72 results on '"Gengyo-Ando K"'
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2. Molecular and functional analysis of the C. elegans Sec1/Munc-18 family, C44C1.4 (CeVps45), involved in membrane traffic
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Gengyo-Ando, K, primary
- Published
- 2000
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3. Single/low-copy integration of transgenes in Caenorhabditis elegans using an ultraviolet trimethylpsoralen method
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Kage-Nakadai Eriko, Kobuna Hiroyuki, Funatsu Osamu, Otori Muneyoshi, Gengyo-Ando Keiko, Yoshina Sawako, Hori Sayaka, and Mitani Shohei
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Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Transgenic strains of Caenorhabditis elegans are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single- or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations. Results We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single- or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in C. elegans. Conclusion Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations.
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- 2012
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4. Functional analysis of epilepsy-linked pathogenic variants of the Munc18-1 gene in the inhibitory nervous system of Caenorhabditis elegans .
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Gengyo-Ando K, Osawa-Noguchi A, Ando H, and Nakai J
- Abstract
Heterozygous de novo mutations in Munc18-1, which is essential for neurotransmitter release, cause early infantile epileptic encephalopathy. Munc18-1-linked epilepsy is currently an untreatable disorder and its precise disease mechanism remains elusive. Here, we investigated how Munc18-1 pathogenic variants affect inhibitory neurons using Caenorhabditis elegans . Expression analysis revealed that three missense mutant proteins form aggregates in the cell body of gamma-aminobutyric-acid (GABA)-ergic motoneurons, resulting in a strong reduction of their expression in axons. Their defects of axonal expression correlated closely with pentylenetetrazol-induced convulsions, suggesting that the degree of instability of each mutant protein account for the severity of the epileptic phenotypes., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)
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- 2024
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5. Domain 3a mutation of VPS33A suppresses larval arrest phenotype in the loss of VPS45 in Caenorhabditis elegans .
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Gengyo-Ando K, Kumagai M, Ando H, and Nakai J
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The Sec1/Munc18 (SM) protein VPS45 is a key regulator of SNARE-mediated membrane fusion in endosomal trafficking, but its precise role remains unknown. To understand the function of VPS45 in vivo , we performed a genetic suppressor screen in Caenorhabditis elegans . We found that the temperature-sensitive lethality caused by the loss of VPS-45 can be suppressed by a mutation in another SM protein, VPS33A. The VPS33A M376I mutation is located in domain 3a, which is predicted to be essential for SNARE complex assembly. These results highlight the functional importance of domain 3a in endosomal SM proteins and its role in specific membrane fusion., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)
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- 2024
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6. Physical Stimulation Methods Developed for In Vitro Neuronal Differentiation Studies of PC12 Cells: A Comprehensive Review.
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Tominami K, Kudo TA, Noguchi T, Hayashi Y, Luo YR, Tanaka T, Matsushita A, Izumi S, Sato H, Gengyo-Ando K, Matsuzawa A, Hong G, and Nakai J
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- Animals, Rats, PC12 Cells, Cell Differentiation, Physical Stimulation, p38 Mitogen-Activated Protein Kinases, Adrenal Gland Neoplasms
- Abstract
PC12 cells, which are derived from rat adrenal pheochromocytoma cells, are widely used for the study of neuronal differentiation. NGF induces neuronal differentiation in PC12 cells by activating intracellular pathways via the TrkA receptor, which results in elongated neurites and neuron-like characteristics. Moreover, the differentiation requires both the ERK1/2 and p38 MAPK pathways. In addition to NGF, BMPs can also induce neuronal differentiation in PC12 cells. BMPs are part of the TGF-β cytokine superfamily and activate signaling pathways such as p38 MAPK and Smad. However, the brief lifespan of NGF and BMPs may limit their effectiveness in living organisms. Although PC12 cells are used to study the effects of various physical stimuli on neuronal differentiation, the development of new methods and an understanding of the molecular mechanisms are ongoing. In this comprehensive review, we discuss the induction of neuronal differentiation in PC12 cells without relying on NGF, which is already established for electrical, electromagnetic, and thermal stimulation but poses a challenge for mechanical, ultrasound, and light stimulation. Furthermore, the mechanisms underlying neuronal differentiation induced by physical stimuli remain largely unknown. Elucidating these mechanisms holds promise for developing new methods for neural regeneration and advancing neuroregenerative medical technologies using neural stem cells.
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- 2024
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7. A humanized Caenorhabditis elegans model for studying pathogenic mutations in VPS45, a protein essential for membrane trafficking, associated with severe congenital neutropenia.
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Gengyo-Ando K, Tateyama M, Mitani S, Ando H, and Nakai J
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VPS45, one of the essential membrane trafficking factors, has been identified as a cause of severe congenital neutropenia 5 (SCN5), but its pathophysiological role remains unknown. Here, we developed a humanized C. elegans model for three pathogenic VPS45 variants. We found that wild-type human VPS45 functionally complemented the loss of C. elegans VPS-45 , and the pathogenic human VPS45 variants functioned almost normally with respect to larval development and endocytosis in C. elegans . These results suggest that SCN5-associated mutations have little effect on the core function of VPS45, and/or that the degree of VPS45 requirement varies, depending on the cell/tissue., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2023 by the authors.)
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- 2023
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8. Role of somite patterning in the formation of Weberian apparatus and pleural rib in zebrafish.
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Akama K, Ebata K, Maeno A, Taminato T, Otosaka S, Gengyo-Ando K, Nakai J, Yamasu K, and Kawamura A
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- Animals, Animals, Genetically Modified, Gene Expression Regulation, Developmental, Ribs diagnostic imaging, Somites diagnostic imaging, T-Box Domain Proteins genetics, Tomography, X-Ray Computed, Zebrafish genetics, Zebrafish Proteins genetics, Body Patterning genetics, Ribs embryology, Somites enzymology, Zebrafish embryology
- Abstract
In the vertebrate body, a metameric structure is present along the anterior-posterior axis. Zebrafish tbx6
-/- larvae, in which somite boundaries do not form during embryogenesis, were shown to exhibit abnormal skeletal morphology such as rib, neural arch and hemal arch. In this study, we investigated the role of somite patterning in the formation of anterior vertebrae and ribs in more detail. Using three-dimensional computed tomography scans, we found that anterior vertebrae including the Weberian apparatus were severely affected in tbx6-/- larvae. In addition, pleural ribs of tbx6 mutants exhibited severe defects in the initial ossification, extension of ossification, and formation of parapophyses. Two-colour staining revealed that bifurcation of ribs was caused by fusion or branching of ribs in tbx6-/- . The parapophyses in tbx6-/- juvenile fish showed irregular positioning to centra and abnormal attachment to ribs. Furthermore, we found that the ossification of the distal portion of ribs proceeded along myotome boundaries even in irregularly positioned myotome boundaries. These results provide evidence of the contribution of somite patterning to the formation of the Weberian apparatus and rib in zebrafish., (© 2019 Anatomical Society.)- Published
- 2020
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9. Confocal and multiphoton calcium imaging of the enteric nervous system in anesthetized mice.
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Motegi Y, Sato M, Horiguchi K, Ohkura M, Gengyo-Ando K, Ikegaya Y, Fusamae Y, Hongo Y, Suzuki M, Ogawa K, Takaki M, and Nakai J
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- Animals, Intestines, Male, Mice, Mice, Transgenic, Microscopy, Fluorescence methods, Neurons metabolism, Neurons physiology, Serotonin pharmacology, Calcium analysis, Calcium metabolism, Enteric Nervous System physiology, Intravital Microscopy methods, Molecular Imaging methods
- Abstract
Most imaging studies of the enteric nervous system (ENS) that regulates the function of the gastrointestinal tract are so far performed using preparations isolated from animals, thus hindering the understanding of the ENS function in vivo. Here we report a method for imaging the ENS cellular network activity in living mice using a new transgenic mouse line that co-expresses G-CaMP6 and mCherry in the ENS combined with the suction-mediated stabilization of intestinal movements. With confocal or two-photon imaging, our method can visualize spontaneous and pharmacologically-evoked ENS network activity in living animals at cellular and subcellular resolutions, demonstrating the potential usefulness for studies of the ENS function in health and disease., (Copyright © 2019 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)
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- 2020
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10. UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase is indispensable for oogenesis, oocyte-to-embryo transition, and larval development of the nematode Caenorhabditis elegans.
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Kanaki N, Matsuda A, Dejima K, Murata D, Nomura KH, Ohkura T, Gengyo-Ando K, Yoshina S, Mitani S, and Nomura K
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- Animals, Caenorhabditis elegans metabolism, Embryo, Nonmammalian metabolism, Oocytes metabolism, Oogenesis genetics, Transferases (Other Substituted Phosphate Groups) metabolism
- Abstract
N-linked glycosylation of proteins is the most common post-translational modification of proteins. The enzyme UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) catalyses the first step of N-glycosylation, and DPAGT1 knockout is embryonic lethal in mice. In this study, we identified the sole orthologue (algn-7) of the human DPAGT1 in the nematode C. elegans. The gene activity was disrupted by RNAi and deletion mutagenesis, which resulted in larval lethality, defects in oogenesis and oocyte-to-embryo transition. Endomitotic oocytes, abnormal fusion of pronuclei, abnormal AB cell rotation, disruption of permeation barriers of eggs, and abnormal expression of chitin and chitin synthase in oocytes and eggs were the typical phenotypes observed. The results indicate that N-glycosylation is indispensable for these processes. We further screened an N-glycosylated protein database of C. elegans, and identified 456 germline-expressed genes coding N-glycosylated proteins. By examining RNAi phenotypes, we identified five germline-expressed genes showing similar phenotypes to the algn-7 (RNAi) animals. They were ribo-1, stt-3, ptc-1, ptc-2, and vha-19. We identified known congenital disorders of glycosylation (CDG) genes (ribo-1 and stt-3) and a recently found CDG gene (vha-19). The results show that phenotype analyses using the nematode could be a powerful tool to detect new CDG candidate genes and their associated gene networks.
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- 2019
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11. Role of tyramine in calcium dynamics of GABAergic neurons and escape behavior in Caenorhabditis elegans .
