RESUMO
Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.
Assuntos
Caenorhabditis elegans/metabolismo , DNA Intergênico/metabolismo , Inativação Gênica , Animais , Composição de Bases , Caenorhabditis elegans/genética , Cromatina , Elementos de DNA Transponíveis , DNA Viral/genética , Células Germinativas/metabolismo , Íntrons , Regiões Promotoras Genéticas , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , TransgenesRESUMO
Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50-100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Caenorhabditis elegans/genética , Edição de Genes/métodos , Genoma Helmíntico , AnimaisRESUMO
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.
Assuntos
Encéfalo/diagnóstico por imagem , Aprendizado de Máquina , Microscopia de Fluorescência/métodos , Neuroimagem/métodos , Animais , Caenorhabditis elegans/citologia , Células Cultivadas , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Procedimentos Cirúrgicos Minimamente Invasivos , Redes Neurais de Computação , Neurônios/citologia , Neurônios/metabolismoRESUMO
Ultrafast endocytosis can retrieve a single, large endocytic vesicle as fast as 50-100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 s, which in turn become small-diameter synaptic vesicles 5-6 s after stimulation. We disrupted clathrin function using RNA interference (RNAi) and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results may explain discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis.
Assuntos
Clatrina/metabolismo , Endossomos/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Membrana Celular/metabolismo , Endocitose , Humanos , Camundongos , TemperaturaRESUMO
To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20 s after fusion by the assembly of clathrin scaffolds or in approximately 1 s by the reversal of fusion pores via 'kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy--'flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs within 50 to 100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster than clathrin-mediated endocytosis. It is likely that 'ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly.
Assuntos
Endocitose , Hipocampo/citologia , Sinapses/metabolismo , Actinas/metabolismo , Actinas/ultraestrutura , Potenciais de Ação , Animais , Dinaminas/metabolismo , Dinaminas/ultraestrutura , Exocitose , Fusão de Membrana , Camundongos , Microscopia Eletrônica , Rodopsina/genética , Rodopsina/metabolismo , Sinapses/ultraestrutura , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Fatores de TempoRESUMO
We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.
Assuntos
Caenorhabditis elegans/genética , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Transgenes , Transposases/genética , Animais , Animais Geneticamente Modificados , Hibridização Genômica Comparativa , Biologia Computacional , Engenharia Genética/métodos , Marcadores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Modelos Genéticos , Mutagênese Insercional , Proteínas Recombinantes/metabolismo , Recombinação GenéticaRESUMO
A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.
Assuntos
Proteínas Luminescentes/análise , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Animais , Caenorhabditis elegans , Elétrons , Histonas/análise , Histonas/ultraestrutura , Proteínas Luminescentes/ultraestrutura , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/ultraestruturaRESUMO
We developed a method, MosDEL, to generate targeted knockouts of genes in Caenorhabditis elegans by injection. We generated a double-strand break by mobilizing a Mos1 transposon adjacent to the region to be deleted; the double-stranded break is repaired using injected DNA as a template. Repair can delete up to 25 kb of DNA and simultaneously insert a positive selection marker.
Assuntos
Caenorhabditis elegans/genética , Elementos de DNA Transponíveis/genética , Deleção de Genes , Animais , Hibridização Genômica Comparativa , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Transposases/fisiologiaRESUMO
A plasmid Editor (ApE) is a free, multi-platform application for visualizing, designing, and presenting biologically relevant DNA sequences. ApE provides a flexible framework for annotating a sequence manually or using a user-defined library of features. ApE can be used in designing plasmids and other constructs via in silico simulation of cloning methods such as PCR, Gibson assembly, restriction-ligation assembly and Golden Gate assembly. In addition, ApE provides a platform for creating visually appealing linear and circular plasmid maps. It is available for Mac, PC, and Linux-based platforms and can be downloaded at https://jorgensen.biology.utah.edu/wayned/ape/.
