Dan Ohtan Wang

Synapse- and Stimulus-Specific Local Translation During Long-Term Neuronal Plasticity

Sensorin Reporter Long-term memory and synaptic plasticity require changes in gene expression and yet can occur in a synapse-specific manner. Messenger RNA (mRNA) localization and regulated translation at synapses have been proposed as mechanisms for spatially restricting gene expression during transcription-dependent, synapse-specific forms of neuronal plasticity. In the sea slug Aplysia, which is a frequently used model system for studying learning and memory, sensorin is a sensory cell–specific peptide neurotransmitter. The mRNA encoding sensorin localizes to distal sensory neurites and further concentrates at sites of synaptic contact between sensory and motor neurons. Wang et al. (p. 1536, published online 14 May; see the Perspective by Korte) demonstrate, using translational reporters of sensorin mRNA expressed in individual cultured Aplysia sensory and motor neurons, that local translation occurs at synapses during transcription-dependent, learning-related forms of synaptic plasticity. Live-cell microscopy reveals local translation during long-term facilitation of Aplysia sensory-motor synapses. Long-term memory and synaptic plasticity require changes in gene expression and yet can occur in a synapse-specific manner. Messenger RNA localization and regulated translation at synapses are thus critical for establishing synapse specificity. Using live-cell microscopy of photoconvertible fluorescent protein translational reporters, we directly visualized local translation at synapses during long-term facilitation of Aplysia sensory-motor synapses. Translation of the reporter required multiple applications of serotonin, was spatially restricted to stimulated synapses, was transcript- and stimulus-specific, and occurred during long-term facilitation but not during long-term depression of sensory-motor synapses. Translational regulation only occurred in the presence of a chemical synapse and required calcium signaling in the postsynaptic motor neuron. Thus, highly regulated local translation occurs at synapses during long-term plasticity and requires trans-synaptic signals.

Spatially restricting gene expression by local translation at synapses

mRNA localization and regulated translation provide a means of spatially restricting gene expression within each of the thousands of subcellular compartments made by a neuron, thereby vastly increasing the computational capacity of the brain. Recent studies reveal that local translation is regulated by stimuli that trigger neurite outgrowth and/or collapse, axon guidance, synapse formation, pruning, activity-dependent synaptic plasticity, and injury-induced axonal regeneration. Impairments in the local regulation of translation result in aberrant signaling, physiology and morphology of neurons, and are linked to neurological disorders. This review highlights current advances in understanding how mRNA translation is repressed during transport and how local translation is activated by stimuli. We address the function of local translation in the context of fragile X mental retardation.

Localized cell stimulation by nitric oxide using a photoactive porous coordination polymer platform

Functional cellular substrates for localized cell stimulation by small molecules provide an opportunity to control and monitor cell signalling networks chemically in time and space. However, despite improvements in the controlled delivery of bioactive compounds, the precise localization of gaseous biomolecules at the single-cell level remains challenging. Here we target nitric oxide, a crucial signalling molecule with site-specific and concentration-dependent activities, and we report a synthetic strategy for developing spatiotemporally controllable nitric oxide-releasing platforms based on photoactive porous coordination polymers. By organizing molecules with poor reactivity into polymer structures, we observe increased photoreactivity and adjustable release using light irradiation. We embed photoactive polymer crystals in a biocompatible matrix and achieve precisely controlled nitric oxide delivery at the cellular level via localized two-photon laser activation. The biological relevance of the exogenous nitric oxide produced by this strategy is evidenced by an intracellular change in calcium concentration, mediated by nitric oxide-responsive plasma membrane channel proteins.

Identification of a cis-acting element that localizes mRNA to synapses

Messenger RNA (mRNA) localization and regulated translation can spatially restrict gene expression to each of the thousands of synaptic compartments formed by a single neuron. Although cis-acting RNA elements have been shown to direct localization of mRNAs from the soma into neuronal processes, less is known about signals that target transcripts specifically to synapses. In Aplysia sensory-motor neuronal cultures, synapse formation rapidly redistributes the mRNA encoding the peptide neurotransmitter sensorin from neuritic shafts into synapses. We find that the export of sensorin mRNA from soma to neurite and the localization to synapse are controlled by distinct signals. The 3′ UTR is sufficient for export into distal neurites, whereas the 5′ UTR is required for concentration of reporter mRNA at synapses. We have identified a 66-nt element in the 5′ UTR of sensorin that is necessary and sufficient for synaptic mRNA localization. Mutational and chemical probing analyses are consistent with a role for secondary structure in this process.

Live‐Cell Imaging of Endogenous mRNAs with a Small Molecule

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin-2, cortactin, and cytoplasmic FMR1-interacting protein 2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules.

A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes

Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO-FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO-FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO-FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO-FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy. An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level. In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Hybridization-sensitive fluorescence control in the near-infrared wavelength range

A series of near-infrared fluorescent probes were designed based on the concept of emission control caused by interdye excitonic interaction. The fluorescent probes showed very weak emission in the unhybridized state, whereas they emitted near-infrared fluorescence after hybridization with the complementary nucleic acid. The hybridization-dependent switching of fluorescence emission made it possible to monitor mRNA in human cells in the range of near-infrared wavelengths.

Hybridization-sensitive fluorescent oligonucleotide probe conjugated with a bulky module for compartment-specific mRNA monitoring in a living cell.

Live-cell RNA imaging at specific intracellular locations is technically limited because of the diffusive nature of small oligonucleotide probes. The bulky fluorescent light-up probes that possess streptavidin or gold nanoparticles at the end of oligonucleotides were designed and synthesized. The bulky probes allowed nucleus- and cytoplasm-selective monitoring of endogenous mRNAs through nuclear and cytoplasmic microinjection, respectively. Simultaneous use of bulky and unbulky probes conjugated with different fluorescent dyes enabled dual color imaging of mRNAs present in nucleus and cytoplasm. Furthermore, we observed that the fluorescence near the cell edge in a living HeLa cell traveled over time in coordination with the dynamic formation and deformation of the pseudopodial protrusions after lipofection of the bulky probes.

Design and Synthesis of Caged Fluorescent Nucleotides and Application to Live-cell RNA Imaging

A binary photocontrolled nucleic acid probe that contains a nucleotide modified with one photolabile nitrobenzyl unit and two hybridization‐sensitive thiazole orange units has been designed for area‐specific fluorescence imaging of RNA in a cell. The synthesized probe emitted very weak fluorescence regardless of the presence of the complementary RNA, whereas it showed hybridization‐sensitive fluorescence emission at 532 nm after photoirradiation at 360 or 405 nm for uncaging. Fluorescence suppression of the caged probe was attributed to a decrease in the duplex‐formation ability. Caged fluorescent nucleotides with other emission wavelengths (622 and 724 nm) were also synthesized in this study; they were uncaged by 360 nm irradiation, and emitted fluorescence in the presence of the complementary RNA. Such probes were applied to area‐specific RNA imaging in a cell. Only probes in the defined irradiation area were activated by uncaging irradiation, and subnuclear mRNA diffusion in a living cell was monitored.