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1.
MicroPubl Biol ; 20242024.
Article in English | MEDLINE | ID: mdl-38716379

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.

2.
MicroPubl Biol ; 20242024.
Article in English | MEDLINE | ID: mdl-38585203

ABSTRACT

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.

3.
Int J Mol Sci ; 25(2)2024 Jan 07.
Article in English | MEDLINE | ID: mdl-38255846

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-ß 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.


Subject(s)
Adrenal Gland Neoplasms , Animals , Rats , PC12 Cells , Cell Differentiation , Physical Stimulation , p38 Mitogen-Activated Protein Kinases
4.
MicroPubl Biol ; 20232023.
Article in English | MEDLINE | ID: mdl-38089934

ABSTRACT

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.

5.
Shokuhin Eiseigaku Zasshi ; 63(2): 51-61, 2022.
Article in Japanese | MEDLINE | ID: mdl-35650029

ABSTRACT

Migration test of food utensils, containers, and packaging is an important test method for confirming the safety and their compliance to the standards. However, there is little report on inter-laboratory study which was performed to evaluate the entire migration test, including migration operations and quantification. An interlaboratory study was performed participating 22 laboratories using 8 types of model synthetic resin samples containing 10 substances with a wide range of Log Pow values to evaluate the accuracy of the entire migration test. As a result, most of HorRat (r) values met the target criteria (0.3

Subject(s)
Food Contamination , Plastics , Food Contamination/analysis , Food Packaging , Laboratories
6.
Neurosci Res ; 151: 53-60, 2020 Feb.
Article in English | MEDLINE | ID: mdl-30790590

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.


Subject(s)
Calcium/analysis , Calcium/metabolism , Enteric Nervous System/physiology , Intravital Microscopy/methods , Molecular Imaging/methods , Animals , Intestines , Male , Mice , Mice, Transgenic , Microscopy, Fluorescence/methods , Neurons/metabolism , Neurons/physiology , Serotonin/pharmacology
7.
J Anat ; 236(4): 622-629, 2020 04.
Article in English | MEDLINE | ID: mdl-31840255

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.


Subject(s)
Body Patterning/genetics , Ribs/embryology , Somites/enzymology , Zebrafish/embryology , 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
8.
Gastrointest Endosc ; 89(6): 1152-1159.e1, 2019 06.
Article in English | MEDLINE | ID: mdl-30825537

ABSTRACT

BACKGROUND AND AIMS: The incidence of metachronous gastric cancer (MGC) in patients whose primary gastric neoplasm is discovered after Helicobacter pylori eradication remains unclear. Here, we evaluated the long-term effect of previous H pylori eradication on development of MGC after endoscopic submucosal dissection (ESD). METHODS: We analyzed prospectively collected data of consecutive patients with successful H pylori eradication more than 1 year before (eradicated group, 180 patients) or after (control group, 602 patients) initial curative ESD. These patients were also followed by endoscopy for over 2 years. Propensity score matching and inverse probability of treatment weighting (IPTW) were used to adjust for confounding variables during data analysis. The main outcome was the incidence of MGC after initial ESD. RESULTS: In a propensity-matched analysis of 174 pairs, the incidence of MGC was similar in the 2 cohorts (33.9 per 1000 person-years vs 40.8 per 1000 person-years in the control group, P = .454) at a median follow-up of 4.1 years (interquartile range, 3.0-5.6). Incidences were also similar in the 2 groups when data were analyzed using IPTW, even after exclusion of 123 patients with successful H pylori eradication <5 years before initial ESD. Multiple Cox regression analysis revealed age, differentiated-type histology, and initial multiplicity were predictors of MGC in patients after initial curative ESD. CONCLUSIONS: The frequency of follow-up surveillance after initial curative ESD should be kept constant, irrespective of whether H pylori eradication is performed before or after initial curative ESD.


Subject(s)
Adenocarcinoma/surgery , Adenoma/surgery , Anti-Bacterial Agents/therapeutic use , Helicobacter Infections/drug therapy , Neoplasms, Second Primary/epidemiology , Stomach Neoplasms/surgery , Adenocarcinoma/epidemiology , Adenocarcinoma/pathology , Adenoma/epidemiology , Adenoma/pathology , Aged , Endoscopic Mucosal Resection , Female , Gastroscopy , Helicobacter pylori , Humans , Incidence , Kaplan-Meier Estimate , Male , Middle Aged , Propensity Score , Proportional Hazards Models , Retrospective Studies , Stomach Neoplasms/epidemiology , Stomach Neoplasms/pathology
9.
Glycobiology ; 29(2): 163-178, 2019 02 01.
Article in English | MEDLINE | ID: mdl-30445613

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.


Subject(s)
Caenorhabditis elegans/metabolism , Embryo, Nonmammalian/metabolism , Oocytes/metabolism , Oogenesis/genetics , Transferases (Other Substituted Phosphate Groups)/metabolism , Animals
10.
Zoological Lett ; 4: 19, 2018.
Article in English | MEDLINE | ID: mdl-30065850

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.

11.
Biomed Opt Express ; 8(9): 4049-4060, 2017 Sep 01.
Article in English | MEDLINE | ID: mdl-28966846

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.