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Kagawa-Nagamura Y, Gengyo-Ando K, Ohkura M, and Nakai J
- Abstract
Background: Tyramine, known as a "trace amine" in mammals, modulates a wide range of behavior in invertebrates; however, the underlying cellular and circuit mechanisms are not well understood. In the nematode Caenorhabditis elegans ( C. elegans ), tyramine affects key behaviors, including foraging, feeding, and escape responses. The touch-evoked backward escape response is often coupled with a sharp omega turn that allows the animal to navigate away in the opposite direction. Previous studies have showed that a metabotropic tyramine receptor, SER-2, in GABAergic body motor neurons controls deep body bending in omega turns. In this study, we focused on the role of tyramine in GABAergic head motor neurons. Our goal is to understand the mechanism by which tyraminergic signaling alters neural circuit activity to control escape behavior., Results: Using calcium imaging in freely moving C. elegans , we found that GABAergic RME motor neurons in the head had high calcium levels during forward locomotion but low calcium levels during spontaneous and evoked backward locomotion. This calcium decrease was also observed during the omega turn. Mutant analyses showed that tbh-1 mutants lacking only octopamine had normal calcium responses, whereas tdc-1 mutants lacking both tyramine and octopamine did not exhibit the calcium decrease in RME. This neuromodulation was mediated by SER-2. Moreover, tyraminergic RIM neuron activity was negatively correlated with RME activity in the directional switch from forward to backward locomotion. These results indicate that tyramine released from RIM inhibits RME via SER-2 signaling. The omega turn is initiated by a sharp head bend when the animal reinitiates forward movement. Interestingly, ser-2 mutants exhibited shallow head bends and often failed to execute deep-angle omega turns. The behavioral defect and the abnormal calcium response in ser-2 mutants could be rescued by SER-2 expression in RME. These results suggest that tyraminergic inhibition of RME is involved in the control of omega turns., Conclusion: We demonstrate that endogenous tyramine downregulates calcium levels in GABAergic RME motor neurons in the head via the tyramine receptor SER-2 during backward locomotion and omega turns. Our data suggest that this neuromodulation allows deep head bending during omega turns and plays a role in the escape behavior in C. elegans ., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Published
- 2018
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12. Fast varifocal two-photon microendoscope for imaging neuronal activity in the deep brain.
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Sato M, Motegi Y, Yagi S, Gengyo-Ando K, Ohkura M, and Nakai J
- Abstract
Fluorescence microendoscopy is becoming a promising approach for deep brain imaging, but the current technology for visualizing neurons on a single focal plane limits the experimental efficiency and the pursuit of three-dimensional functional neural circuit architectures. Here we present a novel fast varifocal two-photon microendoscope system equipped with a gradient refractive index (GRIN) lens and an electrically tunable lens (ETL). This microendoscope enables quasi-simultaneous imaging of the neuronal network activity of deep brain areas at multiple focal planes separated by 85-120 µm at a fast scan rate of 7.5-15 frames per second per plane, as demonstrated in calcium imaging of the mouse dorsal CA1 hippocampus and amygdala in vivo .
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- 2017
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13. A new platform for long-term tracking and recording of neural activity and simultaneous optogenetic control in freely behaving Caenorhabditis elegans.
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Gengyo-Ando K, Kagawa-Nagamura Y, Ohkura M, Fei X, Chen M, Hashimoto K, and Nakai J
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- Animals, Animals, Genetically Modified, Automation, Laboratory, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Calcium metabolism, Channelrhodopsins genetics, Channelrhodopsins metabolism, Green Fluorescent Proteins metabolism, Light, Nerve Net physiology, Neurons physiology, Optogenetics methods, Wakefulness
- Abstract
Background: Real-time recording and manipulation of neural activity in freely behaving animals can greatly advance our understanding of how neural circuits regulate behavior. Ca
2+ imaging and optogenetic manipulation with optical probes are key technologies for this purpose. However, integrating the two optical approaches with behavioral analysis has been technically challenging., New Method: Here, we developed a new imaging system, ICaST (Integrated platform for Ca2+ imaging, Stimulation, and Tracking), which combines an automatic worm tracking system and a fast-scanning laser confocal microscope, to image neurons of interest in freely behaving C. elegans. We optimized different excitation wavelengths for the concurrent use of channelrhodopsin-2 and G-CaMP, a green fluorescent protein (GFP)-based, genetically encoded Ca2+ indicator., Results: Using ICaST in conjunction with an improved G-CaMP7, we successfully achieved long-term tracking and Ca2+ imaging of the AVA backward command interneurons while tracking the head of a moving animal. We also performed all-optical manipulation and simultaneous recording of Ca2+ dynamics from GABAergic motor neurons in conjunction with behavior monitoring., Comparison With Existing Method(s): Our system differs from conventional systems in that it does not require fluorescent markers for tracking and can track any part of the worm's body via bright-field imaging at high magnification. Consequently, this approach enables the long-term imaging of activity from neurons or nerve processes of interest with high spatiotemporal resolution., Conclusion: Our imaging system is a powerful tool for studying the neural circuit mechanisms of C. elegans behavior and has potential for use in other small animals., (Copyright © 2017 Elsevier B.V. All rights reserved.)- Published
- 2017
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14. Calcium dynamics regulating the timing of decision-making in C. elegans .
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Tanimoto Y, Yamazoe-Umemoto A, Fujita K, Kawazoe Y, Miyanishi Y, Yamazaki SJ, Fei X, Busch KE, Gengyo-Ando K, Nakai J, Iino Y, Iwasaki Y, Hashimoto K, and Kimura KD
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- Animals, Behavior, Animal, Decision Making, Smell, Spatial Navigation, Time Factors, Caenorhabditis elegans physiology, Calcium metabolism, Calcium Channels metabolism
- Abstract
Brains regulate behavioral responses with distinct timings. Here we investigate the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in Caenorhabditis elegans . We find that, based on subtle changes in odor concentrations, the animals appear to choose the appropriate migratory direction from multiple trials as a form of behavioral decision-making. Through optophysiological, mathematical and genetic analyses of neural activity under virtual odor gradients, we further find that odor concentration information is temporally integrated for a decision by a gradual increase in intracellular calcium concentration ([Ca
2+ ]i ), which occurs via L-type voltage-gated calcium channels in a pair of olfactory neurons. In contrast, for a reflex-like behavioral response, [Ca2+ ]i rapidly increases via multiple types of calcium channels in a pair of nociceptive neurons. Thus, the timing of neuronal responses is determined by cell type-dependent involvement of calcium channels, which may serve as a cellular basis for decision-making.- Published
- 2017
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15. Distinct roles of the two VPS33 proteins in the endolysosomal system in Caenorhabditis elegans.
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Gengyo-Ando K, Kage-Nakadai E, Yoshina S, Otori M, Kagawa-Nagamura Y, Nakai J, and Mitani S
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- Animals, Animals, Genetically Modified, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Endocytosis genetics, Endosomes genetics, Lysosomes genetics, Male, Microscopy, Confocal, Mutation, Oocytes metabolism, Oocytes ultrastructure, Protein Transport, Spermatogenesis genetics, Spermatogenesis physiology, Spermatozoa metabolism, Spermatozoa ultrastructure, Vesicular Transport Proteins genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Endocytosis physiology, Endosomes metabolism, Lysosomes metabolism, Vesicular Transport Proteins metabolism
- Abstract
Sec1/Munc-18 (SM) family proteins are essential regulators in intracellular transport in eukaryotic cells. The SM protein Vps33 functions as a core subunit of two tethering complexes, class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) in the endocytic pathway in yeast. Metazoan cells possess two Vps33 proteins, VPS33A and VPS33B, but their precise roles remain unknown. Here, we present a comparative analysis of Caenorhabditis elegans null mutants for these proteins. We found that the vps-33.1 (VPS33A) mutants exhibited severe defects in both endocytic function and endolysosomal biogenesis in scavenger cells. Furthermore, vps-33.1 mutations caused endocytosis defects in other tissues, and the loss of maternal and zygotic VPS-33.1 resulted in embryonic lethality. By contrast, vps-33.2 mutants were viable but sterile, with terminally arrested spermatocytes. The spermatogenesis phenotype suggests that VPS33.2 is involved in the formation of a sperm-specific organelle. The endocytosis defect in the vps-33.1 mutant was not restored by the expression of VPS-33.2, which indicates that these proteins have nonredundant functions. Together, our data suggest that VPS-33.1 shares most of the general functions of yeast Vps33 in terms of tethering complexes in the endolysosomal system, whereas VPS-33.2 has tissue/organelle specific functions in C. elegans., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)
- Published
- 2016
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16. Characterization of HAF-4- and HAF-9-localizing organelles as distinct organelles in Caenorhabditis elegans intestinal cells.
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Tanji T, Nishikori K, Haga S, Kanno Y, Kobayashi Y, Takaya M, Gengyo-Ando K, Mitani S, Shiraishi H, and Ohashi-Kobayashi A
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- ATP-Binding Cassette Transporters genetics, Animals, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Female, Male, Organelles genetics, Protein Transport, ATP-Binding Cassette Transporters metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Intestinal Mucosa metabolism, Organelles metabolism
- Abstract
Background: The intestinal cells of Caenorhabditis elegans are filled with heterogeneous granular organelles that are associated with specific organ functions. The best studied of these organelles are lipid droplets and acidified gut granules associated with GLO-1, a homolog of the small GTPase Rab38. In this study, we characterized a subset of the intestinal granules in which HAF-4 and HAF-9 localize on the membrane. HAF-4 and HAF-9 are ATP-binding cassette (ABC) transporter proteins that are homologous to the mammalian lysosomal peptide transporter TAPL (transporter associated with antigen processing-like, ABCB9)., Results: Using transgenic worms expressing fluorescent protein-tagged marker proteins, we demonstrated that the HAF-4- and HAF-9-localizing organelles are not lipid droplets and do not participate in yolk protein transport. They were also ruled out as GLO-1-positive acidified gut granules. Furthermore, we clarified that the late endosomal protein RAB-7 localizes to the HAF-4- and HAF-9-localizing organelles and is required for their biogenesis., Conclusions: Our results indicate that the HAF-4- and HAF-9-localizing organelles are distinct intestinal organelles associated with the endocytic pathway.
- Published
- 2016
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17. REI-1 Is a Guanine Nucleotide Exchange Factor Regulating RAB-11 Localization and Function in C. elegans Embryos.
