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
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/.
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.
Changes in neuronal activity create local and transient changes in energy demands at synapses. Here we discover a metabolic compartment that forms in vivo near synapses to meet local energy demands ...and support synaptic function in Caenorhabditis elegans neurons. Under conditions of energy stress, glycolytic enzymes redistribute from a diffuse localization in the cytoplasm to a punctate localization adjacent to synapses. Glycolytic enzymes colocalize, suggesting the ad hoc formation of a glycolysis compartment, or a “glycolytic metabolon,” that can maintain local levels of ATP. Local formation of the glycolytic metabolon is dependent on presynaptic scaffolding proteins, and disruption of the glycolytic metabolon blocks the synaptic vesicle cycle, impairs synaptic recovery, and affects locomotion. Our studies indicate that under energy stress conditions, energy demands in C. elegans synapses are met locally through the assembly of a glycolytic metabolon to sustain synaptic function and behavior.
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•A metabolic compartment forms in vivo near synapses to meet local energy demands•Under energy stress, glycolytic proteins redistribute to form clusters at synapses•The glycolytic metabolon is needed for the synaptic vesicle cycle•Disruption of glycolytic metabolon impairs synaptic recovery and affects locomotion
Changes in synaptic activity cause local changes in energy demands. Jang and Nelson et al. discover glycolytic microcompartments, or “glycolytic metabolons,” that form dynamically near presynaptic sites to meet local energy demands and support synaptic function.
Regeneration of injured neurons can restore function, but most neurons regenerate poorly or not at all. The failure to regenerate in some cases is due to a lack of activation of cell-intrinsic ...regeneration pathways. These pathways might be targeted for the development of therapies that can restore neuron function after injury or disease. Here, we show that the DLK-1 mitogen-activated protein (MAP) kinase pathway is essential for regeneration in Caenorhabditis elegans motor neurons. Loss of this pathway eliminates regeneration, whereas activating it improves regeneration. Further, these proteins also regulate the later step of growth cone migration. We conclude that after axon injury, activation of this MAP kinase cascade is required to switch the mature neuron from an aplastic state to a state capable of growth.
The cardinal feature of neuronal polarization is the establishment and maintenance of axons and dendrites. How axonal and dendritic proteins are sorted and targeted to different compartments is ...poorly understood. Here, we identified distinct dileucine motifs that are necessary and sufficient to target transmembrane proteins to either the axon or the dendrite through direct interactions with the clathrin-associated adaptor protein complexes (APs) in C. elegans. Axonal targeting requires AP-3, while dendritic targeting is mediated by AP-1. The axonal dileucine motif binds to AP-3 with higher efficiency than to AP-1. Both AP-3 and AP-1 are localized to the Golgi but occupy adjacent domains. We propose that AP-3 and AP-1 directly select transmembrane proteins and target them to axon and dendrite, respectively, by sorting them into distinct vesicle pools.
•AP-3 and AP-1 sort transmembrane proteins to the axon and the dendrite, respectively•AP-3 strongly binds to axonal sorting motifs and select cargos to axonal vesicles•AP-3 and AP-1 are localized and functioning at different domains of Golgi apparatus
Li et al. studied how axonal and dendritic proteins are targeted to their destined compartments from Golgi apparatus in C. elegans. AP-3 strongly binds to axonal sorting motifs and select cargos to the axon. Dendritic targeting is mediated by AP-1.
The human brain is easily the most baffling bit of biology on the planet. How did the nervous system evolve? What came first: neurons or synaptic proteins? A new paper studying the pancake-shaped ...Trichoplax suggests it was not the neurons.
The human brain is easily the most baffling bit of biology on the planet. How did the nervous system evolve? What came first: neurons or synaptic proteins? A new paper studying the pancake-shaped Trichoplax suggests it was not the neurons.
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.
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.
Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi and then sort cargo during maturation before being secreted. ...To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1, and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network.
Pheromones elicit innate sex-specific mating behaviors in many species. We demonstrate that in C. elegans, male-specific sexual attraction behavior is programmed in both sexes but repressed in ...hermaphrodites. Repression requires a single sensory neuron pair, the ASIs. To repress attraction in adults, the ASIs must be present, active, and capable of sensing the environment during development. The ASIs release TGF-β, and ASI function can be bypassed by experimental activation of TGF-β signaling. Sexual attraction in derepressed hermaphrodites requires the same sensory neurons as in males. The sexual identity of both these sensory neurons and a distinct subset of interneurons must be male to relieve repression and release attraction. TGF-β may therefore act to change connections between sensory neurons and interneurons during development to engage repression. Thus, sensation in a single sensory neuron pair during development reprograms a common neural circuit from male to female behavior.
► Male sexual attraction is latent in the hermaphrodite brain but normally repressed ► Two sensory neurons, the ASIs, release TGF-β during development to engage repression ► Males express TGF-β, but repression acts only on the hermaphrodite nervous system ► Sexual attraction requires distributed sex differences in sensory and interneurons
White and Jorgensen show that a male-specific behavior in C. elegans is latent in the opposite sex, the hermaphrodite. Activity in a single sensory neuron pair acts on specific neural connections during development to repress attraction only in hermaphrodites.