Synaptic neurotransmission protein UNC-13 affects RNA interference in neurons

Caenorhabditis elegans UNC-13 is an integral component of the synaptic vesicle cycle, functioning in the priming step. A recent yeast two-hybrid screen against UNC-13 identified three interacting proteins that are thought to function in pathways other than neurotransmitter release. One such protein, ERI-1, negatively regulates exogenous RNA interference in the nervous system and other tissues. This study investigates a role for UNC-13 in RNAi through analysis of RNAi penetrance in unc-13 and eri-1 mutant strains. Feeding these strains double stranded RNA corresponding to a neuronally expressed GFP reporter resulted in a significant reduction of GFP in double mutants compared to GFP expression in eri-1 mutants, indicating that UNC-13 functions in conjunction with ERI-1 in RNAi. There is no evidence for altered neurotransmission in eri-1 mutants.     

The Caenorhabditis elegans NR4A nuclear receptor is required for spermatheca morphogenesis

The gene nhr-6 encodes the Caenorhabditis elegans ortholog of the NR4A nuclear receptor. We determined the biological functions of NHR-6 through the isolation and characterization of a deletion allele of nhr-6, lg6001. We demonstrate that nhr-6 has an essential role in the development of the C. elegans somatic gonad. Specifically, nhr-6 is required for the development of the hermaphrodite spermatheca, a somatic gonad organ that serves as the site of sperm storage and oocyte fertilization. Using a variety of spermatheca cell markers, we have determined that loss of nhr-6 function causes severe morphological defects in the spermatheca and associated spermathecal valves. This appears to be due to specific requirements for nhr-6 in regulating cell proliferation and cell differentiation during development of these structures. The improper development of these structures in nhr-6(lg6001) mutants leads to defects in ovulation and significantly reduced fecundity of C. elegans hermaphrodites. The phenotypes of nhr-6(lg6001) mutants are consistent with a role for nhr-6 in organogenesis, similar to the functions of its mammalian homologs.     

The anchor cell initiates dorsal lumen formation during C. elegans vulval tubulogenesis

Tubulogenesis and lumen formation are critical to the development of most organs. We study Caenorhabditis elegans vulval and uterine development to probe the complex mechanisms that mediate these events. Development of the vulva and the ventral uterus is coordinated by the inductive cell-signaling activity of a gonadal cell called the anchor cell (AC). We demonstrate that in addition to its function in specifying fate, the AC directly promotes dorsal vulval tubulogenesis. Two types of mutants with defective anchor cell behavior reveal that anchor cell invasion of the vulva is important for forming the toroidal shape of the dorsal vulval cell, vulF. In fos-1 mutants, where the AC cannot breakdown the basement membranes between the gonad and the vulva, and in mutants in unc-6 netrin or its receptor unc-40, which cause AC migration defects, the AC fails to invade the vulva and no lumen is formed in vulF. By examining GFP markers of dorsal vulval cell fate, we demonstrate that fate specification defects do not account for the aberrant vulF shape. We propose that the presence of the AC in the center of the developing vulF toroid is required for dorsal vulval lumen formation to complete vulval tubulogenesis.     

SDN-1/syndecan regulates growth factor signaling in distal tip cell migrations in C. elegans

Mutations in the sdn-1/syndecan gene act as genetic enhancers of the ventral-to-dorsal distal tip cell (DTC) migration defects caused by a weak allele of the netrin receptor gene unc-5. The sdn-1(ev697) allele was identified in a genetic screen for enhancers of unc-5 DTC migration defects, and carried a nonsense mutation predicted to truncate the SDN-1 protein prior to the transmembrane domain. The enhancement of unc-5 caused by an sdn-1 mutation was rescued by expression of wild-type sdn-1 in the hypodermis or nervous system rather than the DTCs, indicating a cell non-autonomous function of sdn-1. The enhancement was also partially reversed by mutations in the egl-17/FGF or egl-20/Wnt genes, suggesting that sdn-1 affects UNC-5 function through a mis-regulation of signaling in growth factor pathways. egl-20 reporter constructs exhibited increased and mis-localized EGL-20 distribution in sdn-1 mutants compared to wild-type animals. Finally, using loss of function mutations, we show that egl-17/Fgf and egl-20/Wnt are partially redundant in regulating the migration pattern of the posterior DTC, as double mutants exhibit significant frequencies of defects in migration phases along both the anteroposterior and dorsoventral axes. Together these results suggest that SDN-1 affects UNC-5 function by regulating the proper extracellular distribution of growth factors.     