RESUMO
The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode C. elegans. Transcription is blocked by the presence of an "off cassette" between the promoter and the coding region of the desired product. The "off cassette" is composed of a transcriptional terminator flanked by FLP recognition targets (FRT). This sequence can be excised by FLP recombinase to bring together the promoter and the coding region. We have introduced two fluorescent reporters into the system: a red reporter for promoter activity prior to FLP expression and a green reporter for expression of the gene of interest after FLP expression. The constructs are designed using the multisite Gateway system, so that promoters and coding regions can be quickly mixed and matched. We demonstrate that heat-shock-driven FLP recombinase adds temporal control on top of tissue specific expression provided by the transgene promoter. In addition, the temporal switch is permanent, rather than acute, as is usually the case for heat-shock driven transgenes. Finally, FLP expression can be driven by a tissue specific promoter to provide expression in a subset of cells that can only be addressed as the intersection of two available promoters. As a test of the system, we have driven the light chain of tetanus toxin, a protease that cleaves the synaptic vesicle protein synaptobrevin. We show that we can use this to inactivate synaptic transmission in all neurons or a subset of neurons in a FLP-dependent manner.
Assuntos
Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , DNA Nucleotidiltransferases/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , DNA Nucleotidiltransferases/genética , Primers do DNA/genética , Regulação da Expressão Gênica , Genes de Helmintos , Proteínas de Fluorescência Verde/genética , Resposta ao Choque Térmico/genética , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Recombinação Genética , Toxina Tetânica/genética , Ativação Transcricional , Proteína Vermelha FluorescenteRESUMO
The structural features of a synapse help determine its function. Synapses are extremely small and tightly packed with vesicles and other organelles. Visualizing synaptic structure requires imaging by electron microscopy, and the features in micrographs must be quantified, a process called morphometry. Three parameters are typically assessed from each specimen: (1) the sizes of individual vesicles and organelles; (2) the absolute number and densities of organelles; and (3) distances between organelles and key features at synapses, such as active zone membranes and dense projections. For data to be meaningful, the analysis must be repeated from hundreds to thousands of images from several biological replicates, a daunting task. Here we report a custom computer program to analyze key structural features of synapses: SynapsEM. In short, we developed ImageJ/Fiji macros to record x,y-coordinates of segmented structures. The coordinates are then exported as text files. Independent investigators can reload the images and text files to reexamine the segmentation using ImageJ. The Matlab program then calculates and reports key synaptic parameters from the coordinates. Since the values are calculated from coordinates, rather than measured from each micrograph, other parameters such as locations of docked vesicles relative to the center of an active zone can be extracted in Matlab by additional scripting. Thus, this program can accelerate the morphometry of synapses and promote a more comprehensive analysis of synaptic ultrastructure.
RESUMO
Synaptic vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new vesicles must 'dock' to refill vacated release sites. To capture synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation, then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple vesicles can fuse at a single active zone. Fusions during synchronous release are distributed throughout the active zone, whereas fusions during asynchronous release are biased toward the center of the active zone. After stimulation, the total number of docked vesicles across all synapses decreases by ~40%. Within 14 ms, new vesicles are recruited and fully replenish the docked pool, but this docking is transient and they either undock or fuse within 100 ms. These results demonstrate that the recruitment of synaptic vesicles to release sites is rapid and reversible.
Assuntos
Exocitose/fisiologia , Neurônios/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Células Cultivadas , Feminino , Hipocampo/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , Neurônios/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
Excision of a Mos1 transposon in the germline of Caenorhabditis elegans generates a double-strand break in the chromosome. We demonstrate that breaks are most prominently repaired by gene conversion from the homolog, but also rarely by nonhomologous end-joining. In some cases, gene conversion events are resolved by crossing over. Surprisingly, expression of the transposase using an intestine-specific promoter can induce repair, raising the possibility that activation of transposase expression in somatic cells can lead to transposition of Mos1 in the germline.
Assuntos
Caenorhabditis elegans/genética , Conversão Gênica , Animais , Sequência de Bases , Troca Genética , Dano ao DNA , Reparo do DNA , Elementos de DNA Transponíveis , Genoma Helmíntico , Mutação em Linhagem Germinativa , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Recombinação Genética , Transposases/metabolismoRESUMO
The normal distribution of crossover events on meiotic bivalents depends on homolog recognition, alignment, and interference. We developed a method for precisely locating all crossovers on Caenorhabditis elegans chromosomes and demonstrated that wild-type animals have essentially complete interference, with each bivalent receiving one and only one crossover. A physical break in one homolog has previously been shown to disrupt interference, suggesting that some aspect of bivalent structure is required for interference. We measured the distribution of crossovers in animals heterozygous for a large insertion to determine whether a break in sequence homology would have the same effect as a physical break. Insertions disrupt crossing over locally. However, every bivalent still experiences essentially one and only one crossover, suggesting that interference can act across a large gap in homology. Although insertions did not affect crossover number, they did have an effect on crossover distribution. Crossing over was consistently higher on the side of the chromosome bearing the homolog recognition region and lower on the other side of the chromosome. We suggest that nonhomologous sequences cause heterosynapsis, which disrupts crossovers along the distal chromosome, even when those regions contain sequences that could otherwise align. However, because crossovers are not completely eliminated distal to insertions, we propose that alignment can be reestablished after a megabase-scale gap in sequence homology.
