The role of dynamin during polarity establishment in C. elegans one-cell embryos

C. elegans Embryonen im Einzellstadium legen die anterior-posterior Polaritätsachse bereits kurze Zeit nach der Befruchtung fest. Die Entwicklung der Zellpolarität hängt von den Zentrosomen ab, welche vom Spermium zur Verfügung gestellt werden. Dies führt zu einer Veränderung der Aktivität des Aktin-Myosin-Netzwerkes in einer Hälfte des Embryos. Die Nähe zwischen Zentrosom und Cortex während der Entwicklung der Zellpolarität in C. elegans Einzellembryonen tritt auch in vielen anderen Zellpolarisierungen auf. Darunter wären zum Beispiel die T-Zellpolarisierung und die Spezifizierung hippokampischer Axone. In beiden dieser Beispiele spielt der intrazelluläre Vesikeltransport eine wesentliche Rolle für die Polarisierung der Zelle. Ich habe mich daher gefragt, ob der intrazelluläre Vesikeltransport auch in der Polaritätsentwicklung in C. elegans Embryonen eine grundlegende Rolle spielt. Für meine Experimente benützte ich ein bereits isoliertes temperatur-sensitives Allel von Dynamin, einer GTPase, die für die Vesikelbildung verantwortlich ist. Mit Hilfe von quantitativer Time-Lapse-Mikroskopie werden die Folgen der Inaktivierung von Dynamin zu bestimmten Zeitpunkten während der Entwicklung der Zellpolarität analysiert. Meine anfänglichen Experimente deuten darauf hin, dass Dynamin kurz vor oder während der Einleitung der Polaritätsentwicklung gebraucht wird, ähnlich wie die Zentrosomen. Manchmal jedoch scheint es als ob Dynamin auch noch einige Zeit nach der Einleitung der Zellpolarisierung gebraucht wird. Weitere Experimente sollten Aufschluss über den Effekt der Inaktivierung von Dynamin auf verschiedene Komponenten des Signalweges geben, der zur Entwicklung der Zellpolarität führt. Dafür wurden verschiedene neue Wurmstämme durch Kreuzungen generiert, die ein GFP-getaggtes Protein und das temperatursensitive Allel von Dynamin enthalten. Die GFP-getaggten Proteine sind bestimmte Marker für Komponenten des Vesikeltransportwegs, zum Beispiel RAB-5::GFP als Marker für frühe Endosomen. Manche der GFP-Stämmen standen bereits zur Verfügung, andere wurden durch Klonierungen und Wurmbombardierung erst neu hergestellt. Die mögliche Überexpression von PAR-3, einem Marker für anteriore Polarität, in den PAR-3::GFP Würmern ohne Dynamin führte zu einer stärkeren Ausprägung des Phenotyps gegenüber den temperatur-sensitiven Würmern alleine. Dies könnte möglicherweise durch die Veränderung des anterior-posterior PAR Proteinverhältnisses verursacht werden. In diesem Fall wäre es mögich, dass Dynamin parallel zu PAR-2, einem posterioren Polaritätsprotein, arbeitet. Diese Hypothese wird durch par-2 RNAi Experimente noch verstärkt, denn sie ergaben einen ähnlichen Phenotyp wie PAR-3::GFP in temperatur-sensitiven Würmern. Um noch näher auf das Thema einzugehen, machte ich weitere Versuche mit RNAi von anderen Vesikeltransportproteinen und behandelte Embryonen mit speziellen Vesikeltransportinhibitoren.One-cell C. elegans embryos establish the anterior-posterior polarity axis shortly after fertilization. Polarity establishment depends on a cue from the sperm-derived centrosomes, which subsequently leads to a change in acto-myosin activity in one half of the embryo. Centrosome-cortex proximity during polarity establishment in one-cell C. elegans embryos is paralleled in several other cell polarization events, specifically in T cell polarization and hippocampal axon specification. In both of these polarized cells, vesicle trafficking plays an integral role in polarization. I therefore wondered whether vesicle trafficking also plays an essential role in polarity establishment in C. elegans embryos. To study this question, I am taking advantage of a previously isolated temperature-sensitive allele of dynamin, a GTPase required for vesicle formation. Using quantitative time-lapse microscopy, I am analyzing the effects of dynamin inactivation at different times during polarity establishment. My initial experiments suggest that dynamin is required immediately prior to or during polarity initiation. This is similar to the temporal requirement for centrosomes. In some cases, dynamin is needed for a short time after initiation of polarity establishment. I am continuing to investigate the mechanism by which dynamin contributes to polarity establishment by analyzing the effect of dynamin inactivation on various components of the polarity establishment pathway. Therefore I generated worm strains expressing markers for specific components of the vesicle transport pathway. These strains and existing GFP lines, e.g. RAB-5::GFP, were crossed to the temperature sensitive dynamin strain to investigate the behavior of the endomembrane system in the absence of dynamin function. Overexpression of GFP::PAR-3, a marker of anterior polarity, in embryos lacking dynamin function led to more severe defects during polarity establishment than in the dynamin mutant alone. This might result from the change in the ratio of anterior and posterior PAR proteins, suggesting that dynamin could work in parallel to PAR-2, a posterior polarity protein. This idea is supported by experiments showing that dynamin mutant embryos depleted of PAR-2 have more severe polarity establishment defects than dynamin mutants alone. To further investigate this topic, I looked at the polarity establishment phenotypes of embryos depleted of other vesicle transport proteins by RNAi or treated with specific vesicle transport inhibitors

GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult <i>C. elegans</i> Germ Line

<div><p>To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult <i>Caenorhabditis elegans</i> reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that <i>gld-4</i> activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with <i>glp-1</i> mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of <i>glp-1</i> mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.</p></div

Differential GLD-4 and GLD-2 expression in the proliferative zone is FBF dependent.

<p>(A) GLD-4 expression is equal across the distal germ line. GLD-2 intensities increase from low-to-high in a distal-to-proximal manner. Extruded gonads of indicated genotype stained with DAPI, α-GLD-2, α-GLD-4, and α-GLH-2 as a positive tissue penetration control (not shown). Asterisk, distal tip; arrowhead, mitosis-to-meiosis boundary. (B,C) Distal GLD-2 expression is repressed by <i>fbf</i> activity. (B) Example of an <i>fbf</i>(RNAi) immunostained extruded gonad. For the complete RNAi experiment see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647.s001" target="_blank">Figure S1</a>. (C) Quantification of the complete <i>fbf</i>(RNAi) experiment. Four different regions of nine germ lines per genotype were analyzed in their median, primarily cytoplasmic area. Error bars are SEM. ***, p<0.001; **, p<0.01; *, p<0.05; bars without indicated p value are statistically not significant (Student's t-test). (D, E) FBF binds specifically to at least one of the five predicted sequence elements in the <i>gld-2</i> 3′UTR. (D) Schematic drawing of the 1094 nt long <i>gld-2</i> 3′UTR. Sequence alignment of FBF-binding element consensus (FBE cons.) sequence <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647-Lamont1" target="_blank">[14]</a> and the conserved FBE4 element in three <i>Caenorhabditis</i> species: <i>ce</i>, <i>C. elegans</i>; <i>cb</i>, <i>C. briggsae</i>; <i>cr</i>, <i>C. remanei</i>. pA indicates beginning of the poly(A) tail. (E) Yeast three-hybrid assay. RNA hybrid and Gal4-protein fusions are indicated. FBF-1, FBF-2 and PUF-5 belong to same RNA-binding protein family. Note, the wild-type (wt) and mutant (mut) sequence of FBE4 tested is larger than the given sequences (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#s4" target="_blank">Materials and Methods</a>). A positive and negative control RNA was included (not shown) and protein expression was confirmed by western blotting (not shown). (F) LAP-tagged FBF-2 associates with endogenous <i>gld-2</i> mRNA in RNA-coimmunoprecipitation experiments (RIPs) directed against the GFP portion of the fusion protein.</p

<i>glp-1</i> mRNA associates with GLD-4 and is a likely target of poly(A) tail extension and translational activation.