Two Hox cofactors, the Meis/Hth homolog UNC-62 and the Pbx/Exd homolog CEH-20, function together during C. elegans postembryonic mesodermal development

The TALE homeodomain-containing PBC and MEIS proteins play multiple roles during metazoan development. Mutations in these proteins can cause various disorders, including cancer. In this study, we examined the roles of MEIS proteins in mesoderm development in C. elegans using the postembryonic mesodermal M lineage as a model system. We found that the MEIS protein UNC-62 plays essential roles in regulating cell fate specification and differentiation in the M lineage. Furthermore, UNC-62 appears to function together with the PBC protein CEH-20 in regulating these processes. Both unc-62 and ceh-20 have overlapping expression patterns within and outside of the M lineage, and they share physical and regulatory interactions. In particular, we found that ceh-20 is genetically required for the promoter activity of unc-62, providing evidence for another layer of regulatory interactions between MEIS and PBC proteins.     

The non-neuronal syntaxin SYN-1 regulates defecation behavior and neural activity in C. elegans through interaction with the Munc13-like protein AEX-1

We have previously shown that the AEX-1 protein, which is expressed in postsynaptic muscles, retrogradely regulates presynaptic neural activity at the Caenorhabditis elegans neuromuscular junctions. AEX-1 is similar to vertebrate Munc13-4 protein, suggesting a function for vesicle exocytosis from a kind of cells. Compared to emerging evidences of the role of Munc13 proteins in synaptic vesicle release, however, the precise mechanism for vesicle exocytosis by AEX-1 and Munc13-4 is little understood. Here we have identified SYN-1 as a candidate molecule of AEX-1-dependent vesicle exocytosis from non-neuronal cells. The syn-1 gene encodes a C. elegans syntaxin, which is distantly related to the neuronal syntaxin UNC-64. The syn-1 gene is predominantly expressed in non-neuronal tissues and genetically interacts with aex-1 for presynaptic activity. However, the two proteins did not interact physically in our yeast two-hybrid system and mutational SYN-1 did not bypass the requirement of AEX-1 for the behavioral defects in aex-1 mutants, whereas mutant UNC-64 does in unc-13 mutants. These results suggest that a novel molecular interaction between the AEX-1 and syntaxin may regulate vesicle exocytosis for retrograde signal release.     

CSN-5, a Component of the COP9 Signalosome Complex,Regulates the Levels of UNC-96 and UNC-98, Two Components of M-lines in Caenorhabditis elegans Muscle

In Caenorhabditis elegans two M-line proteins, UNC-98 and UNC-96, are involved in myofibril assembly and/or maintenance, especially myosin thick filaments. We found that CSN-5, a component of the COP9 signalosome complex, binds to UNC-98 and -96 using the yeast two-hybrid method. These interactions were confirmed by biochemical methods. The CSN-5 protein contains a Mov34 domain. Although one other COP9 signalosome component, CSN-6, also has a Mov34 domain, CSN-6 did not interact with UNC-98 or -96. Anti-CSN-5 antibody colocalized with paramyosin at A-bands in wild type and colocalized with abnormal accumulations of paramyosin found in unc-98, -96, and -15 (encodes paramyosin) mutants. Double knockdown of csn-5 and -6 could slightly suppress the unc-96 mutant phenotype. In the double knockdown of csn-5 and -6, the levels of UNC-98 protein were increased and the levels of UNC-96 protein levels were slightly reduced, suggesting that CSN-5 promotes the degradation of UNC-98 and that CSN-5 stabilizes UNC-96. In unc-15 and unc-96 mutants, CSN-5 protein was reduced, implying the existence of feed back regulation from myofibril proteins to CSN-5 protein levels. Taken together, we found that CSN-5 functions in muscle cells to regulate UNC-98 and -96, two M-line proteins.     

