Comparative Analysis of Pdf-Mediated Circadian Behaviors Between Drosophila melanogaster and D. virilis

PAR proteins (partitioning defective) are major regulators of cell polarity and asymmetric cell division. One of the par genes, par-1, encodes a Ser/Thr kinase that is conserved from yeast to mammals. In Caenorhabditis elegans, par-1 governs asymmetric cell division by ensuring the polar distribution of cell fate determinants. However the precise mechanisms by which PAR-1 regulates asymmetric cell division in C. elegans remain to be elucidated. We performed a genomewide RNAi screen and identified six genes that specifically suppress the embryonic lethal phenotype associated with mutations in par-1. One of these suppressors is mpk-1, the C. elegans homolog of the conserved mitogen activated protein (MAP) kinase ERK. Loss of function of mpk-1 restored embryonic viability, asynchronous cell divisions, the asymmetric distribution of cell fate specification markers, and the distribution of PAR-1 protein in par-1 mutant embryos, indicating that this genetic interaction is functionally relevant for embryonic development. Furthermore, disrupting the function of other components of the MAPK signaling pathway resulted in suppression of par-1 embryonic lethality. Our data therefore indicates that MAP kinase signaling antagonizes PAR-1 signaling during early C. elegans embryonic polarization.ASYMMETRIC cell division, a process in which a mother cell divides in two different daughter cells, is a fundamental mechanism to achieve cell diversity during development. We use the early embryo of Caenorhabditis elegans as a model system to study asymmetric cell division. The C. elegans one-cell embryo divides asymmetrically along its anteroposterior axis, generating two cells of different sizes and fates: the larger anterior daughter cell will generate somatic tissues while the smaller posterior daughter cell will generate the germline (Sulston et al. 1983).A group of proteins called PAR proteins (partitioning defective) is required for asymmetric cell division in C. elegans (Kemphues et al. 1988). Depletion of any of the seven par genes (par-1 to -6 and pkc-3) leads to defects in asymmetric cell division and embryonic lethality (Kemphues et al. 1988; Kirby et al. 1990; Tabuse et al. 1998; Hung and Kemphues 1999; Hao et al. 2006). PAR-3 and PAR-6 are conserved proteins that contain PDZ-domains and form a complex with PKC-3 (Etemad-Moghadam et al. 1995; Izumi et al. 1998; Tabuse et al. 1998; Hung and Kemphues 1999). This complex becomes restricted to the anterior cortex of the embryo in response to spatially defined actomyosin contractions occurring in the embryo upon fertilization (Goldstein and Hird 1996; Munro et al. 2004). The posterior cortex of the embryo that becomes devoid of the anterior PAR proteins is occupied by the RING protein PAR-2 and the Ser/Thr kinase PAR-1 (Guo and Kemphues 1995; Boyd et al. 1996; Cuenca et al. 2003). Once polarized, the anterior and posterior PAR proteins mutually exclude each other from their respective cortices (Etemad-Moghadam et al. 1995; Boyd et al. 1996; Cuenca et al. 2003; Hao et al. 2006). Loss of function of the gene par-1, as opposed to loss of most other par genes, results in embryos that exhibit only subtle effects on the polarized cortical domains occupied by the other PAR proteins (Cuenca et al. 2003). However defects in this gene are associated with a more symmetric division in size, an aberrant distribution of cell fate specification markers, altered cell fates of the daughter cells of the embryo, and ultimately embryonic lethality (Kemphues et al. 1988; Guo and Kemphues 1995).PAR-1 controls asymmetric cell division and cell fate specification by regulating the localization of the two highly similar CCCH-type zinc-finger proteins MEX-5 and MEX-6 (referred to as MEX-5/6). MEX-5 and MEX-6 are 70% identical in their amino acid sequence and fulfill partially redundant functions in the embryo (Schubert et al. 2000). In wild-type animals, endogenous MEX-5 and GFP fusions of MEX-6 localize primarily to the anterior of the embryo while both proteins are evenly distributed in par-1 mutant embryos (Schubert et al. 2000; Cuenca et al. 2003). This suggests that in wild-type animals, PAR-1 acts in part by restricting MEX-5 and MEX-6 to the anterior of the embryo. The precise mechanism of this regulation is not known, but an elegant study performed for MEX-5 indicates that differential protein mobility in the anterior and posterior cytoplasm of the one-cell embryo contributes to this asymmetry (Tenlen et al. 2008). While increased mobility in the posterior of the one-cell embryo correlates with a par-1- and par-4-dependent phosphorylation on MEX-5, the kinase directly phosphorylating MEX-5 remains to be identified (Tenlen et al. 2008).Some of the phenotypes associated with loss of par-1 function are dependent on the function of mex-5 and mex-6. First, loss of function of par-1 leads to a decreased stability and aberrant localization of the posterior cell fate specification marker PIE-1, a protein that is usually inherited by the posterior daughter cell in wild-type animals and ensures the correct specification of the germline (Mello et al. 1996; Seydoux et al. 1996). This decreased stability is dependent on mex-5/6 function as PIE-1 levels are restored, albeit with symmetrical distribution, in mex-6(RNAi); mex-5(RNAi); par-1(b274) embryos (Schubert et al. 2000; Cuenca et al. 2003; Derenzo et al. 2003). Second, embryos lacking par-1 function exhibit decreased amounts of P granules in the one-cell embryo, while these markers are present in mex-6(pk440); mex-5(zu199); par-1(RNAi) embryos of comparable age (Cheeks et al. 2004). Third, in par-1(RNAi) one-cell embryos the posterior cortical domain occupied by the polarity protein PAR-2 is extended anteriorly, when compared to wild-type embryos (Cuenca et al. 2003). This anterior extension is rescued in embryos deficient for both par-1 and mex-5/6 (Cuenca et al. 2003). Taken together, these results indicate that par-1 acts in the embryo—at least in part—by regulating the localization and/or activity of the proteins MEX-5 and MEX-6. However, it remains unclear whether other proteins can modulate PAR-1 function to affect MEX-5/6 activity.To gain insight into the mechanisms of par-1 function in the embryo, we sought to identify genes that act together with par-1 during embryonic development. We performed an RNAi-based screen for genetic interactors of the temperature-sensitive allele par-1(zu310), using the embryonic lethal phenotype of this mutant as a readout. This method has proven successful in previous screens to identify genes involved in early embryonic processes (Labbé et al. 2006; O''Rourke et al. 2007). We were able to identify six genes that, upon disruption of their function, suppress the embryonic lethal phenotype of par-1 mutant embryos. One of these genes is mpk-1, the C. elegans homolog of the highly conserved MAP kinase ERK. Closer analysis subsequently showed that reduction of function of mpk-1 not only increases viability of par-1 mutant embryos, but also reverts several polarity phenotypes associated with loss of function of par-1. Our data indicate that mpk-1 antagonizes par-1 activity to regulate polarization and asymmetric cell divisions in the early embryo.     

