The Importance of cGMP Signaling in Sensory Cilia for Body Size Regulation in Caenorhabditis elegans.

The body size of Caenorhabditis elegans is thought to be controlled by sensory inputs because many mutants with sensory cilium structure defects exhibit small body size. The EGL-4 cGMP-dependent protein kinase acts in sensory neurons to reduce body size when animals fail to perceive sensory signals. In addition to body size control, EGL-4 regulates various other behavioral and developmental pathways, including those involved in the regulation of egg laying and chemotaxis behavior. Here we have identified gcy-12, which encodes a receptor-type guanylyl cyclase, as a gene involved in the sensory regulation of body size. Analyses with GFP fusion constructs showed that gcy-12 is expressed in several sensory neurons and localizes to sensory cilia. Genetic analyses indicated that GCY-12 acts upstream of EGL-4 in body size control but does not affect other EGL-4 functions. Our studies indicate that the function of the GCY-12 guanylyl cyclase is to provide cGMP to the EGL-4 cGMP-dependent kinase only for limited tasks including body size regulation. We also found that the PDE-2 cyclic nucleotide phosphodiesterase negatively regulates EGL-4 in controlling body size. Thus, the cGMP level is precisely controlled by GCY-12 and PDE-2 to determine body size through EGL-4, and the defects in the sensory cilium structure may disturb the balanced control of the cGMP level. The large number of guanylyl cyclases encoded in the C. elegans genome suggests that EGL-4 exerts pleiotropic effects by partnering with different guanylyl cyclases for different downstream functions.

Comparative analysis of cells and proteins of pumpkin plants for the control of fruit size.

Common pumpkin plants (Cucurbita maxima) produce fruits of 1-2 kg size on the average, while special varieties of the same species called Atlantic Giant are known to produce a huge fruit up to several hundred kilograms. As an approach to determine the factors controlling the fruit size in C. maxima, we cultivated both AG and control common plants, and found that both the cell number and cell sizes were increased in a large fruit while DNA content of the cell did not change significantly. We also compared protein patterns in the leaves, stems, ripe and young fruits by two-dimensional (2D) gel electrophoresis, and identified those differentially expressed between them with mass spectroscopy. Based on these results, we suggest that factors in photosynthesis such as ribulose-bisphosphate carboxylase, glycolysis pathway enzymes, heat-shock proteins and ATP synthase play positive or negative roles in the growth of a pumpkin fruit. These results provide a step toward the development of plant biotechnology to control fruit size in the future.

Active uptake of artificial particles in the nematode Caenorhabditis elegans.

Feeding and food choice are crucial to the survival of an animal. The nematode Caenorhabditis elegans feeds on various microorganisms in nature, and is usually fed Escherichia coli in the laboratory. To elucidate the mechanisms of food/non-food discrimination in C. elegans, we examined the accumulation of various fluorescent polystyrene microspheres in the absence and presence of bacterial food. In the absence of food and on agar plates, C. elegans worms actively accumulated 0.5 and 1 µm diameter microspheres, whereas those microspheres <0.5 µm or >3 µm were rarely accumulated. Carboxylate microspheres were accumulated more than sulfate or amine microspheres. These results of accumulation in the absence of food probably well simulate uptake of or feeding on the microspheres. Presence of food bacteria even at bacteria:nematode ratios of 1:100 or 1:10 significantly reduced accumulation of 0.5 µm microspheres, and accumulation was reduced to approximately one-fourth of that observed in the absence of bacteria at a ratio of 1:1. When accumulation of microspheres was examined with the chemical sense mutants che-2, tax-2, odr-1 and odr-2, or the feeding mutant eat-1, all the mutants showed less accumulation than the wild type in the absence of food. In the presence of food, the che-2 mutant showed more accumulation than the wild type. It is possible that C. elegans discriminates food both physically, based on size, and chemically, based on taste and olfaction.

