RESUMEN
Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Lisosomas , Animales , Humanos , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/ultraestructura , Homeostasis , Longevidad , Lisosomas/metabolismo , Lisosomas/ultraestructura , Secuencias de Aminoácidos , Microscopía ElectrónicaRESUMEN
Mitochondria transport is crucial for axonal mitochondria distribution and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 binding to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1, and metaxin2. We conclude that transport complexes containing kinesin-1 and RIC-7 polarize at the leading edge of mitochondria and are required for anterograde axonal transport in C. elegans.
Asunto(s)
Transporte Axonal , Cinesinas , Animales , Axones , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Cinesinas/metabolismo , Mitocondrias/metabolismoAsunto(s)
Caenorhabditis elegans , Red Nerviosa , Tejido Nervioso , Neuronas , Transducción de Señal , Caenorhabditis elegans/anatomía & histología , Caenorhabditis elegans/citología , Caenorhabditis elegans/fisiología , Red Nerviosa/anatomía & histología , Red Nerviosa/citología , Red Nerviosa/fisiología , Tejido Nervioso/anatomía & histología , Tejido Nervioso/citología , Tejido Nervioso/fisiología , Neuronas/fisiología , Neuropéptidos/metabolismo , Transmisión Sináptica , AnimalesRESUMEN
Establishing how neural function emerges from network properties is a fundamental problem in neuroscience1. Here, to better understand the relationship between the structure and the function of a nervous system, we systematically measure signal propagation in 23,433 pairs of neurons across the head of the nematode Caenorhabditis elegans by direct optogenetic activation and simultaneous whole-brain calcium imaging. We measure the sign (excitatory or inhibitory), strength, temporal properties and causal direction of signal propagation between these neurons to create a functional atlas. We find that signal propagation differs from model predictions that are based on anatomy. Using mutants, we show that extrasynaptic signalling not visible from anatomy contributes to this difference. We identify many instances of dense-core-vesicle-dependent signalling, including on timescales of less than a second, that evoke acute calcium transients-often where no direct wired connection exists but where relevant neuropeptides and receptors are expressed. We propose that, in such cases, extrasynaptically released neuropeptides serve a similar function to that of classical neurotransmitters. Finally, our measured signal propagation atlas better predicts the neural dynamics of spontaneous activity than do models based on anatomy. We conclude that both synaptic and extrasynaptic signalling drive neural dynamics on short timescales, and that measurements of evoked signal propagation are crucial for interpreting neural function.
Asunto(s)
Caenorhabditis elegans , Vías Nerviosas , Neuronas , Animales , Caenorhabditis elegans/anatomía & histología , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Calcio/análisis , Calcio/metabolismo , Modelos Neurológicos , Mutación , Vías Nerviosas/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Neuropéptidos/metabolismo , Sinapsis/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Cellular homeostasis requires the robust control of biomolecule concentrations, but how do millions of mRNAs coordinate their stoichiometries in the face of dynamic translational changes? Here, we identified a two-tiered mechanism controlling mRNA:mRNA and mRNA:protein stoichiometries where mRNAs super-assemble into condensates with buffering capacity and sorting selectivity through phase-transition mechanisms. Using C. elegans oogenesis arrest as a model, we investigated the transcriptome cytosolic reorganization through the sequencing of RNA super-assemblies coupled with single mRNA imaging. Tightly repressed mRNAs self-assembled into same-sequence nanoclusters that further co-assembled into multiphase condensates. mRNA self-sorting was concentration dependent, providing a self-buffering mechanism that is selective to sequence identity and controls mRNA:mRNA stoichiometries. The cooperative sharing of limiting translation repressors between clustered mRNAs prevented the disruption of mRNA:repressor stoichiometries in the cytosol. Robust control of mRNA:mRNA and mRNA:protein stoichiometries emerges from mRNA self-demixing and cooperative super-assembly into multiphase multiscale condensates with dynamic storage capacity.