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Sakaguchi A, Sato M, Sato K, Gengyo-Ando K, Yorimitsu T, Nakai J, Hara T, Sato K, and Sato K
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- Animals, Caenorhabditis elegans embryology, Caenorhabditis elegans Proteins biosynthesis, Embryo, Nonmammalian, Endosomes metabolism, Gene Expression Regulation, Developmental, Humans, RNA Interference, Vesicular Transport Proteins biosynthesis, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Cytokinesis genetics, Embryonic Development genetics, Guanine Nucleotide Exchange Factors genetics, Vesicular Transport Proteins genetics
- Abstract
The small GTPase Rab11 dynamically changes its location to regulate various cellular processes such as endocytic recycling, secretion, and cytokinesis. However, our knowledge of its upstream regulators is still limited. Here, we identify the RAB-11-interacting protein-1 (REI-1) as a unique family of guanine nucleotide exchange factors (GEFs) for RAB-11 in Caenorhabditis elegans. Although REI-1 and its human homolog SH3-binding protein 5 do not contain any known Rab-GEF domains, they exhibited strong GEF activity toward Rab11 in vitro. In C. elegans, REI-1 is expressed in the germline and co-localizes with RAB-11 on the late-Golgi membranes. The loss of REI-1 specifically impaired the targeting of RAB-11 to the late-Golgi compartment and the recycling endosomes in embryos and further reduced the RAB-11 distribution to the cleavage furrow, which resulted in cytokinesis delay. These results suggest that REI-1 is a GEF specifically regulating the RAB-11 localization and functions in early embryos., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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18. Generation and Imaging of Transgenic Mice that Express G-CaMP7 under a Tetracycline Response Element.
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Sato M, Kawano M, Ohkura M, Gengyo-Ando K, Nakai J, and Hayashi Y
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- Animals, Calcium metabolism, Fluorescence Resonance Energy Transfer methods, Gene Expression genetics, Green Fluorescent Proteins genetics, Mice, Mice, Transgenic, Microscopy, Fluorescence methods, Tetracycline pharmacology, CA1 Region, Hippocampal metabolism, Diagnostic Imaging methods, Membrane Proteins genetics, Pyramidal Cells metabolism, Response Elements genetics
- Abstract
The spatiotemporally controlled expression of G-CaMP fluorescent calcium indicator proteins can facilitate reliable imaging of brain circuit activity. Here, we generated a transgenic mouse line that expresses G-CaMP7 under a tetracycline response element. When crossed with a forebrain-specific tetracycline-controlled transactivator driver line, the mice expressed G-CaMP7 in defined cell populations in a tetracycline-controlled manner, notably in pyramidal neurons in layer 2/3 of the cortex and in the CA1 area of the hippocampus; this expression allowed for imaging of the in vivo activity of these circuits. This mouse line thus provides a useful genetic tool for controlled G-CaMP expression in vivo.
- Published
- 2015
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19. Rational design of a high-affinity, fast, red calcium indicator R-CaMP2.
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Inoue M, Takeuchi A, Horigane S, Ohkura M, Gengyo-Ando K, Fujii H, Kamijo S, Takemoto-Kimura S, Kano M, Nakai J, Kitamura K, and Bito H
- Subjects
- Action Potentials physiology, Animals, Caenorhabditis elegans radiation effects, Calcium metabolism, Calcium Signaling physiology, Calmodulin-Binding Proteins, Cells, Cultured, Cerebral Cortex cytology, Fluorescent Dyes metabolism, HEK293 Cells, Hippocampus cytology, Humans, Light, Mice, Neurons physiology, Patch-Clamp Techniques, Peptide Fragments chemistry, Peptide Fragments metabolism, Calcium-Calmodulin-Dependent Protein Kinase Kinase metabolism, Indicators and Reagents chemical synthesis
- Abstract
Fluorescent Ca(2+) reporters are widely used as readouts of neuronal activities. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-β in lieu of an M13 sequence resulted in threefold faster rise and decay times of Ca(2+) transients than R-CaMP1.07. These features allowed resolving single action potentials (APs) and recording fast AP trains up to 20-40 Hz in cortical slices. Somatic and synaptic activities of a cortical neuronal ensemble in vivo were imaged with similar efficacy as with previously reported sensitive green GECIs. Combining green and red GECIs, we successfully achieved dual-color monitoring of neuronal activities of distinct cell types, both in the mouse cortex and in freely moving Caenorhabditis elegans. Dual imaging using R-CaMP2 and green GECIs provides a powerful means to interrogate orthogonal and hierarchical neuronal ensembles in vivo.
- Published
- 2015
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20. Amino- and carboxyl-terminal domains of Filamin-A interact with CRMP1 to mediate Sema3A signalling.
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Nakamura F, Kumeta K, Hida T, Isono T, Nakayama Y, Kuramata-Matsuoka E, Yamashita N, Uchida Y, Ogura K, Gengyo-Ando K, Mitani S, Ogino T, and Goshima Y
- Subjects
- Actins metabolism, Animals, Caenorhabditis elegans genetics, HEK293 Cells, Humans, Mice, Inbred C57BL, Nerve Tissue Proteins metabolism, Phosphoproteins metabolism, Rats, Wistar, Semaphorin-3A metabolism, Actin Cytoskeleton metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Filamins metabolism, Growth Cones metabolism, Nerve Growth Factors metabolism
- Abstract
Reorganization of the actin cytoskeleton is an early cellular response to various extracellular signals. Sema3A, a repulsive axon guidance molecule, induces the reorganization of actin cytoskeleton in the growth cones. Collapsin response mediator protein 1 (CRMP1) mediates the intracellular Sema3A signalling through its Ser522 phosphorylation. Here we show that UNC-33, CRMP1 C. elegans homologue, interacts with FLN-1, an actin-binding Filamin-A orthologue. In nematodes, this interaction participates in the projection of DD/VD motor neurons. CRMP1 binds both the actin-binding domain and the last immunoglobulin-like repeat of Filamin-A. The alanine mutants of Filamin-A or CRMP1 in their interacting residues suppress the Sema3A repulsion in neurons. Conversely, a phosphor-mimicking mutant CRMP1(Ser522Asp) enhances the Sema3A response. Atomic-force microscopy analysis reveals that the V-shaped Filamin-A changes to a condensed form with CRMP1(Ser522Asp). CRMP1(Ser522Asp) weakens the F-actin gelation crosslinked by Filamin-A. Thus, phosphorylated CRMP1 may remove Filamin-A from the actin cytoskeleton to facilitate its remodelling.
- Published
- 2014
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21. Arl8/ARL-8 functions in apoptotic cell removal by mediating phagolysosome formation in Caenorhabditis elegans.
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Sasaki A, Nakae I, Nagasawa M, Hashimoto K, Abe F, Saito K, Fukuyama M, Gengyo-Ando K, Mitani S, Katada T, and Kontani K
- Subjects
- Animals, Caenorhabditis elegans cytology, Germ Cells physiology, Gonads cytology, Gonads enzymology, Lysosomes enzymology, Protein Transport, Time-Lapse Imaging, Vesicular Transport Proteins metabolism, rab GTP-Binding Proteins metabolism, rab7 GTP-Binding Proteins, Apoptosis, Caenorhabditis elegans enzymology, Caenorhabditis elegans Proteins physiology, GTP Phosphohydrolases physiology, Phagosomes enzymology
- Abstract
Efficient clearance of apoptotic cells by phagocytes is important for development, tissue homeostasis, and the prevention of autoimmune responses. Phagosomes containing apoptotic cells undergo acidification and mature from Rab5-positive early to Rab7-positive late stages. Phagosomes finally fuse with lysosomes to form phagolysosomes, which degrade apoptotic cells; however, the molecular mechanism underlying phagosome-lysosome fusion is not fully understood. Here we show that the Caenorhabditis elegans Arf-like small GTPase Arl8 (ARL-8) is involved in phagolysosome formation and is required for the efficient removal of apoptotic cells. Loss of function of arl-8 results in the accumulation of apoptotic germ cells. Both the engulfment of the apoptotic cells by surrounding somatic sheath cells and the phagosomal maturation from RAB-5- to RAB-7-positive stages occur in arl-8 mutants. However, the phagosomes fail to fuse with lysosomes in the arl-8 mutants, leading to the accumulation of RAB-7-positive phagosomes and the delayed degradation of apoptotic cells. ARL-8 localizes primarily to lysosomes and physically interacts with the homotypic fusion and protein sorting complex component VPS-41. Collectively our findings reveal that ARL-8 facilitates apoptotic cell removal in vivo by mediating phagosome-lysosome fusion during phagocytosis.
- Published
- 2013
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22. Neuronally expressed Ras-family GTPase Di-Ras modulates synaptic activity in Caenorhabditis elegans.
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Tada M, Gengyo-Ando K, Kobayashi T, Fukuyama M, Mitani S, Kontani K, and Katada T
- Subjects
- Acetylcholine genetics, Acetylcholine metabolism, Aldicarb pharmacology, Amino Acid Sequence, Animals, Caenorhabditis elegans drug effects, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Cholinergic Neurons drug effects, Cholinergic Neurons physiology, Cholinesterase Inhibitors pharmacology, Drug Resistance, GTP Phosphohydrolases genetics, GTP-Binding Protein alpha Subunits, Gi-Go genetics, GTP-Binding Protein alpha Subunits, Gi-Go metabolism, Guanine Nucleotide Exchange Factors genetics, Guanine Nucleotide Exchange Factors metabolism, Immunoprecipitation methods, Neuromuscular Junction drug effects, Neuromuscular Junction genetics, Neuromuscular Junction metabolism, Phenotype, Protein Interaction Mapping, Sequence Deletion, Caenorhabditis elegans enzymology, Cholinergic Neurons enzymology, GTP Phosphohydrolases metabolism, Synaptic Transmission
- Abstract
Ras-family GTPases regulate a wide variety of cellular functions including cell growth and differentiation. Di-Ras, which belongs to a distinct subfamily of Ras-family GTPases, is expressed predominantly in brain, but the role of Di-Ras in nervous systems remains totally unknown. Here, we report that the Caenorhabditis elegans Di-Ras homologue drn-1 is expressed specifically in neuronal cells and involved in synaptic function at neuromuscular junctions. Loss of function of drn-1 conferred resistance to the acetylcholinesterase inhibitor aldicarb and partially suppressed the aldicarb-hypersensitive phenotypes of heterotrimeric G-protein mutants, in which acetylcholine release is up-regulated. drn-1 mutants displayed no apparent defects in the axonal distribution of the membrane-bound second messenger diacylglycerol (DAG), which is a key stimulator of acetylcholine release. Finally, we have identified EPAC-1, a C. elegans Epac homologue, as a binding partner for DRN-1. Deletion mutants of epac-1 displayed an aldicarb-resistant phenotype as drn-1 mutants. Genetic analysis of drn-1 and epac-1 showed that they acted in the same pathway to control acetylcholine release. Furthermore, DRN-1 and EPAC-1 were co-immunoprecipitated. These findings suggest that DRN-1 may function cooperatively with EPAC-1 to modulate synaptic activity in C. elegans., (© 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Wiley Publishing Ltd.)
