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In JoVE (1)

Other Publications (6)

Articles by Gunther Hollopeter in JoVE

 JoVE Biology

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

1Department of Biology, Howard Hughes Medical Institute, University of Utah


JoVE 3995

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

Other articles by Gunther Hollopeter on PubMed

The P2Y12 Receptor Regulates Microglial Activation by Extracellular Nucleotides

Microglia are primary immune sentinels of the CNS. Following injury, these cells migrate or extend processes toward sites of tissue damage. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia express several purinoceptors, including a G(i)-coupled subtype that has been implicated in ATP- and ADP-mediated migration in vitro. Here we show that microglia from mice lacking G(i)-coupled P2Y(12) receptors exhibit normal baseline motility but are unable to polarize, migrate or extend processes toward nucleotides in vitro or in vivo. Microglia in P2ry(12)(-/-) mice show significantly diminished directional branch extension toward sites of cortical damage in the living mouse. Moreover, P2Y(12) expression is robust in the 'resting' state, but dramatically reduced after microglial activation. These results imply that P2Y(12) is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury.

Graded Synaptic Transmission at the Caenorhabditis Elegans Neuromuscular Junction

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.

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.

Molecular Basis of Infrared Detection by Snakes

Snakes possess a unique sensory system for detecting infrared radiation, enabling them to generate a 'thermal image' of predators or prey. Infrared signals are initially received by the pit organ, a highly specialized facial structure that is innervated by nerve fibres of the somatosensory system. How this organ detects and transduces infrared signals into nerve impulses is not known. Here we use an unbiased transcriptional profiling approach to identify TRPA1 channels as infrared receptors on sensory nerve fibres that innervate the pit organ. TRPA1 orthologues from pit-bearing snakes (vipers, pythons and boas) are the most heat-sensitive vertebrate ion channels thus far identified, consistent with their role as primary transducers of infrared stimuli. Thus, snakes detect infrared signals through a mechanism involving radiant heating of the pit organ, rather than photochemical transduction. These findings illustrate the broad evolutionary tuning of transient receptor potential (TRP) channels as thermosensors in the vertebrate nervous system.

Membrane Tension Regulates Motility by Controlling Lamellipodium Organization

Many cell movements proceed via a crawling mechanism, where polymerization of the cytoskeletal protein actin pushes out the leading edge membrane. In this model, membrane tension has been seen as an impediment to filament growth and cell motility. Here we use a simple model of cell motility, the Caenorhabditis elegans sperm cell, to test how membrane tension affects movement and cytoskeleton dynamics. To enable these analyses, we create transgenic worm strains carrying sperm with a fluorescently labeled cytoskeleton. Via osmotic shock and deoxycholate treatments, we relax or tense the cell membrane and quantify apparent membrane tension changes by the membrane tether technique. Surprisingly, we find that membrane tension reduction is correlated with a decrease in cell displacement speed, whereas an increase in membrane tension enhances motility. We further demonstrate that apparent polymerization rates follow the same trends. We observe that membrane tension reduction leads to an unorganized, rough lamellipodium, composed of short filaments angled away from the direction of movement. On the other hand, an increase in tension reduces lateral membrane protrusions in the lamellipodium, and filaments are longer and more oriented toward the direction of movement. Overall we propose that membrane tension optimizes motility by streamlining polymerization in the direction of movement, thus adding a layer of complexity to our current understanding of how membrane tension enters into the motility equation.

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.

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