Assuntos
Caenorhabditis elegans/genética , Troca Genética/fisiologia , Triagem de Portadores Genéticos , Animais , Caenorhabditis elegans/metabolismo , Cromátides/metabolismo , Marcadores Genéticos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Single-nucleotide polymorphism (SNP) mapping is the easiest and most reliable way to map genes in Caenorhabditis elegans. SNPs are extremely dense and usually have no associated phenotype, making them ideal markers for mapping. SNP mapping has three steps. First, recombinant mutant animals are generated over a polymorphic strain (usually CB4856) using standard genetic techniques. Second, the genotype of these animals at SNP loci is determined using one of a variety of SNP detection technologies. Third, linkage between the mutant and one or more SNPs is used to position the mutant on the chromosome relative to the SNPs. This chapter presents a detailed procedure for generating recombinant animals, for assaying SNPs using restriction enzymes, and for analyzing mapping data.
Assuntos
Caenorhabditis elegans/genética , Mapeamento Cromossômico , Genes de Helmintos/genética , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , Animais , Animais Geneticamente Modificados/genética , Mapeamento Cromossômico/métodosRESUMO
Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells.
Assuntos
Baculoviridae/genética , Sistemas CRISPR-Cas , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Transfecção/métodos , Animais , Células COS , Sistemas CRISPR-Cas/genética , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Células Sf9 , Transgenes/genéticaRESUMO
BACKGROUND: In C. elegans, single nucleotide polymorphisms (SNPs) can function as silent genetic markers, with applications ranging from classical two- and three-factor mapping to measuring recombination across whole chromosomes. RESULTS: Here, we describe a set of 48 primer pairs that flank SNPs evenly spaced across the C. elegans genome and that work under identical PCR conditions. Each SNP in this set alters a DraI site, enabling rapid and parallel scoring. We describe a procedure using these reagents to quickly and reliably map mutations. We show that these techniques correctly map a known gene, dpy-5. We then use these techniques to map mutations in an uncharacterized strain, and show that its behavioral phenotype can be simultaneously mapped to three loci. CONCLUSION: Together, the reagents and methods described represent a significant advance in the accurate, rapid and inexpensive mapping of genes in C. elegans.
Assuntos
Caenorhabditis elegans/genética , Mapeamento Cromossômico/métodos , Polimorfismo de Nucleotídeo Único , Animais , Primers do DNA/química , Feminino , Ligação Genética , Marcadores Genéticos , Genoma , Masculino , Modelos Genéticos , Mutação , Mapeamento de Nucleotídeos , Fenótipo , Mapeamento Físico do Cromossomo , Reação em Cadeia da Polimerase , Recombinação GenéticaRESUMO
Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis. DOI:http://dx.doi.org/10.7554/eLife.00723.001.
Assuntos
Caenorhabditis elegans/metabolismo , Endocitose , Junção Neuromuscular/metabolismo , AnimaisRESUMO
UNC119 is widely expressed among vertebrates and other phyla. We found that UNC119 recognized the acylated N terminus of the rod photoreceptor transducin α (Tα) subunit and Caenorhabditis elegans G proteins ODR-3 and GPA-13. The crystal structure of human UNC119 at 1.95-Å resolution revealed an immunoglobulin-like ß-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. The structure of co-crystals of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound Tα-GTP, inhibiting its GTPase activity, thereby providing a stable UNC119-Tα-GTP complex capable of diffusing from the inner segment back to the outer segment after light-induced translocation. UNC119 deletion in both mouse and C. elegans led to G protein mislocalization. Thus, UNC119 is a Gα subunit cofactor essential for G protein trafficking in sensory cilia.