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- 2012
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23. Depletion of mboa-7, an enzyme that incorporates polyunsaturated fatty acids into phosphatidylinositol (PI), impairs PI 3-phosphate signaling in Caenorhabditis elegans.
- Author
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Lee HC, Kubo T, Kono N, Kage-Nakadai E, Gengyo-Ando K, Mitani S, Inoue T, and Arai H
- Subjects
- Acyltransferases genetics, Acyltransferases metabolism, Alleles, Animals, Animals, Genetically Modified genetics, Animals, Genetically Modified growth & development, Animals, Genetically Modified metabolism, Autophagy, Caenorhabditis elegans genetics, Caenorhabditis elegans growth & development, Caenorhabditis elegans Proteins genetics, Cloning, Molecular, Endosomes genetics, Endosomes metabolism, Fatty Acids, Unsaturated genetics, Gene Deletion, Gene Knockdown Techniques, Genetic Vectors genetics, Genetic Vectors metabolism, Phosphatidylinositol 3-Kinases genetics, Phosphatidylinositol 3-Kinases metabolism, Phosphatidylinositol Phosphates genetics, Promoter Regions, Genetic, RNA Interference, X Chromosome genetics, X Chromosome metabolism, Caenorhabditis elegans enzymology, Caenorhabditis elegans Proteins metabolism, Fatty Acids, Unsaturated metabolism, Phosphatidylinositol Phosphates metabolism, Phosphatidylinositols metabolism, Signal Transduction
- Abstract
Phosphatidylinositol (PI) is a constituent of biomembranes and a precursor of all phosphoinositides (PIPs). A prominent characteristic of PI is that its sn-2 position is highly enriched in polyunsaturated fatty acids (PUFAs), such as arachidonic acid or eicosapentaenoic acid. However, the biological significance of PUFA-containing PI remains unknown. We previously identified Caenorhabditis elegans (C. elegans) mboa-7 as an acyltransferase that incorporates PUFAs into the sn-2 position of PI. In this study, we performed an RNAi enhancer screen against PI kinases and phosphatases using mboa-7 mutants that have a reduced PUFA content in PI. Among the genes tested, knockdown of vps-34, a catalytic subunit of class III PI 3-kinase that produces PI 3-phosphate (PI3P) from PI, caused severe growth defects in mboa-7 mutants. In both vps-34 RNAi-treated wild-type worms and mboa-7 mutants, the size of PI3P-positive early endosomes was significantly decreased. We also performed an RNAi enhancer screen against PI3P-related genes and found that, like knockdown of vps-34, knockdown of autophagy-related genes caused severe growth defects in mboa-7 mutants. Finally, we showed that autophagic clearance of protein aggregates is impaired in mboa-7 mutants. Taken together, these results suggest that the PUFA chain in PI has a role in some PI3P signaling., (© 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Wiley Publishing Ltd.)
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- 2012
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24. Physiological function, expression pattern, and transcriptional regulation of a Caenorhabditis elegans insulin-like peptide, INS-18.
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Matsunaga Y, Gengyo-Ando K, Mitani S, Iwasaki T, and Kawano T
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- Animals, Caenorhabditis elegans growth & development, Caenorhabditis elegans Proteins antagonists & inhibitors, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Forkhead Transcription Factors, Larva genetics, Larva growth & development, Longevity genetics, RNA Interference, Receptor, Insulin antagonists & inhibitors, Receptor, Insulin genetics, Transcription Factors genetics, Transcription Factors metabolism, Caenorhabditis elegans genetics, Gene Expression Regulation, Neurons metabolism, Peptide Hormones genetics, Transcription, Genetic
- Abstract
In Caenorhabditis elegans, insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an important pathway that controls larval diapause and adult lifespan. The IIS pathway is modulated by many insulin-like peptides (ILPs) through the DAF-2 receptor, the sole insulin/IGF-1 receptor-like protein in C. elegans. We previously identified the ILP, INS-18, and predicted its tertiary structure to be similar to the crystal structures of human insulin and IGF-1. In this study, the physiological function of INS-18 was first examined by gene disruption and overexpression, and we identified INS-18 as a DAF-2 antagonist required for larval diapause and longevity. Analysis of the INS-18 expression pattern using a reporter gene showed it to be expressed in nerve cells, including hermaphrodite-specific neurons (HSNs) at the adult stage. Other ILP expressions have not been previously observed in HSNs, and we believe that INS-18 expression in these cells may contribute to longevity by regulating reproduction. Loss of the DAF-16 transcription factor located downstream of the IIS pathway completely blocked ins-18 expression. We propose a positive feedback model for the regulation of ins-18 expression in which an antagonist binding to the DAF-2 receptor increases ins-18 gene expression, thus leading to increased INS-18 protein levels and increased DAF-2 receptor binding. Thus, this study provides a new insight into the hormonal regulation of insulin, an important and widespread process in the animal kingdom., (Copyright © 2012 Elsevier Inc. All rights reserved.)
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- 2012
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25. Identification of a novel ADAMTS9/GON-1 function for protein transport from the ER to the Golgi.
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Yoshina S, Sakaki K, Yonezumi-Hayashi A, Gengyo-Ando K, Inoue H, Iino Y, and Mitani S
- Subjects
- ADAM Proteins genetics, ADAMTS9 Protein, Animals, Animals, Genetically Modified, Caenorhabditis elegans, Caenorhabditis elegans Proteins genetics, Cell Line, Conserved Sequence, HEK293 Cells, Humans, Metalloendopeptidases genetics, Protein Transport, ADAM Proteins metabolism, Caenorhabditis elegans Proteins metabolism, Endoplasmic Reticulum metabolism, Golgi Apparatus metabolism, Metalloendopeptidases metabolism
- Abstract
A disintegrin-like and metalloprotease with thrombospondin type I motif (ADAMTS9) is a member of the secreted metalloprotease family that is believed to digest extracellular matrix (ECM) proteins outside of cells. Its Caenorhabditis elegans orthologue, GON-1, is involved in ECM degradation and is required for gonad morphogenesis. ADAMTS9 and GON-1 have similar domain structures, and both have a unique C-terminal domain called the "GON domain," whose function remains unknown. Here we show that down-regulation of human ADAMTS9 and C. elegans GON-1 results in the inhibition of protein transport from the endoplasmic reticulum (ER) to the Golgi. This phenotype was rescued by the expression of the GON domain localizing in the ER in human cells and C. elegans. We propose a novel function of ADAMTS9 and GON-1 in the ER that promotes protein transport from the ER to the Golgi. This function is GON-domain dependent but protease activity independent.
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- 2012
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26. GPI-anchor synthesis is indispensable for the germline development of the nematode Caenorhabditis elegans.
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Murata D, Nomura KH, Dejima K, Mizuguchi S, Kawasaki N, Matsuishi-Nakajima Y, Ito S, Gengyo-Ando K, Kage-Nakadai E, Mitani S, and Nomura K
- Subjects
- Animals, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins genetics, Gene Knockdown Techniques, Gonads metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Caenorhabditis elegans embryology, Caenorhabditis elegans Proteins metabolism, Germ Cells metabolism, Glycosylphosphatidylinositols metabolism
- Abstract
Glycosylphosphatidylinositol (GPI)-anchor attachment is one of the most common posttranslational protein modifications. Using the nematode Caenorhabditis elegans, we determined that GPI-anchored proteins are present in germline cells and distal tip cells, which are essential for the maintenance of the germline stem cell niche. We identified 24 C. elegans genes involved in GPI-anchor synthesis. Inhibition of various steps of GPI-anchor synthesis by RNA interference or gene knockout resulted in abnormal development of oocytes and early embryos, and both lethal and sterile phenotypes were observed. The piga-1 gene (orthologue of human PIGA) codes for the catalytic subunit of the phosphatidylinositol N-acetylglucosaminyltransferase complex, which catalyzes the first step of GPI-anchor synthesis. We isolated piga-1-knockout worms and found that GPI-anchor synthesis is indispensable for the maintenance of mitotic germline cell number. The knockout worms displayed 100% lethality, with decreased mitotic germline cells and abnormal eggshell formation. Using cell-specific rescue of the null allele, we showed that expression of piga-1 in somatic gonads and/or in germline is sufficient for normal embryonic development and the maintenance of the germline mitotic cells. These results clearly demonstrate that GPI-anchor synthesis is indispensable for germline formation and for normal development of oocytes and eggs.
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- 2012
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27. Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals.
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Ohkura M, Sasaki T, Sadakari J, Gengyo-Ando K, Kagawa-Nagamura Y, Kobayashi C, Ikegaya Y, and Nakai J
- Subjects
- Animals, Cells, Cultured, Dendritic Spines metabolism, Dentate Gyrus cytology, Dentate Gyrus metabolism, HeLa Cells, Humans, Indicators and Reagents analysis, Indicators and Reagents metabolism, Neuroanatomical Tract-Tracing Techniques, Neurons cytology, Neurons metabolism, Rats, Calcium chemistry, Calcium isolation & purification, Calcium metabolism, Calcium Signaling genetics, Green Fluorescent Proteins chemistry, Green Fluorescent Proteins genetics, Pyramidal Cells cytology, Pyramidal Cells metabolism
- Abstract
Imaging the activities of individual neurons with genetically encoded Ca(2+) indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca(2+) signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (F(max)/F(min) = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca(2+) imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca(2+) responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.
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- 2012
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28. A Caenorhabditis elegans insulin-like peptide, INS-17: its physiological function and expression pattern.
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Matsunaga Y, Nakajima K, Gengyo-Ando K, Mitani S, Iwasaki T, and Kawano T
- Subjects
- Animals, Caenorhabditis elegans genetics, Insulins genetics, Sequence Deletion, Transcription, Genetic, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Gene Expression Regulation, Insulins metabolism
- Abstract
The insulin/insulin-like growth factor-1 signaling pathway of Caenorhabditis elegans regulates larval diapause and adult lifespan through the sole insulin receptor-like protein, DAF-2. In the present study, the physiological function and expression pattern of INS-17, one of the C. elegans insulin-like peptides, were examined by disruption and overexpression of the gene, and by the use of a reporter gene. INS-17 might function as a DAF-2 antagonist in the regulation of larval diapause, but not of the adult lifespan. The reporter protein was intensively expressed during larval diapause. It showed a drastic decrease in amount after larval diapause, which matches well the physiological function of INS-17.