Body size change in various nematodes depending on bacterial food, sex and growth temperature.

We previously reported significant body size change in the nematode Caenorhabditis elegans, depending on the food strain of E. coli. Here, we examined this body size change in 11 other nematode species as well, and found that it is common to most of these nematodes. Furthermore, this food-dependent body size change is influenced by sex and growth temperature.

Does a DNA-less cellular organism exist on Earth?

All the self-reproducing cellular organisms so far examined have DNA as the genome. However, a DNA-less organism carrying an RNA genome is suggested by the fact that many RNA viruses exist and the widespread view that an RNA world existed before the present DNA world. Such a possibility is most plausible in the microbial world where biological diversity is enormous and most organisms have not been identified. We have developed experimental methodology to search DNA-less microorganisms, which is based on cultivation with drugs that inhibit replication or expression of DNA, detection of DNA in colonies with a fluorescent dye and double staining for DNA and RNA at a cellular level. These methods have been applied for about 100 microbial samples from various waters including hot springs, soils including deep sea sediments, and organisms. We found many colonies and cells which apparently looked DNA-less and examined them further. So far, all such colonies that reformed colonies on isolation were identified to be DNA-positive. However, considering the difficulty in cultivation, we think it possible for DNA-less microorganisms to live around us. We believe that our ideas and results will be of interest and useful to discover one in the future.

Control of body size in C. elegans dependent on food and insulin/IGF-1 signal.

The body size of an organism is governed by genetic and environmental factors. As an environmental factor, food appears to be the most important for body size control in animals. C. elegans worms are usually grown on an E. coli strain OP50. We show that the wild-type worms fed on another E. coli strain HB101 grow 1.6 times as large as those fed on OP50. The regression line representing the relationship between the sizes of worms grown on each food for over 30 mutants was drawn, indicating that small mutants tend to be more affected by the change in food. Mutants for the DAF-2 insulin/IGF-1 receptor and downstream SGK-1, a homolog of the serum- and glucocorticoid-inducible kinase, grow less or little larger on HB101, indicating control of body size by these factors. Results on the suppression of mutations in these factors by a mutation in the DAF-16/FOXO transcription factor indicate both DAF-16-dependent and DAF-16-independent control. Furthermore, we show that the food-dependent body size change is because of a change in cell size that is closely related to the protein content per cell.

Control of lifespan by food bacteria, nutrient limitation and pathogenicity of food in C. elegans.

The increased lifespan caused by food limitation has been observed in a wide range of animals including the nematode Caenorhabditis elegans. We show here that the lifespans of eat-2 and eat-5 feeding-defective mutants and a mutant of dbl-1 encoding a TGFß ligand significantly change between the cultures fed on Escherichia coli strain OP50 or a more nutrient-rich strain HB101. On HB101 food, the eat-2, eat-5 and dbl-1 mutants show increased lifespan compared to that of the wild type. This result is probably due to nutrient limitation because the eat mutations reduce food uptake and the mutation of dbl-1 that regulates expression of several digestive enzymes leads to nutrient limitation. In contrast, the lifespans of the eat-2 and dbl-1 mutants decreased from that of the wild type on OP50 food. We found that live OP50 cells within a worm were markedly more in these mutants than in the wild type, which suggests that impaired digestion of pathogenic OP50 decreased lifespan in the eat-2 and dbl-1 mutants.

A novel zf-MYND protein, CHB-3, mediates guanylyl cyclase localization to sensory cilia and controls body size of Caenorhabditis elegans.

Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.