hunchback and Ikaros-like zinc finger genes control reproductive system development in Caenorhabditis elegans

Here we provide evidence for a C2H2 zinc finger gene family with similarity to Ikaros and hunchback. The founding member of this family is Caenorhabditis elegans ehn-3, which has important and poorly understood functions in somatic gonad development. We examined the expression and function of four additional hunchback/Ikaros-like (HIL) genes in C. elegans reproductive system development. Two genes, ehn-3 and R08E3.4, are expressed in somatic gonadal precursors (SGPs) and have overlapping functions in their development. In ehn-3; R08E3.4 double mutants, we find defects in the generation of distal tip cells, anchor cells, and spermatheca; three of the five tissues derived from the SGPs. We provide in vivo evidence that C. elegans HIL proteins have functionally distinct zinc finger domains, with specificity residing in the N-terminal set of four zinc fingers and a likely protein-protein interaction domain provided by the C-terminal pair of zinc fingers. In addition, we find that a chimeric human Ikaros protein containing the N-terminal zinc fingers of EHN-3 functions in C. elegans. Together, these results lend support to the idea that the C. elegans HIL genes and Ikaros have similar functional domains. We propose that hunchback, Ikaros, and the HIL genes arose from a common ancestor that was present prior to the divergence of protostomes and deuterostomes.     

Wnt signaling controls temporal identities of seam cells in Caenorhabditis elegans

The Wnt signaling pathway regulates multiple aspects of the development of stem cell-like epithelial seam cells in Caenorhabditis elegans, including cell fate specification and symmetric/asymmetric division. In this study, we demonstrate that lit-1, encoding the Nemo-like kinase in the Wnt/β-catenin asymmetry pathway, plays a role in specifying temporal identities of seam cells. Loss of function of lit-1 suppresses defects in retarded heterochronic mutants and enhances defects in precocious heterochronic mutants. Overexpressing lit-1 causes heterochronic defects opposite to those in lit-1(lf) mutants. LIT-1 exhibits a periodic expression pattern in seam cells within each larval stage. The kinase activity of LIT-1 is essential for its role in the heterochronic pathway. lit-1 specifies the temporal fate of seam cells likely by modulating miRNA-mediated silencing of target heterochronic genes. We further show that loss of function of other components of Wnt signaling, including mom-4, wrm-1, apr-1, and pop-1, also causes heterochronic defects in sensitized genetic backgrounds. Our study reveals a novel function of Wnt signaling in controlling the timing of seam cell development in C. elegans.     

The role of C. elegans Ena/VASP homolog UNC-34 in neuronal polarity and motility

Ena/VASP proteins mediate the effects of guidance cues on the actin cytoskeleton. The single C. elegans homolog of the Ena/VASP family of proteins, UNC-34, is required for the migrations of cells and growth cones. Here we show that unc-34 mutant alleles also interact genetically with Wnt mutants to reveal a role for unc-34 in the establishment of neuronal polarity along the C. elegans anterior-posterior axis. Our mutant analysis shows that eliminating UNC-34 function results in neuronal migration and polarity phenotypes that are enhanced at higher temperatures, revealing a heat-sensitive process that is normally masked by the presence of UNC-34. Finally, we show that the UNC-34 protein is expressed broadly and accumulates in axons and at the apical junctions of epithelial cells. While most mutants lacked detectable UNC-34, three unc-34 mutants that contained missense mutations in the EVH1 domain produced full-length UNC-34 that failed to localize to apical junctions and axons, supporting the role for the EVH1 domain in localizing Ena/VASP family members.     