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Condensados Biomoleculares , Caenorhabditis elegans , ARN Mensajero , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Oogénesis , Biosíntesis de Proteínas , Transporte de ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas/química , Proteínas/metabolismo , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismoRESUMEN
Aging is characterized by a global decline in physiological function. However, by constructing a complete single-cell gene expression atlas, we find that Caenorhabditis elegans aging is not random in nature but instead is characterized by coordinated changes in functionally related metabolic, proteostasis, and stress-response genes in a cell-type-specific fashion, with downregulation of energy metabolism being the only nearly universal change. Similarly, the rates at which cells age differ significantly between cell types. In some cell types, aging is characterized by an increase in cell-to-cell variance, whereas in others, variance actually decreases. Remarkably, multiple resilience-enhancing transcription factors known to extend lifespan are activated across many cell types with age; we discovered new longevity candidates, such as GEI-3, among these. Together, our findings suggest that cells do not age passively but instead react strongly, and individualistically, to events that occur during aging. This atlas can be queried through a public interface.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Envejecimiento , Senescencia Celular , Metabolismo Energético , Longevidad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Homeostasis , Análisis de Expresión Génica de una Sola Célula , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fenómenos Fisiológicos CelularesRESUMEN
Bub1 and Polo kinases are well-known multitasking regulators of mitosis. New work shows that they team up at kinetochores to determine the mitotic duration of embryonic divisions in nematodes. As is often the case, the key effector is Cdc20 activity.
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Ciclo Celular , Embrión no Mamífero , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Cdc20/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Mitosis , Factores de Tiempo , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismoRESUMEN
The human AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 are multipass plasma membrane proteins that protect cells against membrane rigidification. However, how AdipoR2 promotes membrane fluidity mechanistically is not clear. Using 13C-labeled fatty acids, we show that AdipoR2 can promote the elongation and incorporation of membrane-fluidizing polyunsaturated fatty acids into phospholipids. To elucidate the molecular basis of these activities, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified coimmunoprecipitated proteins using mass spectrometry. We found that several of the evolutionarily conserved AdipoR2/PAQR-2 interactors are important for fatty acid elongation and incorporation into phospholipids. We experimentally verified some of these interactions, namely, with the dehydratase HACD3 that is essential for the third of four steps in long-chain fatty acid elongation and ACSL4 that is important for activation of unsaturated fatty acids and their channeling into phospholipids. We conclude that AdipoR2 and PAQR-2 can recruit protein interactors to promote the production and incorporation of unsaturated fatty acids into phospholipids.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Membrana Celular , Ácidos Grasos , Fluidez de la Membrana , Receptores de Adiponectina , Animales , Humanos , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Ácidos Grasos/metabolismo , Células HEK293 , Fluidez de la Membrana/fisiología , Fosfolípidos/metabolismo , Receptores de Adiponectina/metabolismo , Unión ProteicaRESUMEN
Cytokinesis requires the constriction of an actomyosin-based contractile ring and involves multiple F-actin crosslinkers. We show that partial depletion of the C. elegans cytokinetic formin generates contractile rings with low F-actin levels that constrict but are structurally fragile, and we use this background to investigate the roles of the crosslinkers plastin/PLST-1 and ß-heavy-spectrin/SMA-1 during ring constriction. We show that the removal of PLST-1 or SMA-1 has opposite effects on the structural integrity of fragile rings. PLST-1 loss reduces cortical tension that resists ring constriction and makes fragile rings less prone to ruptures and regressions, whereas SMA-1 loss exacerbates structural defects, leading to frequent ruptures and cytokinesis failure. Fragile rings without SMA-1 or containing a shorter SMA-1, repeatedly rupture at the same site, and SMA-1::GFP accumulates at repair sites in fragile rings and in rings cut by laser microsurgery. These results establish that ß-heavy-spectrin stabilizes the constricting ring and reveals the importance of ß-heavy-spectrin size for network connectivity at low F-actin density.