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- 2012
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29. Two Golgi-resident 3'-Phosphoadenosine 5'-phosphosulfate transporters play distinct roles in heparan sulfate modifications and embryonic and larval development in Caenorhabditis elegans.
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Dejima K, Murata D, Mizuguchi S, Nomura KH, Izumikawa T, Kitagawa H, Gengyo-Ando K, Yoshina S, Ichimiya T, Nishihara S, Mitani S, and Nomura K
- Subjects
- Alleles, Animals, Caenorhabditis elegans, Gene Deletion, Gene Expression Profiling, Genes, Reporter, Glycosaminoglycans chemistry, Green Fluorescent Proteins chemistry, Mutation, Subcellular Fractions, Substrate Specificity, Transgenes, Caenorhabditis elegans Proteins physiology, Gene Expression Regulation, Developmental, Golgi Apparatus metabolism, Heparitin Sulfate metabolism, Nucleotide Transport Proteins physiology
- Abstract
Synthesis of extracellular sulfated molecules requires active 3'-phosphoadenosine 5'-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.
- Published
- 2010
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30. Multivesicular body formation requires OSBP-related proteins and cholesterol.
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Kobuna H, Inoue T, Shibata M, Gengyo-Ando K, Yamamoto A, Mitani S, and Arai H
- Subjects
- Animals, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, HeLa Cells, Humans, Multigene Family, Receptors, Steroid genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Cholesterol metabolism, Multivesicular Bodies metabolism, Receptors, Steroid metabolism
- Abstract
In eukaryotes, different subcellular organelles have distinct cholesterol concentrations, which is thought to be critical for biological functions. Oxysterol-binding protein-related proteins (ORPs) have been assumed to mediate nonvesicular cholesterol trafficking in cells; however, their in vivo functions and therefore the biological significance of cholesterol in each organelle are not fully understood. Here, by generating deletion mutants of ORPs in Caenorhabditis elegans, we show that ORPs are required for the formation and function of multivesicular bodies (MVBs). In an RNAi enhancer screen using obr quadruple mutants (obr-1; -2; -3; -4), we found that MVB-related genes show strong genetic interactions with the obr genes. In obr quadruple mutants, late endosomes/lysosomes are enlarged and membrane protein degradation is retarded, although endocytosed soluble proteins are normally delivered to lysosomes and degraded. We also found that the cholesterol content of late endosomes/lysosomes is reduced in the mutants. In wild-type worms, cholesterol restriction induces the formation of enlarged late endosomes/lysosomes, as observed in obr quadruple mutants, and increases embryonic lethality upon knockdown of MVB-related genes. Finally, we show that knockdown of ORP1L, a mammalian ORP family member, induces the formation of enlarged MVBs in HeLa cells. Our in vivo findings suggest that the proper cholesterol level of late endosomes/lysosomes generated by ORPs is required for normal MVB formation and MVB-mediated membrane protein degradation., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2010
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31. The arf-like GTPase Arl8 mediates delivery of endocytosed macromolecules to lysosomes in Caenorhabditis elegans.
- Author
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Nakae I, Fujino T, Kobayashi T, Sasaki A, Kikko Y, Fukuyama M, Gengyo-Ando K, Mitani S, Kontani K, and Katada T
- Subjects
- Animals, Caenorhabditis elegans cytology, Caenorhabditis elegans embryology, Cell Compartmentation, Embryonic Development, Endosomes enzymology, Intracellular Membranes enzymology, Mutation genetics, Protein Transport, Serum Albumin, Bovine metabolism, Subcellular Fractions metabolism, Time Factors, Caenorhabditis elegans enzymology, Caenorhabditis elegans Proteins metabolism, Endocytosis, GTP Phosphohydrolases metabolism, Lysosomes metabolism
- Abstract
Late endocytic organelles including lysosomes are highly dynamic acidic organelles. Late endosomes and lysosomes directly fuse for content mixing to form hybrid organelles, from which lysosomes are reformed. It is not fully understood how these processes are regulated and maintained. Here we show that the Caenorhabditis elegans ARL-8 GTPase is localized primarily to lysosomes and involved in late endosome-lysosome fusion in the macrophage-like coelomocytes. Loss of arl-8 results in an increase in the number of late endosomal/lysosomal compartments, which are smaller than wild type. In arl-8 mutants, late endosomal compartments containing endocytosed macromolecules fail to fuse with lysosomal compartments enriched in the aspartic protease ASP-1. Furthermore, loss of arl-8 strongly suppresses formation of enlarged late endosome-lysosome hybrid organelles caused by mutations of cup-5, which is the orthologue of human mucolipin-1. These findings suggest that ARL-8 mediates delivery of endocytosed macromolecules to lysosomes by facilitating late endosome-lysosome fusion.
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- 2010
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32. An Arf-like small G protein, ARL-8, promotes the axonal transport of presynaptic cargoes by suppressing vesicle aggregation.
- Author
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Klassen MP, Wu YE, Maeder CI, Nakae I, Cueva JG, Lehrman EK, Tada M, Gengyo-Ando K, Wang GJ, Goodman M, Mitani S, Kontani K, Katada T, and Shen K
- Subjects
- ADP-Ribosylation Factors genetics, Animals, Axonal Transport genetics, Caenorhabditis elegans, Membrane Proteins genetics, Multiprotein Complexes antagonists & inhibitors, Multiprotein Complexes genetics, Multiprotein Complexes metabolism, Presynaptic Terminals ultrastructure, Protein Transport genetics, Synaptic Vesicles genetics, Synaptic Vesicles ultrastructure, Vesicular Neurotransmitter Transport Proteins genetics, Vesicular Neurotransmitter Transport Proteins metabolism, ADP-Ribosylation Factors physiology, Axonal Transport physiology, Membrane Proteins physiology, Presynaptic Terminals metabolism, Synaptic Vesicles metabolism, Vesicular Neurotransmitter Transport Proteins antagonists & inhibitors
- Abstract
Presynaptic assembly requires the packaging of requisite proteins into vesicular cargoes in the cell soma, their long-distance microtubule-dependent transport down the axon, and, finally, their reconstitution into functional complexes at prespecified sites. Despite the identification of several molecules that contribute to these events, the regulatory mechanisms defining such discrete states remain elusive. We report the characterization of an Arf-like small G protein, ARL-8, required during this process. arl-8 mutants prematurely accumulate presynaptic cargoes within the proximal axon of several neuronal classes, with a corresponding failure to assemble presynapses distally. This proximal accumulation requires the activity of several molecules known to catalyze presynaptic assembly. Dynamic imaging studies reveal that arl-8 mutant vesicles exhibit an increased tendency to form immotile aggregates during transport. Together, these results suggest that arl-8 promotes a trafficking identity for presynaptic cargoes, facilitating their efficient transport by repressing premature self-association., (Copyright 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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33. Protein phosphatase 2A cooperates with the autophagy-related kinase UNC-51 to regulate axon guidance in Caenorhabditis elegans.
- Author
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Ogura K, Okada T, Mitani S, Gengyo-Ando K, Baillie DL, Kohara Y, and Goshima Y
- Subjects
- Amino Acid Sequence, Animals, Animals, Genetically Modified, Autophagy genetics, Autophagy physiology, Axonal Transport genetics, Axonal Transport physiology, Axons metabolism, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Molecular Sequence Data, Neurogenesis genetics, Neurogenesis physiology, Phosphorylation genetics, Protein Binding physiology, Protein Kinases metabolism, Protein Kinases physiology, Protein Phosphatase 2 genetics, Protein Phosphatase 2 metabolism, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Protein Subunits genetics, Protein Subunits metabolism, Sequence Homology, Amino Acid, Synaptic Transmission genetics, Synaptic Transmission physiology, Axons physiology, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins physiology, Protein Phosphatase 2 physiology, Protein Serine-Threonine Kinases physiology
- Abstract
UNC-51 is a serine/threonine protein kinase conserved from yeast to humans. The yeast homolog Atg1 regulates autophagy (catabolic membrane trafficking) required for surviving starvation. In C. elegans, UNC-51 regulates the axon guidance of many neurons by a different mechanism than it and its homologs use for autophagy. UNC-51 regulates the subcellular localization (trafficking) of UNC-5, a receptor for the axon guidance molecule UNC-6/Netrin; however, the molecular details of the role for UNC-51 are largely unknown. Here, we report that UNC-51 physically interacts with LET-92, the catalytic subunit of serine/threonine protein phosphatase 2A (PP2A-C), which plays important roles in many cellular functions. A low allelic dose of LET-92 partially suppressed axon guidance defects of weak, but not severe, unc-51 mutants, and a low allelic dose of PP2A regulatory subunits A (PAA-1/PP2A-A) and B (SUR-6/PP2A-B) partially enhanced the weak unc-51 mutants. We also found that LET-92 can work cell-non-autonomously on axon guidance in neurons, and that LET-92 colocalized with UNC-51 in neurons. In addition, PP2A dephosphorylated phosphoproteins that had been phosphorylated by UNC-51. These results suggest that, by forming a complex, PP2A cooperates with UNC-51 to regulate axon guidance by regulating phosphorylation. This is the first report of a serine/threonine protein phosphatase functioning in axon guidance in vivo.
- Published
- 2010
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34. Two very long chain fatty acid acyl-CoA synthetase genes, acs-20 and acs-22, have roles in the cuticle surface barrier in Caenorhabditis elegans.
- Author
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Kage-Nakadai E, Kobuna H, Kimura M, Gengyo-Ando K, Inoue T, Arai H, and Mitani S
- Subjects
- Animals, Caenorhabditis elegans genetics, Coenzyme A Ligases metabolism, Coenzyme A Ligases physiology, Fatty Acid Transport Proteins physiology, Mutation, Sphingomyelins metabolism, Caenorhabditis elegans enzymology, Coenzyme A Ligases genetics
- Abstract
In multicellular organisms, the surface barrier is essential for maintaining the internal environment. In mammals, the barrier is the stratum corneum. Fatty acid transport protein 4 (FATP4) is a key factor involved in forming the stratum corneum barrier. Mice lacking Fatp4 display early neonatal lethality with features such as tight, thick, and shiny skin, and a defective skin barrier. These symptoms are strikingly similar to those of a human skin disease called restrictive dermopathy. FATP4 is a member of the FATP family that possesses acyl-CoA synthetase activity for very long chain fatty acids. How Fatp4 contributes to skin barrier function, however, remains to be elucidated. In the present study, we characterized two Caenorhabditis elegans genes, acs-20 and acs-22, that are homologous to mammalian FATPs. Animals with mutant acs-20 exhibited defects in the cuticle barrier, which normally prevents the penetration of small molecules. acs-20 mutant animals also exhibited abnormalities in the cuticle structure, but not in epidermal cell fate or cell integrity. The acs-22 mutants rarely showed a barrier defect, whereas acs-20;acs-22 double mutants had severely disrupted barrier function. Moreover, the barrier defects of acs-20 and acs-20;acs-22 mutants were rescued by acs-20, acs-22, or human Fatp4 transgenes. We further demonstrated that the incorporation of exogenous very long chain fatty acids into sphingomyelin was reduced in acs-20 and acs-22 mutants. These findings indicate that C. elegans Fatp4 homologue(s) have a crucial role in the surface barrier function and this model might be useful for studying the fundamental molecular mechanisms underlying human skin barrier and relevant diseases.