Molecular Characterization of a Soybean Cyst Nematode (Heterodera glycines) Homolog of unc-87

In Caenorhabditis elegans the unc-87 gene encodes a protein that binds to actin at the I band and is important in nematodes for maintenance of the body-wall muscle. Caenorhabditis elegans mutant phenotypes of unc-87 exhibit severe paralysis in larvae and limp paralysis in the adult. We cloned and characterized a full-length cDNA representing a Heterodera glycines homolog of the unc-87 gene from C. elegans that encodes a protein that contains a region of seven repeats similar to CLIK-23 from C-elegans and has 81% amino acid identity with that of C. elegans unc-87 variant A. In the EST database clones labeled "unc-87'''' encode mainly the 3'' portion of unc-87, while clones labeled "calponin homolog OV9M'''' contain mainly DNA sequence representing the 5'' and middle transcribed regions of unc-87. A 1770 nucleotide cDNA encoding H. glycines unc-87 was cloned and encodes a predicted UNC-87 protein product of 375 amino acids. The expression of unc-87 was determined using RT-PCR and, in comparison to its expression in eggs, unc-87 was expressed 6-fold higher in J2 juveniles and 20-fold and 13-fold (P = 0.05) higher in nematodes 15 and 30 days after inoculation, respectively. In situ hybridization patterns confirmed the expression patterns observed with RT-PCR.     

Copulation in C. elegans males requires a nuclear hormone receptor

In Caenorhabditis elegans, uncoordinated (unc)-55 encodes a nuclear hormone receptor that is necessary for coordinated movement and male mating. An unc-55 reporter gene revealed a sexually dimorphic pattern: early in post-embryonic motor neurons in both sexes; and later in a subset of male-specific cells that included an interneuron and eight muscle cells. A behavioral analysis coupled with RNA interference (RNAi) revealed that males require UNC-55 to execute copulatory motor programs. Two mRNA isoforms (unc-55a and unc-55b) were detected throughout post-embryonic development in males, whereas only one, unc-55a, was detected in hermaphrodites. In unc-55 mutant males isoform a rescued the locomotion and mating defect, whereas isoform b rescued the mating defect only. Isoform b represents the first report of male-specific splicing in C. elegans. In addition, isoform b extended the number of days that transgenic unc-55 mutant males mated when compared to males rescued with isoform a, suggesting an anabolic role for the nuclear hormone receptor. The male-specific expression and splicing is part of a regulatory hierarchy that includes two key genes, male abnormal (mab)-5 and mab-9, required for the generation and differentiation of male-specific cells. We suggest that UNC-55 acts as an interface between genes involved in male tail pattern formation and those responsible for function.     

ced-10 Rac and mig-2 function redundantly and act with unc-73 trio to control the orientation of vulval cell divisions and migrations in Caenorhabditis elegans.

Vulval development in the nematode Caenorhabditis elegans can be divided into a fate specification phase controlled in part by let-60 Ras, and a fate execution phase involving stereotypical patterns of cell division and migration controlled in part by lin-17 Frizzled. Since the small GTPase Rac has been implicated as a downstream target of both Ras and Frizzled and influences cytoskeletal dynamics, we investigated the role of Rac signaling during each phase of vulval development. We show that the Rac gene ced-10 and the Rac-related gene mig-2 are redundantly required for the proper orientation of certain vulval cell divisions, suggesting a role in spindle positioning. ced-10 Rac and mig-2 are also redundantly required for vulval cell migrations and play a minor role in vulval fate specification. Constitutively active and dominant-negative mutant forms of mig-2 cause vulval defects that are very similar to those seen in ced-10;mig-2 double loss-of-function mutants, indicating that they interfere with the functions of both ced-10 Rac and mig-2. Mutations in unc-73 (a Trio-like guanine nucleotide exchange factor) cause similar vulval defects, suggesting that UNC-73 is an exchange factor for both CED-10 and MIG-2. We discuss the similarities and differences between the cellular defects seen in Rac mutants and let-60 Ras or lin-17 Frizzled mutants.     

UNC-10在致密核心囊泡分泌过程中的作用

Rim是囊泡分泌活性区中的重要组成蛋白,它与细胞分泌和突触可塑性相关．在秀丽隐感线虫中只存在一种编码Rim的基因即unc-10．我们的研究发现,在线虫中Rim的基因突变unc-10(md1117)会导致致密核心囊泡的分泌缺陷．在活体中,unc-10突变虫系的神经多肽分泌显著下降．此外,在主要分泌致密核心囊泡的ALA神经元内,钙光解释放促发的快相分泌也比野生型减少．运用全内反射荧光显微成像技术,我们观察在unc-10缺失的情况下ALA 神经元中致密核心囊泡的锚定过程,结果显示在细胞膜附近停留的囊泡数目减少,表明囊泡锚定受到阻碍．上述试验结果表明,UNC-10能够影响致密核心囊泡的分泌过程,其机制可能是影响了囊泡的锚定过程．     

