Characterizing the localization and role of lin-5 and era-1 mRNAs in early C. elegans embryos

Asymmetric cell division is essential for the generation of diversity during development and the function of stem cell lineages. The Caenorhabditis elegans zygote is an attractive model to investigate the mechanisms of spindle positioning during asymmetric cell division. In this polarized cell, the asymmetric distribution of cortical force generators along the antero-posterior axis and pulling on astral microtubules leads to the unequal cleavage of the one-cell embryo. The mechanisms underlying such cortical force generation are thought to act strictly at the protein level. In this thesis work we report that the mRNA encoding the cortical force generator component LIN-5 is enriched around centrosomes in early embryos, in a manner that depends on microtubules and dynein. We established that the lin-5 coding sequence is necessary and sufficient for mRNA enrichment around centrosomes in C. elegans. In addition, we found that lin-5 mRNA is mislocalized in lin-5(ev571) mutant embryos, which harbor a 9 nucleotide insertion in the coding sequence. Moreover, an intragenic revertant of lin-5(ev571), lin-5(ev571he63), also exhibits mislocalized lin-5 mRNA distribution. We demonstrated that this is accompanied by diminished pulling forces on the posterior spindle pole, suggesting that centrosomal localization of lin-5 mRNA is important for robust pulling forces. We found also that lin-5 mRNA centrosomal enrichment is slightly asymmetric during anaphase, with more transcripts present on the anterior side. We developed a novel FRAP-based assay, which revealed that lin-5 is translated/folded preferentially in the cytoplasm compared to centrosomes. Furthermore, we found that morpholino-mediated inhibition of lin-5 translation diminishes pulling forces on the posterior side during anaphase. Together, these findings lead us to propose that preferential translation/folding of lin-5 in the posterior cytoplasm following release of the mRNA from the posterior centrosome contributes to asymmetric cortical distribution of force generators, and thus to proper spindle positioning. Moreover, we found that the mRNA of an uncharacterized gene, era-1 is enriched on the anterior side of the zygote and is inherited by the anterior blastomeres. Similar to era-1 mRNA, a YFP fusion of ERA-1 protein is also asymmetrically distributed. Moreover, asymmetric distribution of both era-1 mRNA and YFP-ERA-1 protein requires the era-1 3'UTR. Furthermore, the RNA-binding protein MEX-5 is needed for both asymmetric era-1 mRNA localization and for its translational activation. Furthermore, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain normal levels of CHC-1. We demonstrate that CHC-1 is necessary for proper organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network results in excess centrosome movements. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension

Polarity-Dependent Asymmetric Distribution and MEX-5/6-Mediated Translational Activation of the Era-1 mRNA in C. elegans Embryos

The early C. elegans embryo is an attractive model system to investigate fundamental developmental processes. With the exception of mex-3 mRNA, maternally contributed mRNAs are thought to be distributed uniformly in the one-cell embryo. Here, we report and characterize the striking distribution of the mRNA encoding the novel protein ERA-1. We found that era-1 mRNA is enriched in the anterior of the one-cell embryo and present solely in anterior blastomeres thereafter. Although era-1 is not an essential gene, we uncovered that era-1 null mutant embryos are sensitive to slight impairment of embryonic polarity. We found that the asymmetric distribution of era-1 mRNA depends on anterior-posterior polarity cues and on the era-1 3'UTR. Similarly to the era-1 mRNA, the YFP-ERA-1 protein is enriched in anterior blastomeres. Interestingly, we found that the RNA-binding protein MEX-5 is required for era-1 mRNA asymmetry. Furthermore, we show that MEX-5, together with its partially redundant partner MEX-6, are needed to activate era-1 mRNA translation in anterior blastomeres. These findings lead us to propose that MEX-5/6-mediated regulation of era-1 mRNA contributes to robust embryonic development

Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos

Regulation of centrosome and spindle positioning is crucial for spatial cell division control. The one-cell Caenorhabditis elegans embryo has proven attractive for dissecting the mechanisms underlying centrosome and spindle positioning in a metazoan organism. Previous work revealed that these processes rely on an evolutionarily conserved force generator complex located at the cell cortex. This complex anchors the motor protein dynein, thus allowing cortical pulling forces to be exerted on astral microtubules emanating from microtubule organizing centers (MTOCs). Here, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain proper levels of CHC-1. We demonstrate that CHC-1 is necessary for normal organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network in otherwise wild-type embryos results in excess centrosome movements resembling those in chc-1(RNAi) embryos. We developed a 2D computational model to simulate cortical rigidity-dependent pulling forces, which recapitulates the experimental data and further demonstrates that excess centrosome movements are produced at medium cortical rigidity values. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension

Uniformly curated signaling pathways reveal tissue-specific cross-talks and support drug target discovery

Motivation: Signaling pathways control a large variety of cellular processes. However, currently, even within the same database signaling pathways are often curated at different levels of detail. This makes comparative and cross-talk analyses difficult. Results: We present SignaLink, a database containing 8 major signaling pathways from Caenorhabditis elegans, Drosophila melanogaster, and humans. Based on 170 review and approx. 800 research articles, we have compiled pathways with semi-automatic searches and uniform, well-documented curation rules. We found that in humans any two of the 8 pathways can cross-talk. We quantified the possible tissue- and cancer-specific activity of cross-talks and found pathway-specific expression profiles. In addition, we identified 327 proteins relevant for drug target discovery. Conclusions: We provide a novel resource for comparative and cross-talk analyses of signaling pathways. The identified multi-pathway and tissue-specific cross-talks contribute to the understanding of the signaling complexity in health and disease and underscore its importance in network-based drug target selection. Availability: http://SignaLink.orgComment: 9 pages, 4 figures, 2 tables and a supplementary info with 5 Figures and 13 Table

MEX-5/6-mediated translational activation of <i>era-1</i>.

