ApE, A Plasmid Editor: A Freely Available DNA Manipulation and Visualization Program.

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/.

Scan-less machine-learning-enabled incoherent microscopy for minimally-invasive deep-brain imaging.

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.

High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome.

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.

Synaptic vesicles transiently dock to refill release sites.

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.

SynapsEM: Computer-Assisted Synapse Morphometry.

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.

An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline.

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.

Highly efficient baculovirus-mediated multigene delivery in primary cells.

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.

Clathrin regenerates synaptic vesicles from endosomes.

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.

Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon.

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.

Ultrafast endocytosis at mouse hippocampal synapses.

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.

Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions.

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.

UNC119 is required for G protein trafficking in sensory neurons.

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.

Protein localization in electron micrographs using fluorescence nanoscopy.

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.

Targeted gene deletions in C. elegans using transposon excision.

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.

Single-copy insertion of transgenes in Caenorhabditis elegans.

At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.

Gene conversion and end-joining-repair double-strand breaks in the Caenorhabditis elegans germline.

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.

Gene activation using FLP recombinase in C. elegans.

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.

Protons act as a transmitter for muscle contraction in C. elegans.

Muscle contraction is normally mediated by the release of neurotransmitters from motor neurons. Here we demonstrate that protons can act as a direct transmitter from intestinal cells to stimulate muscle contraction. During the C. elegans defecation motor program the posterior body muscles contract even in the absence of neuronal inputs or vesicular neurotransmission. In this study, we demonstrate that the space between the intestine and the muscle is acidified just prior to muscle contraction and that the release of caged protons is sufficient to induce muscle contraction. PBO-4 is a putative Na+/H+ ion exchanger expressed on the basolateral membrane of the intestine, juxtaposed to the posterior body muscles. In pbo-4 mutants the extracellular space is not acidified and the muscles fail to contract. The pbo-5 and pbo-6 genes encode subunits of a "cys-loop" proton-gated cation channel required for muscles to respond to acidification. In heterologous expression assays the PBO receptor is half-maximally activated at a pH of 6.8. The identification of the mechanisms for release and reception of proton signals establishes a highly unusual mechanism for intercellular communication.

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells.

Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, approximately 20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.