<p>(A,B) RNA-coimmunoprecipitation experiments (RIPs) of GLD-4 and GLS-1 proteins specifically enrich <i>glp-1</i> mRNA and the positive control <i>gld-1</i> mRNA. <i>eft-3</i> and <i>rpl-11.1</i> mRNA served as negative controls. (A) A representative ethidium bromide-stained agarose gel of semiquantitative RT-PCR products from three independent biological replicates. (B) Quantitative RT-PCR measurements of three additional RIPs. Error bars are SEM. ***, p<0.001; **, p<0.01; n.s., not significant (Student's t-test). (C,D) Translational efficiency of <i>glp-1</i> mRNA depends on <i>gld-4</i> activity. The data are representative of three independent biological experiments. (C) Polysome gradient. Top is to the right; grey peaks represent optical density read of 258 nm; the peaks of the large ribosomal subunit (60S), monosomes (80S), and polysomes are indicated. Relative <i>glp-1</i> mRNA levels are lower in polysome fractions of <i>gld-4</i>(RNAi) as measured by RT-qPCR. (D) Quantification and comparison of <i>glp-1</i> mRNA in pooled polysomal (polys.) and non-polysomal (non-polys.) fractions. Each measurement was normalized to an internal spike-in control (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#s4" target="_blank">Materials and Methods</a>). Error bars are SEM. *, p<0.05; n.s., not significant (Student's t-test). (E,F) poly(A) tails of <i>glp-1</i> mRNA are reduced upon <i>gld-4</i>(RNAi). (E) Representative PAT assay (n = 2) of the <i>glp-1</i> mRNA material from (C) and the gradient input material. Nucleotide size marker to the left. Lane 7 reflects a 3′UTR with a strongly reduced poly(A) tail (pA) after RNAase H and oligo dT treatment (H/dT). (F) Line scans of PAT assay from (E).</p

<i>gld-4</i> and <i>gls-1</i> promote onset of differentiation in parallel to <i>gld-2</i> and <i>gld-1</i>.

<p>(A–F) Distal region of extruded gonads stained with DAPI, and with α-REC-8, α-HIM-3 (A–E), and α-pSUN-1 (F) antibodies. Asterisk, distal tip; arrowhead, mitosis-to-meiosis boundary. Scale bars: 50 µm. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen-1004647-t001" target="_blank">Table 1</a> for the total number of analyzed germ lines. (E–F) <i>gld-1 gls-1</i> double mutant germ lines possess a mitosis-to-meiosis boundary, albeit HIM-3 fails to be detected in E. A strong reduction of nucleoplasmic REC-8, the appearance of crescent-shaped nuclei in meiotic prophase (circles in E), and the abundant expression of pSUN-1 (F) reveal onset of differentiation.</p

Translational regulators maintain a robust proliferative zone in the adult germ line.

<p>(A) Expanded genetic circuitry of primarily translation regulators that fine-tunes the balance between proliferation and differentiation. A light grey box highlights pathway members that regulate differentiation onset. A dark grey box highlights redundant activities that promote GLD-1 expression, primarily when cells commit into meiotic progression (dashed line). See text for details. Note, this simplified circuitry focuses on the RNA regulatory network downstream of GLP-1/Notch and does neither include other known downstream RNA targets nor potential upstream protein regulators. (B) Diagram of translational control examples in proliferative germ cells. Next to <i>glp-1</i> mRNA, GLD-4 may also translationally activate additional mRNAs, encoding proliferation-promoting genes. Additional FBF-regulated mRNAs are known that promote the meiotic program. See text for details. (C) Diagram of translational control examples in differentiating germ cells. Additional GLD-regulated mRNAs are known that promote the proliferative fate. See text for details.</p

<i>gld-4</i> and <i>gls-1</i> promote onset of differentiation in parallel to <i>gld-2</i> and <i>nos-3</i>.