- Published
- 2010
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35. FLR-2, the glycoprotein hormone alpha subunit, is involved in the neural control of intestinal functions in Caenorhabditis elegans.
- Author
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Oishi A, Gengyo-Ando K, Mitani S, Mohri-Shiomi A, Kimura KD, Ishihara T, and Katsura I
- Subjects
- Amino Acid Sequence, Animals, Caenorhabditis elegans genetics, Caenorhabditis elegans microbiology, Caenorhabditis elegans Proteins genetics, Carrier Proteins genetics, Carrier Proteins metabolism, Cloning, Molecular, DNA, Complementary chemistry, DNA, Complementary genetics, Escherichia coli physiology, Glycoprotein Hormones, alpha Subunit genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Host-Pathogen Interactions, Life Expectancy, Microscopy, Fluorescence, Molecular Sequence Data, Mutation, Neurons cytology, Neurons metabolism, Pigmentation, Protein Binding, RNA Interference, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins metabolism, Glycoprotein Hormones, alpha Subunit metabolism, Intestines innervation, Intestines physiology
- Abstract
The intestine plays an essential role in organism-wide regulatory networks in both vertebrates and invertebrates. In Caenorhabditis elegans, class 1 flr genes (flr-1, flr-3 and flr-4) act in the intestine and control growth rates and defecation cycle periods, while class 2 flr genes (flr-2, flr-5, flr-6 and flr-7) are characterized by mutations that suppress the slow growth of class 1 flr mutants. This study revealed that flr-2 gene controls antibacterial defense and intestinal color, confirming that flr-2 regulates intestinal functions. flr-2 encoded the only glycoprotein hormone alpha subunit in C. elegans and was expressed in certain neurons. Furthermore, FLR-2 bound to another secretory protein GHI-1, which belongs to a family of lipid- and lipopolysaccharide-binding proteins. A ghi-1 deletion mutation partially suppressed the short defecation cycle periods of class 1 flr mutants, and this effect was enhanced by flr-2 mutations. Thus, FLR-2 acts as a signaling molecule for the neural control of intestinal functions, which is achieved in a functional network involving class 1 and class 2 flr genes as well as ghi-1. These results are informative to studies of glycoprotein hormone signaling in higher animals.
- Published
- 2009
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36. A Caenorhabditis elegans glycolipid-binding galectin functions in host defense against bacterial infection.
- Author
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Ideo H, Fukushima K, Gengyo-Ando K, Mitani S, Dejima K, Nomura K, and Yamashita K
- Subjects
- Animals, Bacillus thuringiensis metabolism, Bacillus thuringiensis physiology, Bacillus thuringiensis Toxins, Bacterial Proteins metabolism, Bacterial Proteins toxicity, Caenorhabditis elegans genetics, Caenorhabditis elegans microbiology, Caenorhabditis elegans Proteins drug effects, Caenorhabditis elegans Proteins genetics, Chromatography, High Pressure Liquid, Endotoxins metabolism, Endotoxins toxicity, Enzyme-Linked Immunosorbent Assay, Galectins genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Hemolysin Proteins metabolism, Hemolysin Proteins toxicity, Host-Pathogen Interactions, Intestinal Mucosa metabolism, Mutation, Protein Binding, Protein Isoforms genetics, Protein Isoforms metabolism, Recombinant Proteins metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Galectins metabolism, Glycolipids metabolism
- Abstract
Galectins are a family of beta-galactoside-binding proteins that are widely found among animal species and that regulate diverse biological phenomena. To study the biological function of glycolipid-binding galectins, we purified recombinant Caenorhabditis elegans galectins (LEC-1-11) and studied their binding to C. elegans glycolipids. We found that LEC-8 binds to glycolipids in C. elegans through carbohydrate recognition. It has been reported that Cry5B-producing Bacillus thuringiensis strains can infect C. elegans and that the C. elegans Cry5B receptor molecules are glycolipids. We found that Cry5B and LEC-8 bound to C. elegans glycolipid-coated plates in a dose-dependent manner and that Cry5B binding to glycolipids was inhibited by the addition of LEC-8. LEC-8 is usually expressed strongly in the pharyngeal-intestinal valve and intestinal-rectal valve and is expressed weakly in intestine. However, when C. elegans were fed Escherichia coli expressing Cry5B, intestinal LEC-8::EGFP protein levels increased markedly. In contrast, LEC-8::EGFP expression triggered by Cry5B was reduced in toxin-resistant C. elegans mutants, which had mutations in genes involved in biosynthesis of glycolipids. Moreover, the LEC-8-deficient mutant was more susceptible to Cry5B than wild-type worms. These results suggest that the glycolipid-binding lectin LEC-8 contributes to host defense against bacterial infection by competitive binding to target glycolipid molecules.
- Published
- 2009
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37. Functional analysis of GS28, an intra-Golgi SNARE, in Caenorhabditis elegans.
- Author
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Maekawa M, Inoue T, Kobuna H, Nishimura T, Gengyo-Ando K, Mitani S, and Arai H
- Subjects
- Animals, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins genetics, Cell Differentiation, Cell Proliferation, Epithelial Cells cytology, Gene Deletion, Humans, Mice, R-SNARE Proteins genetics, RNA Interference, SNARE Proteins genetics, Stem Cells cytology, Caenorhabditis elegans embryology, Caenorhabditis elegans Proteins metabolism, Gene Expression Regulation, Developmental, Golgi Apparatus metabolism, R-SNARE Proteins metabolism, SNARE Proteins metabolism
- Abstract
Intra-Golgi retrograde transport is assumed to maintain Golgi function by recycling Golgi-resident proteins to younger cisternae in the progression of entire Golgi stack from cis to trans. GS28 (Golgi SNARE of 28 kDa, also known as GOS28) is a Golgi-localized SNARE protein and has been implicated in intra-Golgi retrograde transport. However, the in vivo functions of GS28, and consequently, the roles of the intra-Golgi retrograde transport in animal development are largely unknown. In this study, we generated deletion mutants of Caenorhabditis elegans GS28 and performed a synthetic lethal RNAi screen using GS28 mutants. We found that another Golgi-localized SNARE, Ykt6, functions cooperatively with GS28 in embryonic development. During post-embryonic development, GS28 mutants exhibited reduced seam cell numbers and a missing ray phenotype under Ykt6 knockdown conditions, suggesting that cell proliferation and/or differentiation of stem cell-like seam cells are impaired in GS28- and Ykt6-depleted worms. We also demonstrated that GS28 and Ykt6 act redundantly for the proper expression of Golgi-resident proteins in adult intestinal cells. This study reveals the in vivo importance of the Golgi-localized SNAREs GS28 and Ykt6.
- Published
- 2009
- Full Text
- View/download PDF
38. The ortholog of human solute carrier family 35 member B1 (UDP-galactose transporter-related protein 1) is involved in maintenance of ER homeostasis and essential for larval development in Caenorhabditis elegans.
- Author
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Dejima K, Murata D, Mizuguchi S, Nomura KH, Gengyo-Ando K, Mitani S, Kamiyama S, Nishihara S, and Nomura K
- Subjects
- Animals, Gene Expression Regulation, Genes, Reporter, Nucleotide Transport Proteins, Phenotype, RNA, Small Interfering pharmacology, Sequence Deletion, Caenorhabditis elegans physiology, Endoplasmic Reticulum physiology, Homeostasis, Larva growth & development, Monosaccharide Transport Proteins physiology
- Abstract
Although the solute carrier 35B1 (SLC35B1) is evolutionarily conserved, its functions in metazoans remain unknown. To elucidate its function, we examined developmental roles of an SLC35B1 family gene (HUT-1: homolog of UDP-Gal transporter) in Caenorhabditis elegans. We isolated a deletion mutant of the gene and characterized phenotypes of the mutant and hut-1 RNAi-treated worms. GFP-HUT-1 reporter analysis was performed to examine gene expression patterns. We also tested whether several nucleotide sugar transporters can compensate for hut-1 deficiency. The hut-1 deletion mutant and RNAi worms showed larval growth defect and lethality with disrupted intestinal morphology. Inactivation of hut-1 induced chronic endoplasmic reticulum (ER) stress, and hut-1 showed genetic interactions with the atf-6, pek-1, and ire-1 genes involved in unfolded protein response signaling. ER ultrastructure and ER marker distribution in hut-1-deficient animals showed that HUT-1 is required for maintenance of ER structure. Reporter analysis revealed that HUT-1 is an ER protein ubiquitously expressed in tissues, including the intestine. Lethality and the ER stress phenotype of the mutant were rescued with the human hut-1 ortholog UGTrel1. These results indicate important roles for hut-1 in development and maintenance of ER homeostasis in C. elegans.
- Published
- 2009
- Full Text
- View/download PDF
39. Normal formation of a subset of intestinal granules in Caenorhabditis elegans requires ATP-binding cassette transporters HAF-4 and HAF-9, which are highly homologous to human lysosomal peptide transporter TAP-like.
- Author
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Kawai H, Tanji T, Shiraishi H, Yamada M, Iijima R, Inoue T, Kezuka Y, Ohashi K, Yoshida Y, Tohyama K, Gengyo-Ando K, Mitani S, Arai H, Ohashi-Kobayashi A, and Maeda M
- Subjects
- ATP-Binding Cassette Transporters chemistry, Amino Acid Sequence, Animals, Caenorhabditis elegans cytology, Caenorhabditis elegans growth & development, Caenorhabditis elegans ultrastructure, Cytoplasmic Granules ultrastructure, Gene Deletion, Green Fluorescent Proteins metabolism, Humans, Intestines cytology, Intestines ultrastructure, Lysosomes metabolism, Membrane Glycoproteins metabolism, Molecular Sequence Data, Organ Specificity, Phenotype, Protein Transport, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, ATP-Binding Cassette Transporters metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Cytoplasmic Granules metabolism, Intestinal Mucosa metabolism
- Abstract
TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects.