The Jaw of the Worm: GTPase-activating Protein EAT-17 Regulates Grinder Formation in Caenorhabditis elegans

Constitutive transport of cellular materials is essential for cell survival. Although multiple small GTPase Rab proteins are required for the process, few regulators of Rabs are known. Here we report that EAT-17, a novel GTPase-activating protein (GAP), regulates RAB-6.2 function in grinder formation in Caenorhabditis elegans. We identified EAT-17 as a novel RabGAP that interacts with RAB-6.2, a protein that presumably regulates vesicle trafficking between Golgi, the endoplasmic reticulum, and plasma membrane to form a functional grinder. EAT-17 has a canonical GAP domain that is critical for its function. RNA interference against 25 confirmed and/or predicted RABs in C. elegans shows that RNAi against rab-6.2 produces a phenotype identical to eat-17. A directed yeast two-hybrid screen using EAT-17 as bait and each of the 25 RAB proteins as prey identifies RAB-6.2 as the interacting partner of EAT-17, confirming that RAB-6.2 is a specific substrate of EAT-17. Additionally, deletion mutants of rab-6.2 show grinder defects identical to those of eat-17 loss-of-function mutants, and both RAB-6.2 and EAT-17 are expressed in the terminal bulb of the pharynx where the grinder is located. Collectively, these results suggest that EAT-17 is a specific GTPase-activating protein for RAB-6.2. Based on the conserved function of Rab6 in vesicular transport, we propose that EAT-17 regulates the turnover rate of RAB-6.2 activity in cargo trafficking for grinder formation.     

Translational Control of the Oogenic Program by Components of OMA Ribonucleoprotein Particles in Caenorhabditis elegans

The oocytes of most sexually reproducing animals arrest in meiotic prophase I. Oocyte growth, which occurs during this period of arrest, enables oocytes to acquire the cytoplasmic components needed to produce healthy progeny and to gain competence to complete meiosis. In the nematode Caenorhabditis elegans, the major sperm protein hormone promotes meiotic resumption (also called meiotic maturation) and the cytoplasmic flows that drive oocyte growth. Prior work established that two related TIS11 zinc-finger RNA-binding proteins, OMA-1 and OMA-2, are redundantly required for normal oocyte growth and meiotic maturation. We affinity purified OMA-1 and identified associated mRNAs and proteins using genome-wide expression data and mass spectrometry, respectively. As a class, mRNAs enriched in OMA-1 ribonucleoprotein particles (OMA RNPs) have reproductive functions. Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3′-untranslated regions (3′-UTRs). Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2. We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs. In the accompanying article in this issue, we show that LIN-41 prevents meiotic maturation and promotes oocyte growth in opposition to OMA-1/2. Taken together, these data support a model in which the conserved regulators of mRNA translation LIN-41 and OMA-1/2 coordinately control oocyte growth and the proper spatial and temporal execution of the meiotic maturation decision.     

