RESUMO
Ryanodine receptors (RyR) are essential regulators of cellular calcium homeostasis and signaling. Vertebrate genomes contain multiple RyR gene isoforms, expressed in different tissues and executing different functions. In contrast, invertebrate genomes contain a single RyR-encoding gene and it has long been proposed that different transcripts generated by alternative splicing may diversify their functions. Here, we analyze the expression and function of alternative exons in the C. elegans RyR gene unc-68. We show that specific isoform subsets are created via alternative promoters and via alternative splicing in unc-68 Divergent Region 2 (DR2), which actually corresponds to a region of high sequence variability across vertebrate isoforms. The expression of specific unc-68 alternative exons is enriched in different tissues, such as in body wall muscle, neurons and pharyngeal muscle. In order to infer the function of specific alternative promoters and alternative exons of unc-68, we selectively deleted them by CRISPR/Cas9 genome editing. We evaluated pharyngeal function, as well as locomotor function in swimming and crawling with high-content computer-assisted postural and behavioral analysis. Our data provide a comprehensive map of the pleiotropic impact of isoform-specific mutations and highlight that tissue-specific unc-68 isoforms fulfill distinct functions. As a whole, our work clarifies how the C. elegans single RyR gene unc-68 can fulfill multiple tasks through tissue-specific isoforms, and provide a solid foundation to further develop C. elegans as a model to study RyR channel functions and malfunctions.
Assuntos
Processamento Alternativo/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Contração Muscular/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Caenorhabditis elegans/crescimento & desenvolvimento , Sinalização do Cálcio/genética , Modelos Animais de Doenças , Éxons , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação/genética , Especificidade de Órgãos/genética , Isoformas de Proteínas/genética , Rianodina/metabolismoRESUMO
In the last two decades, it has been shown that bacterial chromosomes have remarkable spatial organization at various scales, and they display well-defined movements during the cell cycle, for example to reliably segregate daughter chromosomes. More recently, various labs have begun investigating also the short time dynamics (displacements during time intervals of 0.1 s-100 s), which should be related to the molecular structure. Probing these dynamics is analogous to "microrheology" approaches that have been applied successfully to study mechanical response of complex fluids. These studies of chromosome fluctuation dynamics have revealed differences of fluctuation amplitude across the chromosome, and different characters of motion depending on the time window of interest. Different fluctuation amplitudes have also been observed for the same chromosomal loci under antibiotic treatments, with magnitudes that are correlated to changes in intracellular density and thus crowding. We describe how to carry out tracking experiments of single loci and how to analyze locus motility. We point out the importance of considering in the analysis the number of GFP molecules per fluorescent locus, as well as the nature of the protein they are fused to, and also how to measure intracellular density.
Assuntos
Cromossomos Bacterianos , Ciclo Celular , Divisão Celular , Cromossomos Bacterianos/genética , Microscopia de Fluorescência , Movimento (Física)RESUMO
Changes in tissue architecture and multicellular organisation contribute to many diseases, including cancer and cardiovascular diseases. Scratch wound assay is a commonly used tool that assesses cells' migratory ability based on the area of a wound they cover over a certain time. However, analysis of changes in the organisational patterns formed by migrating cells following genetic or pharmacological perturbations are not well explored in these assays, in part because analysing the resulting imaging data is challenging. Here we present DeepScratch, a neural network that accurately detects the cells in scratch assays based on a heterogeneous set of markers. We demonstrate the utility of DeepScratch by analysing images of more than 232,000 lymphatic endothelial cells. In addition, we propose various topological measures of cell connectivity and local cell density (LCD) to characterise tissue remodelling during wound healing. We show that LCD-based metrics allow classification of CDH5 and CDC42 genetic perturbations that are known to affect cell migration through different biological mechanisms. Such differences cannot be captured when considering only the wound area. Taken together, single-cell detection using DeepScratch allows more detailed investigation of the roles of various genetic components in tissue topology and the biological mechanisms underlying their effects on collective cell migration.