- Published
- 2009
- Full Text
- View/download PDF
40. Member of the membrane-bound O-acyltransferase (MBOAT) family encodes a lysophospholipid acyltransferase with broad substrate specificity.
- Author
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Matsuda S, Inoue T, Lee HC, Kono N, Tanaka F, Gengyo-Ando K, Mitani S, and Arai H
- Subjects
- 1-Acylglycerophosphocholine O-Acyltransferase genetics, Amino Acid Sequence, Animals, Humans, Lysophosphatidylcholines metabolism, Lysophospholipids metabolism, Molecular Sequence Data, Sequence Alignment, Substrate Specificity, 1-Acylglycerophosphocholine O-Acyltransferase chemistry, 1-Acylglycerophosphocholine O-Acyltransferase metabolism, Caenorhabditis elegans enzymology
- Abstract
Glycerophospholipids in biological membranes are metabolically active and participate in a series of deacylation-reacylation reactions, which may lead to accumulation of polyunsaturated fatty acids (PUFAs) at the sn-2 position of the glycerol backbone. The reacylation reaction is believed to be catalyzed by acyl-coenzyme A (acyl-CoA):lysophospholipid acyltransferase. Very recently, we have shown that Caenorhabditis elegans mboa-7, which belongs to the membrane-bound O-acyltransferase (MBOAT) family, encodes lysophosphatidylinositol (LPI)-specific acyltransferase (LPIAT). In this study, we found that knockdown of another member of the MBOAT family in C. elegans, named mboa-6, reduced incorporation of exogenous PUFAs into phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) in C. elegans. Knockdown of a human mboa-6 homologue, referred to as MBOAT5, also impaired the incorporation of PUFAs into PC, PS and PE in HeLa cells. In in vitro assays, lysoPC (LPC), lysoPS (LPS) and lysoPE (LPE) acyltransferase activities using [(14)C]arachidonoyl-CoA were significantly reduced in the microsomes of MBOAT5 knockdown cells. Conversely, over-expression of MBOAT5 in human embryonic kidney (HEK) 293 cells resulted in great increases in LPC, LPS and LPE acyltransferase activities but not in LPIAT or lysophosphatidic acid (LPA) acyltransferase (LPAAT) activities. These results indicate that human MBOAT5 is a lysophospholipid acyltransferase acting preferentially on LPC, LPS and LPE.
- Published
- 2008
- Full Text
- View/download PDF
41. Beta-catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions.
- Author
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Kanamori T, Inoue T, Sakamoto T, Gengyo-Ando K, Tsujimoto M, Mitani S, Sawa H, Aoki J, and Arai H
- Subjects
- Animals, Biological Transport, Caenorhabditis elegans genetics, Cell Count, Cell Lineage, Cell Polarity, Cytoskeletal Proteins metabolism, Female, Genes, Helminth, Genes, Suppressor, Guanine Nucleotide Exchange Factors, Mutant Proteins isolation & purification, Mutant Proteins metabolism, Mutation genetics, Phenotype, Spindle Apparatus metabolism, Subcellular Fractions metabolism, Vulva cytology, rab GTP-Binding Proteins metabolism, Caenorhabditis elegans cytology, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Cell Division, Phospholipases A1 metabolism, Stem Cells cytology, beta Catenin metabolism
- Abstract
Asymmetric division is an important property of stem cells. In Caenorhabditis elegans, the Wnt/beta-catenin asymmetry pathway determines the polarity of most asymmetric divisions. The Wnt signalling components such as beta-catenin localize asymmetrically to the cortex of mother cells to produce two distinct daughter cells. However, the molecular mechanism to polarize them remains to be elucidated. Here, we demonstrate that intracellular phospholipase A(1) (PLA(1)), a poorly characterized lipid-metabolizing enzyme, controls the subcellular localizations of beta-catenin in the terminal asymmetric divisions of epithelial stem cells (seam cells). In mutants of ipla-1, a single C. elegans PLA(1) gene, cortical beta-catenin is delocalized and the asymmetry of cell-fate specification is disrupted in the asymmetric divisions. ipla-1 mutant phenotypes are rescued by expression of ipla-1 in seam cells in a catalytic activity-dependent manner. Furthermore, our genetic screen utilizing ipla-1 mutants reveals that reduction of endosome-to-Golgi retrograde transport in seam cells restores normal subcellular localization of beta-catenin to ipla-1 mutants. We propose that membrane trafficking regulated by ipla-1 provides a mechanism to control the cortical asymmetry of beta-catenin.
- Published
- 2008
- Full Text
- View/download PDF
42. Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry.
- Author
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Darland-Ransom M, Wang X, Sun CL, Mapes J, Gengyo-Ando K, Mitani S, and Xue D
- Subjects
- Animals, Animals, Genetically Modified, Apoptosis, Caenorhabditis elegans cytology, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Germ Cells cytology, Germ Cells metabolism, Muscle Cells cytology, Muscle Cells metabolism, Neurons cytology, Neurons metabolism, Phagocytosis, Phospholipid Transfer Proteins genetics, RNA Interference, Recombinant Fusion Proteins metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Cell Membrane metabolism, Phosphatidylserines metabolism, Phospholipid Transfer Proteins metabolism
- Abstract
The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.
- Published
- 2008
- Full Text
- View/download PDF
43. Caenorhabditis elegans mboa-7, a member of the MBOAT family, is required for selective incorporation of polyunsaturated fatty acids into phosphatidylinositol.
- Author
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Lee HC, Inoue T, Imae R, Kono N, Shirae S, Matsuda S, Gengyo-Ando K, Mitani S, and Arai H
- Subjects
- Acyltransferases chemistry, Amino Acid Sequence, Animals, Arachidonic Acids metabolism, Caenorhabditis elegans cytology, Caenorhabditis elegans enzymology, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins chemistry, Cell Membrane enzymology, Fatty Acids, Unsaturated biosynthesis, Fatty Acids, Unsaturated chemistry, Genes, Helminth, HeLa Cells, Humans, Mice, Molecular Sequence Data, Mutation genetics, Phenotype, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Acyltransferases metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Fatty Acids, Unsaturated metabolism, Phosphatidylinositols metabolism
- Abstract
Phosphatidylinositol (PI) is a component of membrane phospholipids, and it functions both as a signaling molecule and as a compartment-specific localization signal in the form of polyphosphoinositides. Arachidonic acid (AA) is the predominant fatty acid in the sn-2 position of PI in mammals. LysoPI acyltransferase (LPIAT) is thought to catalyze formation of AA-containing PI; however, the gene that encodes this enzyme has not yet been identified. In this study, we established a screening system to identify genes required for use of exogenous polyunsaturated fatty acids (PUFAs) in Caenorhabditis elegans. In C. elegans, eicosapentaenoic acid (EPA) instead of AA is the predominant fatty acid in PI. We showed that an uncharacterized gene, which we named mboa-7, is required for incorporation of PUFAs into PI. Incorporation of exogenous PUFA into PI of the living worms and LPIAT activity in the microsomes were greatly reduced in mboa-7 mutants. Furthermore, the membrane fractions of transgenic worms expressing recombinant MBOA-7 and its human homologue exhibited remarkably increased LPIAT activity. mboa-7 encodes a member of the membrane-bound O-acyltransferase family, suggesting that mboa-7 is LPIAT. Finally, mboa-7 mutants had significantly lower EPA levels in PI, and they exhibited larval arrest and egg-laying defects.
- Published
- 2008
- Full Text
- View/download PDF
44. Control of sex-specific apoptosis in C. elegans by the BarH homeodomain protein CEH-30 and the transcriptional repressor UNC-37/Groucho.
- Author
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Peden E, Kimberly E, Gengyo-Ando K, Mitani S, and Xue D
- Subjects
- Amino Acid Sequence, Animals, Animals, Genetically Modified, Apoptosis genetics, Base Sequence, Caenorhabditis elegans embryology, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Caspases genetics, Caspases physiology, DNA, Helminth genetics, DNA-Binding Proteins genetics, DNA-Binding Proteins physiology, Female, Gene Expression Regulation, Developmental, Genes, Helminth, Homeodomain Proteins genetics, Male, Models, Biological, Mutation, POU Domain Factors genetics, POU Domain Factors physiology, Repressor Proteins genetics, Repressor Proteins physiology, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid, Sex Characteristics, Transcription Factors genetics, X Chromosome genetics, Apoptosis physiology, Caenorhabditis elegans cytology, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins physiology, Homeodomain Proteins physiology, Transcription Factors physiology
- Abstract
Apoptosis is essential for proper development and tissue homeostasis in metazoans. It plays a critical role in generating sexual dimorphism by eliminating structures that are not needed in a specific sex. The molecular mechanisms that regulate sexually dimorphic apoptosis are poorly understood. Here we report the identification of the ceh-30 gene as a key regulator of sex-specific apoptosis in Caenorhabditis elegans. Loss-of-function mutations in ceh-30 cause the ectopic death of male-specific CEM neurons. ceh-30 encodes a BarH homeodomain protein that acts downstream from the terminal sex determination gene tra-1, but upstream of, or in parallel to, the cell-death-initiating gene egl-1 to protect CEM neurons from undergoing apoptosis in males. The second intron of the ceh-30 gene contains two adjacent cis-elements that are binding sites for TRA-1A and a POU-type homeodomain protein UNC-86 and acts as a sensor to regulate proper specification of the CEM cell fate. Surprisingly, the N terminus of CEH-30 but not its homeodomain is critical for CEH-30's cell death inhibitory activity in CEMs and contains a conserved eh1/FIL domain that is important for the recruitment of the general transcriptional repressor UNC-37/Groucho. Our study suggests that ceh-30 defines a critical checkpoint that integrates the sex determination signal TRA-1 and the cell fate determination and survival signal UNC-86 to control the sex-specific activation of the cell death program in CEMs through the general transcription repressor UNC-37.
- Published
- 2007
- Full Text
- View/download PDF
45. The PLEXIN PLX-2 and the ephrin EFN-4 have distinct roles in MAB-20/Semaphorin 2A signaling in Caenorhabditis elegans morphogenesis.