ADBP-1 regulates an ADAR RNA-editing enzyme to antagonize RNA-interference-mediated gene silencing in Caenorhabditis elegans.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) mediate gene silencing through evolutionarily conserved pathways. In Caenorhabditis elegans, the siRNA/miRNA pathways are also known to affect transgene expression. To identify genes that regulate the efficiencies of the siRNA/miRNA pathways, we used the expression level of a transgene as an indicator of gene silencing and isolated a transgene-silencing mutant, adbp-1 (ADR-2 binding protein). The adbp-1 mutation caused transgene silencing in hypodermal and intestinal cells in a cell-autonomous manner, depending on the RNA interference (RNAi) machinery. The adbp-1 gene encodes a protein with no conserved domains that is localized in the nucleus. Yeast two-hybrid screening and co-immunoprecipitation analysis demonstrated that ADBP-1 physically interacts with ADR-2, an RNA-editing enzyme from the ADAR (adenosine deaminase acting on dsRNA) family. In the adbp-1 mutant, as previously shown in adr-2 mutants, A-to-I RNA editing was not detected, suggesting that ADBP-1 is required for the RNA-editing activity of ADR-2. We found that ADBP-1 facilitates the nuclear localization of ADR-2. ADBP-1 may regulate ADR-2 activity and the consequent RNA editing and thereby antagonize RNAi-mediated transgene silencing in C. elegans.

Mutants carrying two sma mutations are super small in the nematode C. elegans.

Body size determination is critical for multicellular organisms; however, the mechanisms remain largely unknown. Mutations that alter body size were studied to solve the mechanisms, for example, in mouse, fruit fly and the nematode Caenorhabditis elegans. In C. elegans, a large mutant and several small body size (sma) mutants are known. Of the latter, sma-2, sma-3, sma-4, sma-6, dbl-1 and daf-4 have a mutation in the components of the DBL-1/TGFbeta signal pathway, and sma-5 in a MAP kinase homologue. We have constructed double mutants carrying two of such small body size mutations, sma-5 and sma-4 or sma-2. They are much smaller than either of the parental single mutants, indicating that the sma-5 gene functions independently of the DBL-1/TGFbeta pathway. We show that their body volumes are as small as 1/10 of that of the wild-type, and that the sizes of major organs are much reduced, by the methods previously developed by us. But the numbers of cells are not changed, suggesting that the cells are very small. These results highlight surprising flexibility of body size and cell size in a multicellular organism, which will give a novel insight into the mechanisms of body size control.

Sensory ciliogenesis in Caenorhabditis elegans: assignment of IFT components into distinct modules based on transport and phenotypic profiles.

Sensory cilium biogenesis within Caenorhabditis elegans neurons depends on the kinesin-2-dependent intraflagellar transport (IFT) of ciliary precursors associated with IFT particles to the axoneme tip. Here we analyzed the molecular organization of the IFT machinery by comparing the in vivo transport and phenotypic profiles of multiple proteins involved in IFT and ciliogenesis. Based on their motility in wild-type and bbs (Bardet-Biedl syndrome) mutants, IFT proteins were classified into groups with similar transport profiles that we refer to as "modules." We also analyzed the distribution and transport of fluorescent IFT particles in multiple known ciliary mutants and 49 new ciliary mutants. Most of the latter mutants were snip-SNP mapped and one, namely dyf-14(ks69), was cloned and found to encode a conserved protein essential for ciliogenesis. The products of these ciliogenesis genes could also be assigned to the aforementioned set of modules or to specific aspects of ciliogenesis, based on IFT particle dynamics and ciliary mutant phenotypes. Although binding assays would be required to confirm direct physical interactions, the results are consistent with the hypothesis that the C. elegans IFT machinery has a modular design, consisting of modules IFT-subcomplex A, IFT-subcomplex B, and a BBS protein complex, in addition to motor and cargo modules, with each module contributing to distinct functional aspects of IFT or ciliogenesis.

Participation of XPB/Ptr8p, a component of TFIIH, in nucleocytoplasmic transport of mRNA in fission yeast.