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Citoesqueleto de Actina , Citocinesis , Espectrina , Actinas , Actomiosina , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/metabolismo , Forminas , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Espectrina/metabolismoRESUMEN
Haem is an iron-containing tetrapyrrole that is critical for a variety of cellular and physiological processes1-3. Haem binding proteins are present in almost all cellular compartments, but the molecular mechanisms that regulate the transport and use of haem within the cell remain poorly understood2,3. Here we show that haem-responsive gene 9 (HRG-9) (also known as transport and Golgi organization 2 (TANGO2)) is an evolutionarily conserved haem chaperone with a crucial role in trafficking haem out of haem storage or synthesis sites in eukaryotic cells. Loss of Caenorhabditis elegans hrg-9 and its paralogue hrg-10 results in the accumulation of haem in lysosome-related organelles, the haem storage site in worms. Similarly, deletion of the hrg-9 homologue TANGO2 in yeast and mammalian cells induces haem overload in mitochondria, the site of haem synthesis. We demonstrate that TANGO2 binds haem and transfers it from cellular membranes to apo-haemoproteins. Notably, homozygous tango2-/- zebrafish larvae develop pleiotropic symptoms including encephalopathy, cardiac arrhythmia and myopathy, and die during early development. These defects partially resemble the symptoms of human TANGO2-related metabolic encephalopathy and arrhythmias, a hereditary disease caused by mutations in TANGO24-8. Thus, the identification of HRG-9 as an intracellular haem chaperone provides a biological basis for exploring the aetiology and treatment of TANGO2-related disorders.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Hemo , Animales , Humanos , Arritmias Cardíacas/metabolismo , Encefalopatías/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Hemo/metabolismo , Mitocondrias/metabolismo , Chaperonas Moleculares/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
The classification of neurons into distinct types reveals hierarchical taxonomic relationships that reflect the extent of similarity between neuronal cell types. At the base of such taxonomies are neuronal cells that are very similar to one another but differ in a small number of reproducible and select features. How are very similar members of a neuron class that share many features instructed to diversify into distinct subclasses? We show here that the six very similar members of the Caenorhabditis elegans IL2 sensory neuron class, which are all specified by a homeobox terminal selector, unc-86/BRN3, differentiate into two subtly distinct subclasses, a dorsoventral subclass and a lateral subclass, by the toggle switch-like action of the sine oculis/SIX homeobox gene unc-39. unc-39 is expressed only in the lateral IL2 neurons, and loss of unc-39 leads to a homeotic transformation of the lateral into the dorsoventral class; conversely, ectopic unc-39 expression converts the dorsoventral subclass into the lateral subclass. Hence, a terminal selector homeobox gene controls both class- as well as subclass-specific features, while a subordinate homeobox gene determines the ability of the class-specific homeobox gene to activate subtype-specific target genes. We find a similar regulatory mechanism operating in a distinct class of six motor neurons. Our findings underscore the importance of homeobox genes in neuronal identity control and invite speculations about homeotic identity transformations as potential drivers of evolutionary novelty during cell-type evolution in the brain.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Genes Homeobox , Proteínas de Homeodominio , Células Receptoras Sensoriales , Factores de Transcripción , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Neuronas Motoras/clasificación , Neuronas Motoras/citología , Células Receptoras Sensoriales/clasificación , Células Receptoras Sensoriales/citología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
How cells spatially organize their plasma membrane, cytoskeleton, and cytoplasm remains a central question for cell biologists. In this issue of JCB, Calvi et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202201048) identify PP1 phosphatases as key regulators of C. elegans anterior-posterior polarity, by counterbalancing aPKC-mediated phosphorylation of PAR-2.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Polaridad Celular , Fosfoproteínas Fosfatasas , Proteína Quinasa C , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular , Citoplasma , Microtúbulos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismoRESUMEN
A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. Reporter constructs, cell culture assays and computational modeling have made major contributions to answering this question, but analysis of elements in their natural context is an important complement. Here, we mutate Notch-dependent LAG-1 binding sites (LBSs) in the endogenous Caenorhabditis elegans sygl-1 gene, which encodes a key stem cell regulator, and analyze the consequences on sygl-1 expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of one LBS in a three-element cluster approximately halved both expression and stem cell pool size, whereas mutation of two LBSs essentially abolished them. Heterozygous LBS mutant clusters provided intermediate values. Our results lead to two major conclusions. First, both LBS number and configuration impact cluster activity: LBSs act additively in trans and synergistically in cis. Second, the SYGL-1 gradient promotes self-renewal above its functional threshold and triggers differentiation below the threshold. Our approach of coupling CRISPR/Cas9 LBS mutations with effects on both molecular and biological readouts establishes a powerful model for in vivo analyses of DNA cis-regulatory elements.
