein, mRNAs, and other spindle-associated molecules as well as translation machinery for ectopic on-site translation on the plasm membrane, which results in cell division and developmental defects.

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