Caenorhabditis elegans PIG-1/MELK Acts in a Conserved PAR-4/LKB1 Polarity Pathway to Promote Asymmetric Neuroblast Divisions

Asymmetric cell divisions produce daughter cells with distinct sizes and fates, a process important for generating cell diversity during development. Many Caenorhabditis elegans neuroblasts, including the posterior daughter of the Q cell (Q.p), divide to produce a larger neuron or neuronal precursor and a smaller cell that dies. These size and fate asymmetries require the gene pig-1, which encodes a protein orthologous to vertebrate MELK and belongs to the AMPK-related family of kinases. Members of this family can be phosphorylated and activated by the tumor suppressor kinase LKB1, a conserved polarity regulator of epithelial cells and neurons. In this study, we present evidence that the C. elegans orthologs of LKB1 (PAR-4) and its partners STRAD (STRD-1) and MO25 (MOP-25.2) regulate the asymmetry of the Q.p neuroblast division. We show that PAR-4 and STRD-1 act in the Q lineage and function genetically in the same pathway as PIG-1. A conserved threonine residue (T169) in the PIG-1 activation loop is essential for PIG-1 activity, consistent with the model that PAR-4 (or another PAR-4-regulated kinase) phosphorylates and activates PIG-1. We also demonstrate that PIG-1 localizes to centrosomes during cell divisions of the Q lineage, but this localization does not depend on T169 or PAR-4. We propose that a PAR-4-STRD-1 complex stimulates PIG-1 kinase activity to promote asymmetric neuroblast divisions and the generation of daughter cells with distinct fates. Changes in cell fate may underlie many of the abnormal behaviors exhibited by cells after loss of PAR-4 or LKB1.     

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-of-function mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-to-arginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/VAChT and synaptobrevin.     

A Genome-Wide RNAi Screen for Enhancers of par Mutants Reveals New Contributors to Early Embryonic Polarity in Caenorhabditis elegans

The par genes of Caenorhabditis elegans are essential for establishment and maintenance of early embryo polarity and their homologs in other organisms are crucial polarity regulators in diverse cell types. Forward genetic screens and simple RNAi depletion screens have identified additional conserved regulators of polarity in C. elegans; genes with redundant functions, however, will be missed by these approaches. To identify such genes, we have performed a genome-wide RNAi screen for enhancers of lethality in conditional par-1 and par-4 mutants. We have identified 18 genes for which depletion is synthetically lethal with par-1 or par-4, or both, but produces little embryo lethality in wild type. Fifteen of the 18 genes identified in our screen are not previously known to function in C. elegans embryo polarity and 11 of them also increase lethality in a par-2 mutant. Among the strongest synthetic lethal genes, polarity defects are more apparent in par-2 early embryos than in par-1 or par-4, except for strd-1(RNAi), which enhances early polarity phenotypes in all three mutants. One strong enhancer of par-1 and par-2 lethality, F25B5.2, corresponds to nop-1, a regulator of actomyosin contractility for which the molecular identity was previously unknown. Other putative polarity enhancers identified in our screen encode cytoskeletal and membrane proteins, kinases, chaperones, and sumoylation and deubiquitylation proteins. Further studies of these genes should give mechanistic insight into pathways regulating establishment and maintenance of cell polarity.     

Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase’s role in germline development in higher eukaryotes.     

Asymmetric Neuroblast Divisions Producing Apoptotic Cells Require the Cytohesin GRP-1 in Caenorhabditis elegans

Cytohesins are Arf guanine nucleotide exchange factors (GEFs) that regulate membrane trafficking and actin cytoskeletal dynamics. We report here that GRP-1, the sole Caenorhabditis elegans cytohesin, controls the asymmetric divisions of certain neuroblasts that divide to produce a larger neuronal precursor or neuron and a smaller cell fated to die. In the Q neuroblast lineage, loss of GRP-1 led to the production of daughter cells that are more similar in size and to the transformation of the normally apoptotic daughter into its sister, resulting in the production of extra neurons. Genetic interactions suggest that GRP-1 functions with the previously described Arf GAP CNT-2 and two other Arf GEFs, EFA-6 and BRIS-1, to regulate the activity of Arf GTPases. In agreement with this model, we show that GRP-1’s GEF activity, mediated by its SEC7 domain, is necessary for the posterior Q cell (Q.p) neuroblast division and that both GRP-1 and CNT-2 function in the Q.posterior Q daughter cell (Q.p) to promote its asymmetry. Although functional GFP-tagged GRP-1 proteins localized to the nucleus, the extra cell defects were rescued by targeting the Arf GEF activity of GRP-1 to the plasma membrane, suggesting that GRP-1 acts at the plasma membrane. The detection of endogenous GRP-1 protein at cytokinesis remnants, or midbodies, is consistent with GRP-1 functioning at the plasma membrane and perhaps at the cytokinetic furrow to promote the asymmetry of the divisions that require its function.     