Asunto(s)
Caenorhabditis elegans , Elementos Reguladores de la Transcripción , Células Madre , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Autorrenovación de las Células , ADN/metabolismo , Proteínas de Unión al ADN/genética , Receptores Notch , Células Madre/citologíaRESUMEN
Deep-brain microscopy is strongly limited by the size of the imaging probe, both in terms of achievable resolution and potential trauma due to surgery. Here, we show that a segment of an ultra-thin multi-mode fiber (cannula) can replace the bulky microscope objective inside the brain. By creating a self-consistent deep neural network that is trained to reconstruct anthropocentric images from the raw signal transported by the cannula, we demonstrate a single-cell resolution (< 10µm), depth sectioning resolution of 40 µm, and field of view of 200 µm, all with green-fluorescent-protein labelled neurons imaged at depths as large as 1.4 mm from the brain surface. Since ground-truth images at these depths are challenging to obtain in vivo, we propose a novel ensemble method that averages the reconstructed images from disparate deep-neural-network architectures. Finally, we demonstrate dynamic imaging of moving GCaMp-labelled C. elegans worms. Our approach dramatically simplifies deep-brain microscopy.
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Encéfalo/diagnóstico por imagen , Aprendizaje Automático , Microscopía Fluorescente/métodos , Neuroimagen/métodos , Animales , Caenorhabditis elegans/citología , Células Cultivadas , Proteínas Fluorescentes Verdes/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Procedimientos Quirúrgicos Mínimamente Invasivos , Redes Neurales de la Computación , Neuronas/citología , Neuronas/metabolismoRESUMEN
Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.
Asunto(s)
Caenorhabditis elegans/genética , Ciclo Celular/genética , Movimiento Celular/genética , Replicación del ADN/genética , Origen de Réplica/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Larva/citología , Larva/genética , Larva/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/genética , Vulva/citología , Vulva/metabolismoRESUMEN
Cell identity is characterized by a distinct combination of gene expression, cell morphology, and cellular function established as progenitor cells divide and differentiate. Following establishment, cell identities can be unstable and require active and continuous maintenance throughout the remaining life of a cell. Mechanisms underlying the maintenance of cell identities are incompletely understood. Here, we show that the gene ctbp-1, which encodes the transcriptional corepressor C-terminal binding protein-1 (CTBP-1), is essential for the maintenance of the identities of the two AIA interneurons in the nematode Caenorhabditis elegans. ctbp-1 is not required for the establishment of the AIA cell fate but rather functions cell-autonomously and can act in later larval stage and adult worms to maintain proper AIA gene expression, morphology and function. From a screen for suppressors of the ctbp-1 mutant phenotype, we identified the gene egl-13, which encodes a SOX family transcription factor. We found that egl-13 regulates AIA function and aspects of AIA gene expression, but not AIA morphology. We conclude that the CTBP-1 protein maintains AIA cell identity in part by utilizing EGL-13 to repress transcriptional activity in the AIAs. More generally, we propose that transcriptional corepressors like CTBP-1 might be critical factors in the maintenance of cell identities, harnessing the DNA-binding specificity of transcription factors like EGL-13 to selectively regulate gene expression in a cell-specific manner.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Interneuronas/citología , Proteínas Represoras/metabolismo , Factores de Transcripción SOXD/metabolismo , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Interneuronas/fisiología , Proteínas Represoras/genética , Factores de Transcripción SOXD/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Glucose is the main source of energy for organisms, and it is important to understand the spatiotemporal dynamics of intracellular glucose. Single fluorescent protein-based glucose indicators, named "Red Glifons" have been developed that apply to live-cell and dual-color imaging. These indicators exhibited more than 3-fold increase in fluorescence intensity in the presence of 10 mM glucose. The two Red Glifons developed have different half-maximal effective concentration (EC50) values for glucose (300 µM and 3,000 µM) and are able to monitor a wide range of glucose dynamics. Red Glifon combined with green indicators allowing visualization of the interplay between glucose and ATP, lactate, or pyruvate. Glucose influx in the pharyngeal muscle of Caenorhabditis elegans, enteroendocrine cells, and human iPS cell-derived cardiac myocytes was observed using the Red Glifons. Thus these red glucose indicators serve as a multi-color imaging toolkit for investigating complex interactions in energy metabolism.