To identify novel factors involved in nuclear mRNA export in Schizosaccharomyces pombe, we isolated and characterized the ptr8(+) gene, mutation of which causes nuclear accumulation of poly (A)(+) RNA. The ptr8(+) gene encodes an S. pombe homologue of human XPB, a component of TFIIH involved in nucleotide excision repair (NER) and transcription. A temperature-sensitive mutant of ptr8(+) (ptr8-1) was highly sensitive to UV irradiation, as are human XPB cells. Northern blot analysis demonstrated that the amount of total poly (A)(+) mRNAs does not decrease significantly at the nonpermissive temperature in ptr8-1 cells, whereas a pulse-labeling assay using (35)S-methionine showed that protein synthesis decreases rapidly after incubation of cells at the nonpermissive temperature, suggesting that ptr8-1 cells have a defect in nuclear mRNA export. In Saccharomyces cerevisiae, a mutation in the SSL2 gene encoding a homologue of Ptr8p also causes a block of mRNA export at the nonpermissive temperature. In addition, expression of human XPB in ptr8-1 cells rescued the ts phenotype and the mRNA export defects, suggesting that human XPB may also play a role in mRNA export. Furthermore, we revealed a functional interaction between Ptr8p and Tho2p, a component of the TREX complex involved in mRNA export. These results suggest that XPB/Ptr8p plays roles not only in NER and transcription, but also plays a conserved role in mRNA export.

Nucleocytoplasmic transport of fluorescent mRNA in living mammalian cells: nuclear mRNA export is coupled to ongoing gene transcription.

In eukaryotic cells, export of mRNA from the nucleus to the cytoplasm is one of the essential steps in gene expression. To examine mechanisms involved in the nucleocytoplasmic transport of mRNA, we microinjected fluorescently labeled fushi tarazu (ftz) pre-mRNA into the nuclei of HeLa cells. The injected intron-containing ftz pre-mRNA was distributed to the SC35 speckles and exported to the cytoplasm after splicing by an energy-requiring active process. In contrast, the injected intron-less ftz mRNA was diffusely distributed in the nucleus and then presumably degraded. Interestingly, export of the ftz pre-mRNA was inhibited by treatment with transcriptional inhibitors (actinomycin D, alpha-amanitin or DRB). Cells treated with transcriptional inhibitor showed foci enriched with the injected mRNA, which localize side by side with SC35 speckles. Those nuclear foci, referred to as TIDRs (transcriptional-inactivation dependent RNA domain), do not overlap with paraspeckles. In addition, in situ hybridization analysis revealed that the export of endogenous poly(A)+ mRNA is also affected by transcriptional inactivation. These results suggest that nuclear mRNA export is coupled to ongoing gene transcription in mammalian cells.

SRC-1, a non-receptor type of protein tyrosine kinase, controls the direction of cell and growth cone migration in C. elegans.

Src family tyrosine kinase (SFK) has been implicated in the regulation of cell adhesion and migration during animal development. We show that SRC-1, an ortholog of SFK, plays an essential role in directing cell migration in Caenorhabditis elegans. The mutation in the src-1 gene results in defective distal tip cell (DTC)-directed gonad morphogenesis in an activity-dependent and DTC cell-autonomous manners. In the src-1 mutants, DTCs fail to turn and continue their centrifugal migration along the ventral muscles. The effect of the src-1 mutation is suppressed by mutations in genes that function in the CED/Rac pathway, suggesting that SRC-1 in DTCs is an upstream regulator of a Rac pathway that controls cytoskeletal remodeling. In the src-1 mutant, the expression of unc-5/netrin receptor is normally regulated, and neither the precocious expression of UNC-5 nor the mutation in the unc-5 gene significantly affects the DTC migration defect. These data suggest that SRC-1 acts in the netrin signaling in DTCs. The src-1 mutant also exhibits cell-autonomous defects in the migration and growth cone path-finding of Q neuroblast descendants AVM and PVM. However, these roles of SRC-1 do not appear to involve the CED/Rac pathway. These findings show that SRC-1 functions in responding to various extracellular guidance cues that direct the cell migration via disparate signaling pathways in different cell types.