12.
Leuk Res Rep ; 7: 33-35, 2017.
Article in English | MEDLINE | ID: mdl-28462083

ABSTRACT

It is difficult to predict musculoskeletal pain as a withdrawal syndrome following the discontinuation of imatinib (IM) in patients with chronic myeloid leukemia. We investigated a link between physical size and musculoskeletal pain following IM discontinuation. In total, seven out of 24 patients developed musculoskeletal pain after discontinuing IM. Those with symptoms had a significantly lower body weight (BW) and body mass index (BMI) than those without symptoms. While previous reports indicated that physical size is associated with the pharmacokinetics of IM, our current study suggests that lower BW and BMI may be associated with musculoskeletal pain following IM discontinuation.

13.
J Neurosci Methods ; 286: 56-68, 2017 Jul 15.
Article in English | MEDLINE | ID: mdl-28506879

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. Ca2+ 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.


Subject(s)
Neurons/physiology , Optogenetics/methods , Wakefulness , 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
14.
Elife ; 62017 05 23.
Article in English | MEDLINE | ID: mdl-28532547

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 ([Ca2+]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.


Subject(s)
Caenorhabditis elegans/physiology , Calcium Channels/metabolism , Calcium/metabolism , Animals , Behavior, Animal , Decision Making , Smell , Spatial Navigation , Time Factors
15.
Int J Hematol ; 105(4): 419-422, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28197964

ABSTRACT

We analyzed the exosomal miRNA from peripheral blood from CML patients with musculoskeletal pain after stopping tyrosine kinase inhibitors to identify possible factors related to this manifestation. Exosomal miRNA profiling using TaqMan low-density array revealed that exosomal miR-140-3p was significantly elevated in CML patients showing musculoskeletal pain, when compared to those without such pain (P = 0.0336) or healthy individuals (P = 0.0022). All five CML patients with musculoskeletal pain and increased exosomal miR-140-3p levels sustained deep molecular responses: four of them achieved symptom relief and a significant decrease in exosomal miR-140-3p levels was evident. Because exosomal miR-140-3p is considered to have an inflammation-associated biological function in airway smooth muscle cells and targets Myomarker muscle-specific transmembrane protein, it appears that its overexpression in circulating exosomal miR-140-3p may have some role in the mechanism underlying self-limited musculoskeletal pain.


Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/complications , MicroRNAs/blood , MicroRNAs/physiology , Musculoskeletal Pain/blood , Protein-Tyrosine Kinases/antagonists & inhibitors , Enzyme Inhibitors/therapeutic use , Exosomes/chemistry , Female , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/blood , Male , Up-Regulation , Withholding Treatment
16.
Traffic ; 17(11): 1197-1213, 2016 11.
Article in English | MEDLINE | ID: mdl-27558849

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.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Endocytosis/physiology , Endosomes/metabolism , Lysosomes/metabolism , Vesicular Transport Proteins/metabolism , 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
17.
Rinsho Ketsueki ; 57(7): 873-6, 2016 07.
Article in Japanese | MEDLINE | ID: mdl-27498732

ABSTRACT

We conducted a questionnaire survey to assess the state of patients with CML after discontinuation of TKI therapy. Nine of 27 patients developed musculoskeletal pain after TKI discontinuation. One had discontinued nilotinib and eight had discontinued imatinib therapy. Median time to symptom development after discontinuation was 2 weeks. Four experienced grade 3 symptoms as per the CTCAE ver. 4.0. One had pain persisting over a period of 21 months. There was a significant difference between patients with and without symptoms as regards female gender and the probability of persistent MMR. Awareness of this withdrawal syndrome after TKI discontinuation is imperative.


Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Musculoskeletal Pain/etiology , Protein Kinase Inhibitors/adverse effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Protein Kinase Inhibitors/therapeutic use , Surveys and Questionnaires , Treatment Outcome
18.
BMC Cell Biol ; 17: 4, 2016 Jan 27.
Article in English | MEDLINE | ID: mdl-26817689

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.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Intestinal Mucosa/metabolism , Organelles/metabolism , ATP-Binding Cassette Transporters/genetics , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Female , Male , Organelles/genetics , Protein Transport
19.
Dev Cell ; 35(2): 211-21, 2015 Oct 26.
Article in English | MEDLINE | ID: mdl-26506309

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.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Cytokinesis/genetics , Embryonic Development/genetics , Guanine Nucleotide Exchange Factors/genetics , Vesicular Transport Proteins/genetics , Animals , Caenorhabditis elegans/embryology , Caenorhabditis elegans Proteins/biosynthesis , Embryo, Nonmammalian , Endosomes/metabolism , Gene Expression Regulation, Developmental , Humans , RNA Interference , Vesicular Transport Proteins/biosynthesis
20.
PLoS One ; 10(5): e0125354, 2015.
Article in English | MEDLINE | ID: mdl-25946002

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.


Subject(s)
CA1 Region, Hippocampal/metabolism , Diagnostic Imaging/methods , Membrane Proteins/genetics , Pyramidal Cells/metabolism , Response Elements/genetics , Animals , Calcium/metabolism , Fluorescence Resonance Energy Transfer/methods , Gene Expression/genetics , Green Fluorescent Proteins/genetics , Mice , Mice, Transgenic , Microscopy, Fluorescence/methods , Tetracycline/pharmacology
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