Analysis of PHA-1 Reveals a Limited Role in Pharyngeal Development and Novel Functions in Other Tissues

PHA-1 encodes a cytoplasmic protein that is required for embryonic morphogenesis and attachment of the foregut (pharynx) to the mouth (buccal capsule). Previous reports have in some cases suggested that PHA-1 is essential for the differentiation of most or all pharyngeal cell types. By performing mosaic analysis with a recently acquired pha-1 null mutation (tm3671), we found that PHA-1 is not required within most or all pharyngeal cells for their proper specification, differentiation, or function. Rather, our evidence suggests that PHA-1 acts in the arcade or anterior epithelial cells of the pharynx to promote attachment of the pharynx to the future buccal capsule. In addition, PHA-1 appears to be required in the epidermis for embryonic morphogenesis, in the excretory system for osmoregulation, and in the somatic gonad for normal ovulation and fertility. PHA-1 activity is also required within at least a subset of intestinal cells for viability. To better understand the role of PHA-1 in the epidermis, we analyzed several apical junction markers in pha-1(tm3671) homozygous embryos. PHA-1 regulates the expression of several components of two apical junction complexes including AJM-1–DLG-1/discs large complex and the classical cadherin–catenin complex, which may account for the role of PHA-1 in embryonic morphogenesis.     

Systematic Analyses of rpm-1 Suppressors Reveal Roles for ESS-2 in mRNA Splicing in Caenorhabditis elegans

The PHR (Pam/Highwire/RPM-1) family of ubiquitin E3 ligases plays conserved roles in axon patterning and synaptic development. Genetic modifier analysis has greatly aided the discovery of the signal transduction cascades regulated by these proteins. In Caenorhabditis elegans, loss of function in rpm-1 causes axon overgrowth and aberrant presynaptic morphology, yet the mutant animals exhibit little behavioral deficits. Strikingly, rpm-1 mutations strongly synergize with loss of function in the presynaptic active zone assembly factors, syd-1 and syd-2, resulting in severe locomotor deficits. Here, we provide ultrastructural evidence that double mutants, between rpm-1 and syd-1 or syd-2, dramatically impair synapse formation. Taking advantage of the synthetic locomotor defects to select for genetic suppressors, previous studies have identified the DLK-1 MAP kinase cascade negatively regulated by RPM-1. We now report a comprehensive analysis of a large number of suppressor mutations of this screen. Our results highlight the functional specificity of the DLK-1 cascade in synaptogenesis. We also identified two previously uncharacterized genes. One encodes a novel protein, SUPR-1, that acts cell autonomously to antagonize RPM-1. The other affects a conserved protein ESS-2, the homolog of human ES2 or DGCR14. Loss of function in ess-2 suppresses rpm-1 only in the presence of a dlk-1 splice acceptor mutation. We show that ESS-2 acts to promote accurate mRNA splicing when the splice site is compromised. The human DGCR14/ES2 resides in a deleted chromosomal region implicated in DiGeorge syndrome, and its mutation has shown high probability as a risk factor for schizophrenia. Our findings provide the first functional evidence that this family of proteins regulate mRNA splicing in a context-specific manner.     

A Novel Nondevelopmental Role of the SAX-7/L1CAM Cell Adhesion Molecule in Synaptic Regulation in Caenorhabditis elegans

The L1CAM family of cell adhesion molecules is a conserved set of single-pass transmembrane proteins that play diverse roles required for proper nervous system development and function. Mutations in L1CAMs can cause the neurological L1 syndrome and are associated with autism and neuropsychiatric disorders. L1CAM expression in the mature nervous system suggests additional functions besides the well-characterized developmental roles. In this study, we demonstrate that the gene encoding the Caenorhabditis elegans L1CAM, sax-7, genetically interacts with gtl-2, as well as with unc-13 and rab-3, genes that function in neurotransmission. These sax-7 genetic interactions result in synthetic phenotypes that are consistent with abnormal synaptic function. Using an inducible sax-7 expression system and pharmacological reagents that interfere with cholinergic transmission, we uncovered a previously uncharacterized nondevelopmental role for sax-7 that impinges on synaptic function.