Control of body size by SMA-5, a homolog of MAP kinase BMK1/ERK5, in C. elegans.

We have analyzed the sma-5(n678) mutant in C. elegans to elucidate mechanisms controlling body size. The sma-5 mutant is very small, grows slowly and its intestinal granules look abnormal. We found a 15 kb deletion in the mutant that includes a 226 bp deletion of the 3' end of the W06B3.2-coding sequence. Based on this result, rescue experiments, RNAi experiments and a newly isolated deletion mutant of W06B3.2, we conclude that W06B3.2 is the sma-5 gene. The sma-5 mutant has much smaller intestine, body wall muscles and hypodermis than those of the wild type. However, the number of intestinal cells or body wall muscle cells is not changed, indicating that the sma-5 mutant has much smaller cells. In relation to the smaller cell size, the amount of total protein is drastically decreased; however, the DNA content of the intestinal nuclei is unchanged in the sma-5 mutant. The sma-5 gene is expressed in intestine, excretory cell and hypodermis, and encodes homologs of a mammalian MAP kinase BMK1/ERK5/MAPK7, which was reported to control cell cycle and cell proliferation. Expression of the sma-5 gene in hypodermis is important for body size control, and it can function both organ-autonomously and non-autonomously. We propose that the sma-5 gene functions in a MAP kinase pathway to regulate body size mainly through control of cell growth.

The dyf-3 gene encodes a novel protein required for sensory cilium formation in Caenorhabditis elegans.

Ciliated neurons in animals are important for the reception of environmental stimuli. To understand the mechanism of cilium morphogenesis in Caenorhabditis elegans, we analyzed dyf-3 mutants that are defective in uptake of a fluorescent dye and abnormal in sensory cilium structure. Expression of green fluorescent protein in sensory neurons of a dyf-3 mutant revealed that the mutant has stunted cilia and abnormal posterior projections in some sensory neurons. The dyf-3 gene encodes three proteins with different N-terminals. The largest DYF-3 protein has 404 amino acid residues that are 38% identical with those of a predicted human protein of unknown function. Expression of a functional dyf-3Colon, two colonsgfp fusion gene is detected in 26 chemosensory neurons, including six IL2 neurons, eight pairs of amphid neurons (ASE, ADF, ASG, ASH, ASI, ASJ, ASK and ADL) and two pairs of phasmid neurons (PHA and PHB). Expression of a dyf-3 cDNA in specific neurons of dyf-3 animals indicated that dyf-3 acts cell-autonomously for fluorescent dye uptake. Reduction of dyf-3Colon, two colonsgfp expression in a daf-19 mutant suggests that dyf-3 expression is regulated by DAF-19 transcription factor, and DYF-3 may be involved in the intraflagellar transport system.

cGMP and a germ-line signal control body size in C. elegans through cGMP-dependent protein kinase EGL-4.

Mechanisms involved in the control of body size are largely unknown. In the nematode C. elegans, several small body size mutants were isolated, and the responsible genes were reported to encode putative components of a TGFbeta signalling pathway. Recently, mutants in the egl-4 gene encoding cGMP-dependent protein kinases were found to have a larger body size, and it was suggested that EGL-4 down-regulates the TGFbeta/DBL-1 pathway. We show that a permeable cGMP analogue 8-Br-cGMP significantly reduces body size of the wild-type but not that of an egl-4 mutant, indicating that cGMP controls body size through EGL-4. Laser ablation of germ-line cells revealed that a germ-line signal and EGL-4 function in the same pathway. Targeted expression of EGL-4 indicates that EGL-4 can function in hypodermis, neurones and intestine both cell-autonomously and cell-nonautonomously to control organ and body size. We propose a signal cascade for the control of body size that involves a germ-line signal, cGMP, G-kinase EGL-4 and DBL-1/TGFbeta pathway. It is interesting that two important pathways involving cGMP and TGFbeta, respectively, are related. Also, the results suggest a novel mechanism for the control of organ and body size in which hypodermis plays a key role

The nucleolus is involved in mRNA export from the nucleus in fission yeast.