UNC-31/CAPS docks and primes dense core vesicles in C. elegans neurons

UNC-31 or its mammalian homologue, Ca²⁺-dependent activator protein for secretion (CAPS), is indispensable for exocytosis of dense core vesicle (DCV) and synaptic vesicle (SV). From N- to the C-terminus, UNC-31 contains putative functional domains, including dynactin 1 binding domain (DBD), C2, PH, (M)UNC-13 homology domain (MHD) and DCV binding domain (DCVBD), the last four we examined in this study. We employed UNC-31 null mutant C. elegans worms to examine whether UNC-31 functions could be rescued by ectopic expression of full length UNC-31 vs each of these four domain-deleted mutants. Full length UNC-31 cDNA rescued the phenotypes of C. elegans null mutants in response to Ca²⁺-elevation in ALA neurons. Surprisingly, MHD deletion also rescued UNC-31 exocytotic function in part because the relatively high Ca²⁺ level (pre-flash Ca²⁺ was 450 nM) used in the capacitance study could bypass the MHD defect. Nonetheless, the three other domain-truncation cDNAs had almost no rescue on Ca²⁺ evoked secretion. Importantly, this genetic null mutant rescue strategy enabled physiological studies at levels of whole organism to single cells, such as locomotion assay, pharmacological study of neurotransmission at neuromuscular junction, in vivo neuropeptide release measurement and analysis of vesicular docking. Our results suggest that each of these UNC-31 domains support distinct sequential molecular actions of UNC-31 in vesicular exocytosis, including steps in vesicle tethering and docking that bridge vesicle with plasma membrane, and subsequently priming vesicle by initiating the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex.     

FEZ2 has acquired additional protein interaction partners relative to FEZ1: functional and evolutionary implications

Background

The FEZ (fasciculation and elongation protein zeta) family designation was purposed by Bloom and Horvitz by genetic analysis of C. elegans unc-76. Similar human sequences were identified in the expressed sequence tag database as FEZ1 and FEZ2. The unc-76 function is necessary for normal axon fasciculation and is required for axon-axon interactions. Indeed, the loss of UNC-76 function results in defects in axonal transport. The human FEZ1 protein has been shown to rescue defects caused by unc-76 mutations in nematodes, indicating that both UNC-76 and FEZ1 are evolutionarily conserved in their function. Until today, little is known about FEZ2 protein function.

Methodology/Principal Findings

Using the yeast two-hybrid system we demonstrate here conserved evolutionary features among orthologs and non-conserved features between paralogs of the FEZ family of proteins, by comparing the interactome profiles of the C-terminals of human FEZ1, FEZ2 and UNC-76 from C. elegans. Furthermore, we correlate our data with an analysis of the molecular evolution of the FEZ protein family in the animal kingdom.

Conclusions/Significance

We found that FEZ2 interacted with 59 proteins and that of these only 40 interacted with FEZ1. Of the 40 FEZ1 interacting proteins, 36 (90%), also interacted with UNC-76 and none of the 19 FEZ2 specific proteins interacted with FEZ1 or UNC-76. This together with the duplication of unc-76 gene in the ancestral line of chordates suggests that FEZ2 is in the process of acquiring new additional functions. The results provide also an explanation for the dramatic difference between C. elegans and D. melanogaster unc-76 mutants on one hand, which cause serious defects in the nervous system, and the mouse FEZ1 -/- knockout mice on the other, which show no morphological and no strong behavioural phenotype. Likely, the ubiquitously expressed FEZ2 can completely compensate the lack of neuronal FEZ1, since it can interact with all FEZ1 interacting proteins and additional 19 proteins.     

UNC-41/stonin functions with AP2 to recycle synaptic vesicles in Caenorhabditis elegans

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ～50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the μ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as μ2 adaptin.     

C. elegans RNA-binding proteins PUF-8 and MEX-3 function redundantly to promote germline stem cell mitosis

Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(−) mex-3(−) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.