<p>(A) The current genetic wiring of the core regulatory network that regulates the balance between proliferation and differentiation onset. Two genetic pathways of redundantly acting translational regulators operate downstream of the translational repressor, FBF. A third, yet undefined, pathway has been evoked <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647-Fox1" target="_blank">[4]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647-Hansen1" target="_blank">[6]</a>. Note that not all genes are equivalent in the two pathways; only <i>gld-3 nos-3</i> double mutants lack any signs of differentiation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647-Eckmann1" target="_blank">[7]</a>. (B–H) Complete gonads stained with DAPI (left column), and with α-REC-8 and α-HIM-3 (right column) antibodies. Dashed boxes in B and C are close ups of D and E. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen-1004647-t001" target="_blank">Table 1</a> for the total number of analyzed germ lines. (D–H) Distal region of extruded gonads. Asterisk, distal tip; arrowhead, mitosis-to-meiosis boundary. Scale bars: 50 µm.</p

Separable Roles for a Caenorhabditis elegans RMI1 Homolog in Promoting and Antagonizing Meiotic Crossovers Ensure Faithful Chromosome Inheritance.

During the first meiotic division, crossovers (COs) between homologous chromosomes ensure their correct segregation. COs are produced by homologous recombination (HR)-mediated repair of programmed DNA double strand breaks (DSBs). As more DSBs are induced than COs, mechanisms are required to establish a regulated number of COs and to repair remaining intermediates as non-crossovers (NCOs). We show that the Caenorhabditis elegans RMI1 homolog-1 (RMH-1) functions during meiosis to promote both CO and NCO HR at appropriate chromosomal sites. RMH-1 accumulates at CO sites, dependent on known pro-CO factors, and acts to promote CO designation and enforce the CO outcome of HR-intermediate resolution. RMH-1 also localizes at NCO sites and functions in parallel with SMC-5 to antagonize excess HR-based connections between chromosomes. Moreover, RMH-1 also has a major role in channeling DSBs into an NCO HR outcome near the centers of chromosomes, thereby ensuring that COs form predominantly at off-center positions

RMH-1 promotes the bias for CO formation on chromosome arms.

<p>(A) Schematics of crosses to obtain the progeny of singled F2 individuals subjected to Next Generation Sequencing (NGS) for SNP analysis. White insert indicates the WT (Bristol) background, and black insert indicates the Hawaiian background. (B) Quantification of the overall recombination frequencies for assayed chromosomes; stacked bar graph indicates the fraction of meiotic products with zero, one, or two COs. For WT (<i>n</i> = 36 chromatids), for <i>rmh-1(jf54)</i> (<i>n</i> = 40 chromatids), and for <i>rmh-1(tn309)</i> (<i>n</i> = 45 chromatids). The frequency of COs was not found to be different between WT and both mutants (Chi² test). (C) Scheme of the different chromosomes used during the recombination assay. The chromosome domains (left arm in blue, center in yellow, and right arm in purple) are correlated with the physical map of each chromosome. (D) Locations of the recombination events (assayed for chromosomes X, IV, and V) in WT (<i>n</i> = 17 COs: three events on X, four on II, four on IV, and six on V), for <i>rmh-1(jf54)</i> (<i>n</i> = 20 COs: 11 events on II and 9 on V), and <i>rmh-1(tn309)</i> (<i>n</i> = 21 COs: nine events on X, nine on IV, and three on V); also see the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002412#sec015" target="_blank">Experimental Procedures</a> section. The relative distribution of COs in the center versus arm domains differed from the WT for <i>tn309</i> (<i>p</i> = 0.046, Chi² test) and for <i>jf54</i>, (p = 0.062, Chi² test).</p