To elucidate the mechanism of mRNA export from the nucleus, we isolated five novel temperature-sensitive mutants (ptr7 to ptr11) that accumulate poly(A)(+) RNA in the nuclei at the nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr11 mutation was found in the top2(+) gene encoding DNA topoisomerase II. In addition to the nuclear accumulation of poly(A)(+) RNA, ptr11 exhibited the cut (cell untimely torn) phenotype at the nonpermissive temperature, like the previously isolated mutant, ptr4. In these two mutants, cytokinesis occurred without prior nuclear division, resulting in cleavage of the undivided nuclei by the septum. To investigate the relationship between mRNA export defects and the cut phenotype observed in ptr4 and ptr11, we analyzed 11 other mutants displaying the cut phenotype and found that all these tested mutants accumulate poly(A)(+) mRNA in the aberrantly cleaved nuclei. Interestingly, nuclear accumulation of poly(A)(+) mRNA was observed only in the anucleolate nuclei produced by aberrant cytokinesis. In addition, nuc1, the S. pombe mutant exhibiting a collapsed nucleolus, trapped poly(A)(+) mRNA in the nucleolar region at the nonpermissive temperature. In ptr11 and nuc1, mRNA transcribed from the intron-containing TBP gene showed nuclear accumulation, but not transcripts from the intron-less TBP cDNA, suggesting that the export pathway differs between the spliced and unspliced TBP mRNAs. These findings support the notion that a subset of mRNAs in yeast is exported from the nucleus through transient association with the nucleolus.

The fission yeast ptr1+ gene involved in nuclear mRNA export encodes a putative ubiquitin ligase.

Fission yeast ptr1-1 is one of the mRNA transport mutants that accumulate poly(A)+ RNA in the nuclei at the nonpermissive temperature. We found that the ptr1+ gene encodes a homolog of Saccharomyces cerevisiae Tom1p, a hect type ubiquitin ligase. In ptr1-1, a conserved amino acid in the hect domain of Ptr1p is mutated. The ptr1+ gene is essential for growth and its mutation did not affect nuclear protein export. A ptr1-1 rae1-167 double mutant showed a synthetic effect on a growth defect, indicating that Ptr1p functionally interacts with an essential mRNA export factor Rae1p. We also isolated a multi-copy suppressor for ptr1-1 and found that it is the mpd2+ gene isolated as a multi-copy suppressor of cdc7-PD1.

The C. elegans ceh-36 gene encodes a putative homemodomain transcription factor involved in chemosensory functions of ASE and AWC neurons.

Chemotaxis to water-soluble chemicals such as sodium ion is an important behavior of Caenorhabditis elegans for seeking food, and ASE chemosensory neurons have a major role in this behavior. We isolated mutants defective in chemotaxis to sodium acetate. We show here that among them ks86 had a mutation in the ceh-36 gene. ceh-36 :: gfp reporter constructs were expressed in ASE and AWC neurons. In a mutant of the che-1 gene, which encodes another transcription factor and is required for specification of ASE neurons, expression of the ceh-36 :: gfp reporter in ASE is lost. This indicates that the ceh-36 gene functions downstream of the che-1 gene in ASE. In the ceh-36(ks86) mutant, expression of the tax-2 gene encoding a cyclic nucleotide-gated channel was reduced in ASE and AWC. This affords an explanation for defects of the ceh-36 mutant in the chemotaxis mediated by ASE and AWC. When a ceh-36 cDNA was expressed in an adult ceh-36 mutant by a heat shock promoter, chemotaxis to sodium acetate was recovered. These results suggest that ceh-36 is required for functions, and not for development, of ASE.