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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.
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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.
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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.
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Neoplasias das Glândulas Suprarrenais , Animais , Ratos , Células PC12 , Diferenciação Celular , Estimulação Física , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
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.
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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.
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Padronização Corporal/genética , Costelas/embriologia , Somitos/enzimologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Costelas/diagnóstico por imagem , Somitos/diagnóstico por imagem , Proteínas com Domínio T/genética , Tomografia Computadorizada por Raios X , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
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.
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Cálcio/análise , Cálcio/metabolismo , Sistema Nervoso Entérico/fisiologia , Microscopia Intravital/métodos , Imagem Molecular/métodos , Animais , Intestinos , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Neurônios/metabolismo , Neurônios/fisiologia , Serotonina/farmacologiaRESUMO
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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Caenorhabditis elegans/metabolismo , Embrião não Mamífero/metabolismo , Oócitos/metabolismo , Oogênese/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , AnimaisRESUMO
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.
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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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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.
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Neurônios/fisiologia , Optogenética/métodos , Vigília , Animais , Animais Geneticamente Modificados , Automação Laboratorial , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Luz , Rede Nervosa/fisiologiaRESUMO
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.
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Caenorhabditis elegans/fisiologia , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Animais , Comportamento Animal , Tomada de Decisões , Olfato , Navegação Espacial , Fatores de TempoRESUMO
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.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Endocitose/genética , Endossomos/genética , Lisossomos/genética , Masculino , Microscopia Confocal , Mutação , Oócitos/metabolismo , Oócitos/ultraestrutura , Transporte Proteico , Espermatogênese/genética , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Proteínas de Transporte Vesicular/genéticaRESUMO
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.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Mucosa Intestinal/metabolismo , Organelas/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Feminino , Masculino , Organelas/genética , Transporte ProteicoRESUMO
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.
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Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Citocinese/genética , Desenvolvimento Embrionário/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Transporte Vesicular/genética , Animais , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/biossíntese , Embrião não Mamífero , Endossomos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Interferência de RNA , Proteínas de Transporte Vesicular/biossínteseRESUMO
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.
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Região CA1 Hipocampal/metabolismo , Diagnóstico por Imagem/métodos , Proteínas de Membrana/genética , Células Piramidais/metabolismo , Elementos de Resposta/genética , Animais , Cálcio/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Tetraciclina/farmacologiaRESUMO
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-ß 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.
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Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Indicadores e Reagentes/síntese química , Potenciais de Ação/fisiologia , Animais , Caenorhabditis elegans/efeitos da radiação , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Ligação a Calmodulina , Células Cultivadas , Córtex Cerebral/citologia , Corantes Fluorescentes/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Luz , Camundongos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
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.
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Citoesqueleto de Actina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Filaminas/metabolismo , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/metabolismo , Actinas/metabolismo , Animais , Caenorhabditis elegans/genética , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Ratos Wistar , Semaforina-3A/metabolismoRESUMO
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.
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Apoptose , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/enzimologia , GTP Fosfo-Hidrolases/fisiologia , Fagossomos/enzimologia , Animais , Caenorhabditis elegans/citologia , Células Germinativas/fisiologia , Gônadas/citologia , Gônadas/enzimologia , Lisossomos/enzimologia , Transporte Proteico , Imagem com Lapso de Tempo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
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)â=â38) 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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Sinalização do Cálcio/genética , Cálcio , Proteínas de Fluorescência Verde , Células Piramidais , Animais , Cálcio/química , Cálcio/isolamento & purificação , Cálcio/metabolismo , Células Cultivadas , Espinhas Dendríticas/metabolismo , Giro Denteado/citologia , Giro Denteado/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Indicadores e Reagentes/análise , Indicadores e Reagentes/metabolismo , Técnicas de Rastreamento Neuroanatômico , Neurônios/citologia , Neurônios/metabolismo , Células Piramidais/citologia , Células Piramidais/metabolismo , RatosRESUMO
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.