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Eisosomes are membrane furrows at the cell surface of yeast that have been shown to function in two seemingly distinct pathways, membrane stress response and regulation of nutrient transporters. We found that many stress conditions affect both of these pathways by changing plasma membrane tension and thus the morphology and composition of eisosomes. For example, alkaline stress causes swelling of the cell and an endocytic response, which together increase membrane tension, thereby flattening the eisosomes. The flattened eisosomes affect membrane stress pathways and release nutrient transporters, which aids in their down-regulation. In contrast, glucose starvation or hyperosmotic shock causes cell shrinking, which results in membrane slack and the deepening of eisosomes. Deepened eisosomes are able to trap nutrient transporters and protect them from rapid endocytosis. Therefore, eisosomes seem to coordinate the regulation of both membrane tension and nutrient transporter stability.

Ultrafast endocytosis generates vesicles from the plasma membrane as quickly as 50 ms in hippocampal neurons following synaptic vesicle fusion. The molecular mechanism underlying the rapid maturation of these endocytic pits is not known. Here we demonstrate that synaptojanin-1, and its partner endophilin-A, function in ultrafast endocytosis. In the absence of synaptojanin or endophilin, the membrane is rapidly invaginated, but pits do not become constricted at the base. The 5-phosphatase activity of synaptojanin is involved in formation of the neck, but 4-phosphatase is not required. Nevertheless, these pits are eventually cleaved into vesicles; within a 30-s interval, synaptic endosomes form and are resolved by clathrin-mediated budding. Then synaptojanin and endophilin function at a second step to aid with the removal of clathrin coats from the regenerated vesicles. These data together suggest that synaptojanin and endophilin can mediate membrane remodeling on a millisecond timescale during ultrafast endocytosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Unc13 proteins are required for vesicle docking and priming during exocytosis. In this issue of Neuron, Lai et al. (2017) demonstrate that Unc13 ensures that the SNAREs assemble into functional subcomplexes. In a second manuscript, Michelassi et al. (2017) identify a previously unknown autoinhibited state for Unc13 mediated by the tandem C1 and C2 domains., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The heterotrimeric G protein G q positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of G q that acts in parallel to the canonical G q effector phospholipase C. Here, we examine how Trio and Rho act to stimulate neuronal activity downstream of G q in the nematode Caenorhabditis elegans Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the G q -Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of G q in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal G q -Rho signaling in C. elegans ., (Copyright © 2017 by the Genetics Society of America.)

Localization microscopy techniques-such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM)-provide the highest precision for single-molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 μm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless-steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single-molecule localizations of synaptic vesicle and active zone proteins in three dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Nano-resolution fluorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are first imaged using a super-resolution fluorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present five technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fluorescence, the use of ground-state depletion instead of photoactivation for temporal control of fluorescence, the use of organic fluorophores instead of fluorescent proteins to obtain brighter fluorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.

Synaptic communication in the nervous system is initiated by the fusion of synaptic vesicles with the presynaptic plasma membrane and subsequent neurotransmitter release. In the 1980s, this process was characterized by electron microscopy, albeit without the ability to follow processes in living cells. In the last two decades, fluorescence imaging methods have been developed that report synaptic vesicle fusion, endocytosis and recycling. These probes have provided unprecedented insight into synaptic vesicle trafficking in individual synaptic terminals and revealed heterogeneity in recycling pathways as well as synaptic vesicle populations. These methods either take advantage of uptake of fluorescent probes into recycling vesicles or exogenous expression of synaptic vesicle proteins tagged with a pH-sensitive fluorescent marker at regions facing the vesicle lumen. We provide an overview of these methods, with particular emphasis on the challenges associated with their use and the opportunities for future investigations.

Neuroscientists are developing new imaging techniques and generating large volumes of data in an effort to understand the complex structure of the nervous system. The complexity and size of this data makes human interpretation a labor-intensive task. To aid in the analysis, new segmentation techniques for identifying neurons in these feature rich datasets are required. This paper presents a method for neuron boundary detection and nonbranching process segmentation in electron microscopy images and visualizing them in three dimensions. It combines both automated segmentation techniques with a graphical user interface for correction of mistakes in the automated process. The automated process first uses machine learning and image processing techniques to identify neuron membranes that deliniate the cells in each two-dimensional section. To segment nonbranching processes, the cell regions in each two-dimensional section are connected in 3D using correlation of regions between sections. The combination of this method with a graphical user interface specially designed for this purpose, enables users to quickly segment cellular processes in large volumes.

