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Brief heat shocks delivered to cells by pulsed laser light can evoke action potentials in neurons and contraction in cardiomyocytes, but the primary biophysical mechanism has been elusive. In this report we show in the neuromuscular junction of Caenorhabditis elegans that application of a 500°C/s heat shock for 500 μs evoked ~35 pA of excitatory current and injected ~23 fC(femtocoulomb) of charge into the cell while raising the temperature only 0.25°C. The key variable driving the current was the rate of change of temperature (dT/dt heat shock), not temperature itself. The photothermal heat shock current was voltage-dependent and was from thermally driven displacement of ions near the plasma membrane. The charge movement was rapid during the heat shock and slow during thermal relaxation, thus leading to an asymmetrical capacitive current that briefly depolarized the cell. A simple quantitative model is introduced to describe modulation of the membrane potential and facilitate practical application of optical heat shock stimuli., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. 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.

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.

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.

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.

GABA neurotransmission requires a specialized set of proteins to synthesize, transport or respond to GABA. This article reviews results from a genetic strategy in the nematode Caenorhabditis elegans designed to identify the genes responsible for these activities. These studies identified mutations in genes encoding five different proteins: the biosynthetic enzyme for GABA, the vesicular GABA transporter, a transcription factor that determines GABA neuron identity, a classic inhibitory GABA receptor and a novel excitatory GABA receptor. This review discusses the strategy employed to identify these genes as well as the conclusions about GABA transmission derived from study of the mutant phenotypes.

The nematode Caenorhabditis elegans was chosen as a model genetic organism because its attributes, chiefly its hermaphroditic lifestyle and rapid generation time, make it suitable for the isolation and characterization of genetic mutants. The most important challenge for the geneticist is to design a genetic screen that will identify mutations that specifically disrupt the biological process of interest. Since 1974, when Sydney Brenner published his pioneering genetic screen, researchers have developed increasingly powerful methods for identifying genes and genetic pathways in C. elegans.