<p><b>A-D</b> 4-cell embryos expressing YFP-ERA-1<i>[3’ era-1]</i> in control (A), <i>mex-5/6(RNAi)</i> (B), <i>par-3(RNAi)</i> (C) and <i>par-1(RNAi)</i> (D) conditions, stained with antibodies against GFP (green) and GPA-16 (red); DNA is viewed in blue. <b>E</b> Signal intensities at the cell-cell boundaries of ABa/ABp and EMS/P₂ relative to GPA-16 (Materials and Methods). Number of embryos quantified: control, n = 10; <i>mex-5/6(RNAi)</i>, n = 10; <i>par-3(RNAi)</i>, n = 9; <i>par-1</i>(<i>RNAi</i>), n = 10. Statistical analysis was performed using unpaired Student’s t-test to compare control with RNAi conditions for both anterior and posterior blastomeres, yielding the following p-values: <i>mex-5/6(RNAi)</i>, anterior: p = 4.42×10^–7; posterior: p = 1.46×10^–3; <i>par-3(RNAi)</i>, anterior: p = 9.32×10^–3; posterior: p = 0.04; <i>par-1(RNAi)</i>, anterior: p = 0.741; posterior: p = 1.81×10^–5. <b>F-G</b> 4-cell embryos expressing YFP-ERA-1<i>[3’ pie-1]</i> in control (F) or upon <i>mex-5/6(RNAi)</i> (G). Red arrowheads in F indicate cytoplasmic foci resembling endomembranes, suggesting that ERA-1 traffics through endosomal compartments. <b>H</b> Signal intensities at the cell-cell boundaries of ABa/ABp and EMS/P₂ relative to GPA-16 (Materials and Methods). Number of embryos quantified: control, n = 10<i>; mex-5/6(RNAi)</i>, n = 8. Statistical analysis was performed using unpaired Student’s t-test to compare control with <i>mex-5/6</i>(<i>RNAi</i>) for both anterior and posterior blastomeres, yielding the following p-values: <i>mex-5/6 (RNAi)</i>, anterior: p = 0.113; posterior: p = 0.205. <b>I</b> Working model of MEX-5/6-mediated control of <i>era-1</i> mRNA asymmetric distribution in P₀ and translational activation in 2-cell embryos; see also main text.</p

<i>era-1</i> mRNA is enriched on the anterior of early <i>C</i>. <i>elegans</i> embryos.

<p><b>A-D</b><i>era-1</i> mRNA distribution in the wild-type zygote (P₀), in prophase (A) or prometaphase (B), as well as in 2-cell (C) and 4-cell (D) embryos. Here and in other figures, the mRNA appears dark grey, the DNA light blue and scale bars represent 10 microns. <b>E-H</b><i>era-1</i> mRNA distribution in control (E), <i>era-1(RNAi)</i> (F), <i>par-3(RNAi)</i> (G) and <i>mex-5(RNAi)</i> (H) 2-cell embryos. <b>I</b> Corresponding quantifications of mRNA levels in AB and in P₁ (Materials and Methods). Number of embryos quantified: wild type, n = 9; <i>era-1(RNAi)</i>, n = 12; <i>par-3(RNAi)</i>, n = 10; <i>mex-5(RNAi)</i>, n = 7. In this and other figures, experiments were performed at least twice, mean values ± standard deviations (SD) are shown, with exact values given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120984#pone.0120984.s003" target="_blank">S1 Table</a>. Statistical analysis was performed using unpaired Student’s t-test to compare mRNA levels in AB versus P₁, yielding the following p-values: wild type—p = 5.67×10⁻¹⁰, <i>era-1(RNAi)</i>, p = 0.326; <i>par-3(RNAi)</i>, p = 0.907; <i>mex-5(RNAi)</i>, p = 0.287.</p

<i>yfp</i>-tagged <i>era-1</i> mRNA recapitulates endogenous <i>era-1</i> mRNA distribution in a 3’UTR-dependent manner.

<p><b>A, C, E</b> Schematic representation of <i>yfp-era-1[3’ era-1]</i> (A), <i>yfp[3’era-1]</i> (C) and <i>yfp-era-1[3’pie-1]</i> (E) constructs. <b>B, D, F</b> Localization of these constructs in 2-cell embryos. <b>G</b> Corresponding quantifications of mRNA levels in AB and in P₁ (Materials and Methods). Number of embryos quantified: <i>yfp-era-1[3’era-1]</i>, n = 9;. <i>yfp[3’era-1]</i>, n = 8; <i>yfp-era-1[3’pie-1]</i>, n = 10. Statistical analysis was performed using unpaired Student’s t-test to compare mRNA levels in AB versus P₁, yielding the following p-values: <i>yfp-era-1[3’ era-1]</i>, p = 1.03×10⁻⁷; <i>yfp[3’era-1]</i>, p = 2×10⁻⁴; <i>yfp-era-1[3’pie-1]</i>, p = 0.362. Note that anterior enrichment of <i>yfp-era-1[3’era-1]</i> is somewhat less pronounced than that of endogenous <i>era-1</i> mRNA, perhaps because the <i>era-1</i> 5’UTR contributes to mRNA distribution or because the presence of <i>yfp</i> perturbs localization.</p