To understand protein function, we need a detailed description of the molecular topography of the cell. The subcellular localization of proteins can be revealed using genetically encoded fluorescent proteins or immunofluorescence. However, the precise localization of proteins cannot be resolved due to the diffraction limit of light. Recently, the diffraction barrier has been overcome by employing several microscopy techniques. Using super-resolution fluorescence microscopy, one can pinpoint the location of proteins at a resolution of 20 nm or even less. However, the cellular context is often absent in these images. Recently, we developed a method for visualizing the subcellular structures in super-resolution images. Here we describe the method with two technical improvements. First, we optimize the method to preserve more fluorescence without compromising the morphology. Second, we implement ground-state depletion and single-molecule return (GSDIM) imaging, which does not rely on photoactivatable fluorescent proteins. These improvements extend the utility of nano-resolution fluorescence electron microscopy (nano-fEM)., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

Polarized trafficking of synaptic proteins to axons and dendrites is crucial to neuronal function. Through forward genetic analysis in C. elegans, we identified a cyclin (CYY-1) and a cyclin-dependent Pctaire kinase (PCT-1) necessary for targeting presynaptic components to the axon. Another cyclin-dependent kinase, CDK-5, and its activator p35, act in parallel to and partially redundantly with the CYY-1/PCT-1 pathway. Synaptic vesicles and active zone proteins mostly mislocalize to dendrites in animals defective for both PCT-1 and CDK-5 pathways. Unlike the kinesin-3 motor, unc-104/Kif1a mutant, cyy-1 cdk-5 double mutants have no reduction in anterogradely moving synaptic vesicle precursors (SVPs) as observed by dynamic imaging. Instead, the number of retrogradely moving SVPs is dramatically increased. Furthermore, this mislocalization defect is suppressed by disrupting the retrograde motor, the cytoplasmic dynein complex. Thus, PCT-1 and CDK-5 pathways direct polarized trafficking of presynaptic components by inhibiting dynein-mediated retrograde transport and setting the balance between anterograde and retrograde motors., (Copyright 2010 Elsevier Inc. All rights reserved.)

Most neurotransmission is mediated by action potentials, whereas sensory neurons propagate electrical signals passively and release neurotransmitter in a graded manner. Here, we demonstrate that Caenorhabditis elegans neuromuscular junctions release neurotransmitter in a graded fashion. When motor neurons were depolarized by light-activation of channelrhodopsin-2, the evoked postsynaptic current scaled with the strength of the stimulation. When motor neurons were hyperpolarized by light-activation of halorhodopsin, tonic release of synaptic vesicles was decreased. These data suggest that both evoked and tonic neurotransmitter release is graded in response to membrane potential. Acetylcholine synapses are depressed by high-frequency stimulation, in part due to desensitization of the nicotine-sensitve ACR-16 receptor. By contrast, GABA synapses facilitate before becoming depressed. Graded transmission and plasticity confer a broad dynamic range to these synapses. Graded release precisely transmits stimulation intensity, even hyperpolarizing inputs. Synaptic plasticity alters the balance of excitatory and inhibitory inputs into the muscle in a use-dependent manner.

Synaptic vesicles must be recycled to sustain neurotransmission, in large part via clathrin-mediated endocytosis. Clathrin is recruited to endocytic sites on the plasma membrane by the AP2 adaptor complex. The medium subunit (micro2) of AP2 binds to cargo proteins and phosphatidylinositol-4 ,5-bisphosphate on the cell surface. Here, we characterize the apm-2 gene (also called dpy-23), which encodes the only micro2 subunit in the nematode Caenorhabditis elegans. APM-2 is highly expressed in the nervous system and is localized to synapses; yet specific loss of APM-2 in neurons does not affect locomotion. In apm-2 mutants, clathrin is mislocalized at synapses, and synaptic vesicle numbers and evoked responses are reduced to 60 and 65%, respectively. Collectively, these data suggest AP2 micro2 facilitates but is not essential for synaptic vesicle recycling.

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.

The Galpha(q) pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cbeta [PLCbeta]) and at least one other effector. To find the missing effector, we performed forward genetic screens to suppress the slow growth and hyperactive behaviors of mutants with an overactive Galpha(q) pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCbeta). Strains containing null mutations in both EGL-8 (PLCbeta) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Galpha(q)-null mutants. Using cell-based and biochemical assays, we show that activated C. elegans Galpha(q) synergizes with Trio RhoGEF to activate RhoA. Activated Galpha(q) and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. Our results show that Trio's Rho-specific GEF domain is a major Galpha(q) effector that, together with PLCbeta, mediates the Galpha(q) signaling that drives the locomotion, egg laying, and growth of the animal.

Axons and dendrites can withstand acute mechanical strain despite their small diameter. In this study, we demonstrate that beta-spectrin is required for the physical integrity of neuronal processes in the nematode Caenorhabditis elegans. Axons in beta-spectrin mutants spontaneously break. Breakage is caused by acute strain generated by movement because breakage can be prevented by paralyzing the mutant animals. After breaking, the neuron attempts to regenerate by initiating a new growth cone; this second round of axon extension is error prone compared with initial outgrowth. Because spectrin is a major target of calpain proteolysis, it is possible that some neurodegenerative disorders may involve the cleavage of spectrin followed by the breakage of neural processes.