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Técnicas Biosensibles , Caenorhabditis elegans/metabolismo , Glucosa/análisis , Proteínas Luminiscentes/química , Animales , Caenorhabditis elegans/citología , Glucosa/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Modelos MolecularesRESUMEN
Asymmetric cell division is an essential feature of normal development and certain pathologies. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo, particularly how symmetry of the actomyosin cortical cytoskeleton is broken by a sperm-derived signal at fertilization, upstream of polarity establishment. Diploscapter pachys is the closest parthenogenetic relative to C. elegans, and D. pachys one-cell embryos also divide asymmetrically. However how polarity is triggered in the absence of sperm remains unknown. In post-meiotic embryos, we find that the nucleus inhabits principally one embryo hemisphere, the future posterior pole. When forced to one pole by centrifugation, the nucleus returns to its preferred pole, although poles appear identical as concerns cortical ruffling and actin cytoskeleton. The location of the meiotic spindle also correlates with the future posterior pole and slight actin enrichment is observed at that pole in some early embryos along with microtubule structures emanating from the meiotic spindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachys oocytes. All together our results are consistent with the idea that polarity of the D. pachys embryo is attained during meiosis, seemingly based on the location of the meiotic spindle, by a mechanism that may be present but suppressed in C. elegans.
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División Celular Asimétrica/fisiología , Meiosis/fisiología , Oocitos/citología , Oocitos/fisiología , Partenogénesis/fisiología , Rhabditoidea/citología , Rhabditoidea/embriología , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Núcleo Celular/fisiología , Femenino , Microtúbulos/fisiología , Oviparidad/fisiología , Huso Acromático/fisiologíaRESUMEN
In continuation of the search for new anthelmintic natural products, the study at hand investigated the nematicidal effects of the two naturally occurring quassinoids ailanthone and bruceine A against the reproductive system of the model nematode Caenorhabditis elegans to pinpoint their anthelmintic mode of action by the application of various microscopic techniques. Differential Interference Contrast (DIC) and the epifluorescence microscopy experiments used in the presented study indicated the genotoxic effects of the tested quassinoids (c ailanthone = 50 µM, c bruceine A = 100 µM) against the nuclei of the investigated gonadal and spermathecal tissues, leaving other morphological key features such as enterocytes or body wall muscle cells unimpaired. In order to gain nanoscopic insight into the morphology of the gonads as well as the considerably smaller spermathecae of C. elegans, an innovative protocol of polyethylene glycol embedding, ultra-sectioning, acridine orange staining, tissue identification by epifluorescence, and subsequent AFM-based ultrastructural data acquisition was applied. This sequence allowed the facile and fast assessment of the impact of quassinoid treatment not only on the gonadal but also on the considerably smaller spermathecal tissues of C. elegans. These first-time ultrastructural investigations on C. elegans gonads and spermathecae by AFM led to the identification of specific quassinoid-induced alterations to the nuclei of the reproductive tissues (e.g., highly condensed chromatin, impaired nuclear membrane morphology, as well as altered nucleolus morphology), altogether implying an apoptosis-like effect of ailanthone and bruceine A on the reproductive tissues of C. elegans.
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Antihelmínticos/toxicidad , Caenorhabditis elegans/efectos de los fármacos , Cuassinas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Caenorhabditis elegans/citología , Gónadas/efectos de los fármacos , Infertilidad/inducido químicamente , MasculinoRESUMEN
Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.