- Author
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Nakao F, Hudson ML, Suzuki M, Peckler Z, Kurokawa R, Liu Z, Gengyo-Ando K, Nukazuka A, Fujii T, Suto F, Shibata Y, Shioi G, Fujisawa H, Mitani S, Chisholm AD, and Takagi S
- Subjects
- Animals, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins physiology, Signal Transduction, Caenorhabditis elegans Proteins metabolism, Cell Adhesion Molecules physiology, Ephrin-A4 physiology, Membrane Proteins metabolism, Morphogenesis, Nerve Tissue Proteins metabolism, Nerve Tissue Proteins physiology, Semaphorins metabolism
- Abstract
Semaphorins are extracellular proteins that regulate axon guidance and morphogenesis by interacting with a variety of cell surface receptors. Most semaphorins interact with plexin-containing receptor complexes, although some interact with non-plexin receptors. Class 2 semaphorins are secreted molecules that control axon guidance and epidermal morphogenesis in Drosophila and Caenorhabditis elegans. We show that the C. elegans class 2 semaphorin MAB-20 binds the plexin PLX-2. plx-2 mutations enhance the phenotypes of hypomorphic mab-20 alleles but not those of mab-20 null alleles, indicating that plx-2 and mab-20 act in a common pathway. Both mab-20 and plx-2 mutations affect epidermal morphogenesis during embryonic and in postembryonic development. In both contexts, plx-2 null mutant phenotypes are much less severe than mab-20 null phenotypes, indicating that PLX-2 is not essential for MAB-20 signaling. Mutations in the ephrin efn-4 do not synergize with mab-20, indicating that EFN-4 may act in MAB-20 signaling. EFN-4 and PLX-2 are coexpressed in the late embryonic epidermis where they play redundant roles in MAB-20-dependent cell sorting.
- Published
- 2007
- Full Text
- View/download PDF
46. IFT-81 and IFT-74 are required for intraflagellar transport in C. elegans.
- Author
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Kobayashi T, Gengyo-Ando K, Ishihara T, Katsura I, and Mitani S
- Subjects
- Animals, Animals, Genetically Modified, Biological Transport, Active, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Carrier Proteins genetics, Chemotaxis, Cilia physiology, Genes, Helminth, Mutation, Neurons, Afferent physiology, Phenotype, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins physiology, Carrier Proteins physiology, Flagella physiology
- Abstract
Intraflagellar transport (IFT) is essential machinery for biogenesis and maintenance of cilia in many eukaryotic and prokaryotic cells. A large number of polypeptides are known to be involved in IFT, but the physiological role of each component is not fully elucidated. Here, we identified a C. elegans orthologue of a Chlamydomonas reinhardtii IFT component, IFT-81, and found that its loss-of-function mutants show an unusual behavioral property and small body size. IFT-81 is expressed in sensory neurons, and localized at the base of cilia. The similar phenotypes with ift-81 mutants were also observed in several IFT mutants, suggesting these defects are caused by inability of IFT. We also demonstrated that IFT-81 interacts and co-localizes with IFT-74, which is another putative component of IFT. The ift-74 loss-of-function mutants showed phenocopies with ift-81 mutants, suggesting IFT-81 and IFT-74 play comparable functions. Moreover, ift-81 and ift-74 mutants similarly exhibited weak anomalies in cilia formation and obvious disruptions of transport in mature cilia. Thus, we conclude that IFT-81 and IFT-74 coordinately act in IFT in C. elegans sensory cilia.
- Published
- 2007
- Full Text
- View/download PDF
47. C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1.
- Author
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Wang X, Wang J, Gengyo-Ando K, Gu L, Sun CL, Yang C, Shi Y, Kobayashi T, Shi Y, Mitani S, Xie XS, and Xue D
- Subjects
- Animals, Annexin A5 metabolism, Biological Transport, Caenorhabditis elegans cytology, Caenorhabditis elegans enzymology, Caenorhabditis elegans Proteins genetics, Caspases genetics, Caspases metabolism, Cell Membrane metabolism, Germ Cells enzymology, Membrane Proteins genetics, Membrane Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins genetics, Models, Molecular, Mutation, Phospholipid Transfer Proteins genetics, RNA Interference, Staining and Labeling methods, Time Factors, Apoptosis, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Germ Cells metabolism, Mitochondrial Proteins metabolism, Phosphatidylserines metabolism, Phospholipid Transfer Proteins metabolism
- Abstract
Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.
- Published
- 2007
- Full Text
- View/download PDF
48. Expression of rib-1, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes, is indispensable for heparan sulfate synthesis and embryonic morphogenesis.
- Author
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Kitagawa H, Izumikawa T, Mizuguchi S, Dejima K, Nomura KH, Egusa N, Taniguchi F, Tamura J, Gengyo-Ando K, Mitani S, Nomura K, and Sugahara K
- Subjects
- Amino Acid Sequence, Animals, COS Cells, Caenorhabditis elegans, Caenorhabditis elegans Proteins biosynthesis, Cell Differentiation, Chlorocebus aethiops, Female, Gastrula metabolism, Gene Expression Regulation, Humans, Male, Molecular Sequence Data, Neurons metabolism, Tumor Suppressor Proteins physiology, Caenorhabditis elegans Proteins physiology, Heparitin Sulfate metabolism, Tumor Suppressor Proteins biosynthesis
- Abstract
The proteins encoded by all of the five cloned human EXT family genes (EXT1, EXT2, EXTL1, EXTL2, and EXTL3), members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the biosynthesis of heparan sulfate. In the Caenorhabditis elegans genome, only two genes, rib-1 and rib-2, homologous to the mammalian EXT genes have been identified. Although rib-2 encodes an N-acetylglucosaminyltransferase involved in initiating the biosynthesis and elongation of heparan sulfate, the involvement of the protein encoded by rib-1 in the biosynthesis of heparan sulfate remains unclear. Here we report that RIB-1 is indispensable for the biosynthesis and for embryonic morphogenesis. Despite little individual glycosyltransferase activity by RIB-1, the polymerization of heparan sulfate chains was demonstrated when RIB-1 was coexpressed with RIB-2 in vitro. In addition, RIB-1 and RIB-2 were demonstrated to interact by pulldown assays. To investigate the functions of RIB-1 in vivo, we depleted the expression of rib-1 by deletion mutagenesis. The null mutant worms showed reduced synthesis of heparan sulfate and embryonic lethality. Notably, the null mutant embryos showed abnormality at the gastrulation cleft formation stage or later and arrested mainly at the 1-fold stage. Nearly 100% of the embryos died before L1 stage, although the differentiation of some of the neurons and muscle cells proceeded normally. Similar phenotypes have been observed in rib-2 null mutant embryos. Thus, RIB-1 in addition to RIB-2 is indispensable for the biosynthesis of heparan sulfate in C. elegans, and the two cooperate to synthesize heparan sulfate in vivo. These findings also show that heparan sulfate is essential for post-gastrulation morphogenic movement of embryonic cells and is indispensable for ensuring the normal spatial organization of differentiated tissues and organs.
- Published
- 2007
- Full Text
- View/download PDF
49. ASB-1, a germline-specific isoform of mitochondrial ATP synthase b subunit, is required to maintain the rate of germline development in Caenorhabditis elegans.
- Author
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Kawasaki I, Hanazawa M, Gengyo-Ando K, Mitani S, Maruyama I, and Iino Y
- Subjects
- Amino Acid Sequence, Animals, Caenorhabditis elegans embryology, Female, Male, Molecular Sequence Data, Caenorhabditis elegans enzymology, Mitochondrial Proton-Translocating ATPases physiology, Oogenesis physiology, Protein Subunits physiology, Spermatogenesis physiology
- Abstract
The developmental timing of all types of cells must be synchronized and spatially coordinated to achieve the organized development of a multicellular organism. Previously, we found RNAi of asb-1, encoding a germline-specific isoform of mitochondrial ATP synthase b subunit, caused 100% penetrant sterility in Caenorhabditis elegans. ATP synthase is one of the five complexes of the mitochondrial respiratory chain, and defects in some of the components of the chain are known to slow the growth and extend the lifespan of worms. We found that development of asb-1 mutant germ line was not arrested at any stage, but did slow to half the rate of wild type, whereas the rate of somatic development was the same in asb-1 mutants as that of wild type, indicating that asb-1 is required to maintain the rate of germline development but has no effect on somatic development. Among ATP synthase subunit genes, RNAi of asg-1, encoding a germline-specific isoform of the g subunit, also caused asb-1-like sterility, indicating that some other germline-specific components are also required to maintain the rate of germline development. Both asb-1 and asg-1 are located on autosomes while they possess counterparts, asb-2 and asg-2, respectively, on X chromosome, which are both required for somatic development. Chromosomal locations of the genes may be the basis of the segregation of germline/somatic functions of each gene, as were demonstrated for other autosomal/X-linked duplicated gene pairs.
- Published
- 2007
- Full Text
- View/download PDF
50. The SM protein VPS-45 is required for RAB-5-dependent endocytic transport in Caenorhabditis elegans.
- Author
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Gengyo-Ando K, Kuroyanagi H, Kobayashi T, Murate M, Fujimoto K, Okabe S, and Mitani S
- Subjects
- Animals, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins genetics, Endocytosis genetics, Microscopy, Fluorescence, Munc18 Proteins genetics, Mutation genetics, Vesicular Transport Proteins genetics, rab5 GTP-Binding Proteins metabolism, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins metabolism, Endocytosis physiology, Munc18 Proteins metabolism, Signal Transduction genetics, Vesicular Transport Proteins metabolism
- Abstract
Rab5, a small guanosine triphosphatase, is known to regulate the tethering and docking reaction leading to SNARE (soluble NSF attachment protein receptors)-mediated fusion between endosomes. However, it is uncertain how the signal of the activated Rab5 protein is transduced by its downstream effectors during endosome fusion. Here, we show that the Sec1/Munc18 gene vps-45 is essential for not only viability and development but also receptor-mediated and fluid-phase endocytosis pathways in Caenorhabditis elegans. We found that VPS-45 interacts with a Rab5 effector, Rabenosyn-5 (RABS-5), and the mutants of both vps-45 and rabs-5 show similar endocytic phenotypes. In the macrophage-like cells of vps-45 and rabs-5 mutants, aberrantly small endosomes were accumulated, and the endosome fusion stimulated by the mutant RAB-5 (Q78L) is suppressed by these mutations. Our results indicate that VPS-45 is a key molecule that functions downstream from RAB-5, cooperating with RABS-5, to regulate the dynamics of the endocytic system in multicellular organisms.
- Published
- 2007
- Full Text
- View/download PDF
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