RESUMO
In complex biological systems, simple individual-level behavioral rules can give rise to emergent group-level behavior. While collective behavior has been well studied in cells and larger organisms, the mesoscopic scale is less understood, as it is unclear which sensory inputs and physical processes matter a priori. Here, we investigate collective feeding in the roundworm C. elegans at this intermediate scale, using quantitative phenotyping and agent-based modeling to identify behavioral rules underlying both aggregation and swarming-a dynamic phenotype only observed at longer timescales. Using fluorescence multi-worm tracking, we quantify aggregation in terms of individual dynamics and population-level statistics. Then we use agent-based simulations and approximate Bayesian inference to identify three key behavioral rules for aggregation: cluster-edge reversals, a density-dependent switch between crawling speeds, and taxis towards neighboring worms. Our simulations suggest that swarming is simply driven by local food depletion but otherwise employs the same behavioral mechanisms as the initial aggregation.
Assuntos
Comportamento Animal , Caenorhabditis elegans/fisiologia , Movimento , Animais , Modelos BiológicosRESUMO
Behaviour is a sensitive and integrative readout of nervous system function and therefore an attractive measure for assessing the effects of mutation or drug treatment on animals. Video data provide a rich but high-dimensional representation of behaviour, and so the first step of analysis is often some form of tracking and feature extraction to reduce dimensionality while maintaining relevant information. Modern machine-learning methods are powerful but notoriously difficult to interpret, while handcrafted features are interpretable but do not always perform as well. Here, we report a new set of handcrafted features to compactly quantify Caenorhabditis elegans behaviour. The features are designed to be interpretable but to capture as much of the phenotypic differences between worms as possible. We show that the full feature set is more powerful than a previously defined feature set in classifying mutant strains. We then use a combination of automated and manual feature selection to define a core set of interpretable features that still provides sufficient power to detect behavioural differences between mutant strains and the wild-type. Finally, we apply the new features to detect time-resolved behavioural differences in a series of optogenetic experiments targeting different neural subsets.This article is part of a discussion meeting issue 'Connectome to behaviour: modelling C. elegans at cellular resolution'.
Assuntos
Comportamento Animal/fisiologia , Caenorhabditis elegans/fisiologia , Optogenética/métodos , Animais , Caenorhabditis elegans/genética , Locomoção/genética , Locomoção/fisiologia , FenótipoRESUMO
Bacterial chromosomes have been shown in the last two decades to have remarkable spatial organization at various scales, and also well-defined movements during the cell cycle, for example, to reliably segregate daughter chromosomes. More recently, various labs have begun investigating the short-time dynamics (displacements during time intervals of 0.1-100 s), which one hopes to link to structure, in analogy to "microrheology" approaches applied successfully to study mechanical response of complex fluids. These studies of chromosome fluctuation dynamics have revealed differences of fluctuation amplitude across the chromosome, and different characters of motion depending on the time window of interest. The highly nontrivial motion at the shortest experimentally accessible times is still not fully understood in terms of physical models of DNA and cytosol. We describe how to carry out tracking experiments of single locus and how to analyze locus motility. We point out the importance of considering in the analysis the number of GFP molecules per fluorescent locus.
Assuntos
Cromossomos Bacterianos/genética , Escherichia coli/genética , Ciclo Celular , Loci Gênicos , Microscopia de FluorescênciaRESUMO
The mean size of exponentially dividing Escherichia coli cells in different nutrient conditions is known to depend on the mean growth rate only. However, the joint fluctuations relating cell size, doubling time, and individual growth rate are only starting to be characterized. Recent studies in bacteria reported a universal trend where the spread in both size and doubling times is a linear function of the population means of these variables. Here we combine experiments and theory and use scaling concepts to elucidate the constraints posed by the second observation on the division control mechanism and on the joint fluctuations of sizes and doubling times. We found that scaling relations based on the means collapse both size and doubling-time distributions across different conditions and explain how the shape of their joint fluctuations deviates from the means. Our data on these joint fluctuations highlight the importance of cell individuality: Single cells do not follow the dependence observed for the means between size and either growth rate or inverse doubling time. Our calculations show that these results emerge from a broad class of division control mechanisms requiring a certain scaling form of the "division hazard rate function," which defines the probability rate of dividing as a function of measurable parameters. This "model free" approach gives a rationale for the universal body-size distributions observed in microbial ecosystems across many microbial species, presumably dividing with multiple mechanisms. Additionally, our experiments show a crossover between fast and slow growth in the relation between individual-cell growth rate and division time, which can be understood in terms of different regimes of genome replication control.
Assuntos
Divisão Celular , Crescimento Celular , Escherichia coli/citologia , Escherichia coli/fisiologia , Modelos Biológicos , Tamanho Celular , Microscopia de Fluorescência , TempoRESUMO
The physical nature of the bacterial chromosome has important implications for its function. Using high-resolution dynamic tracking, we observe the existence of rare but ubiquitous 'rapid movements' of chromosomal loci exhibiting near-ballistic dynamics. This suggests that these movements are either driven by an active machinery or part of stress-relaxation mechanisms. Comparison with a null physical model for subdiffusive chromosomal dynamics shows that rapid movements are excursions from a basal subdiffusive dynamics, likely due to driven and/or stress-relaxation motion. Additionally, rapid movements are in some cases coupled with known transitions of chromosomal segregation. They do not co-occur strictly with replication, their frequency varies with growth condition and chromosomal coordinate, and they show a preference for longitudinal motion. These findings support an emerging picture of the bacterial chromosome as off-equilibrium active matter and help developing a correct physical model of its in vivo dynamic structure.
Assuntos
Cromossomos Bacterianos , Replicação do DNA , Escherichia coli , Movimento (Física) , Segregação de Cromossomos , Loci GênicosRESUMO
We designed a microfluidic chemostat consisting of 600 sub-micron trapping/growth channels connected to two feeding channels. The microchemostat traps E. coli cells and forces them to grow in lines for over 50 generations. Excess cells, including the mother cells captured at the start of the process, are removed from both ends of the growth channels by the media flow. With the aid of time-lapse microscopy, we have monitored dynamic properties such as growth rate and GFP expression at the single-cell level for many generations while maintaining a population of bacteria of similar age. We also use the microchemostat to show how the population responds to dynamic changes in the environment. Since more than 100 individual bacterial cells are aligned and immobilized in a single field of view, the microchemostat is an ideal platform for high-throughput intracellular measurements. We demonstrate this capability by tracking with sub-diffraction resolution the movements of fluorescently tagged loci in more than one thousand cells on a single device. The device yields results comparable to conventional agar microscopy experiments with substantial increases in throughput and ease of analysis.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Técnicas Analíticas Microfluídicas/métodos , DNA Girase/genética , DNA Girase/metabolismo , Escherichia coli/isolamento & purificação , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia Confocal , Imagem com Lapso de TempoRESUMO
In bacteria, chromosomal architecture shows strong spatial and temporal organization, and regulates key cellular functions, such as transcription. Tracking the motion of chromosomal loci at short timescales provides information related to both the physical state of the nucleo-protein complex and its local environment, independent of large-scale motions related to genome segregation. Here we investigate the short-time (0.1-10 s) dynamics of fluorescently labelled chromosomal loci in Escherichia coli at different growth rates. At these timescales, we observe for the first time a dependence of the loci's apparent diffusion on both their subcellular localization and chromosomal coordinate, and we provide evidence that the properties of the chromosome are similar in the tested growth conditions. Our results indicate that either non-equilibrium fluctuations due to enzyme activity or the organization of the genome as a polymer-protein complex vary as a function of the distance from the origin of replication.