Imaging Translation in Single Cells Using Fluorescent Microscopy

Chao, Jeffrey A.; Yoon, Yong‐Dal; Singer, Robert H.

doi:10.1101/cshperspect.a012310

Cited by 35 publications

(31 citation statements)

References 69 publications

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“…To achieve spatiotemporal control of translation, dendritic translation must follow mRNA transport to stimulated spines (37). The use of translation reporters in neurons to observe discrete enrichment of proteins takes advantage of the polarized morphology in which dendrites and axons provide subcellular compartments away from the soma (38). Local translational output can be detected within dendrites (10) or in a subpopulation of newly translated proteins (11, 12) after stimulation.…”

Section: Resultsmentioning

confidence: 99%

“…The application of fluorescent protein-based translation reporters in neurons has allowed significant progress in visualizing local protein synthesis within dendrites and axons (38). The modular design of translation reporters has opened a way for investigators to incorporate specific features into a reporter to enhance the spatial and temporal resolution of detection (10).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Glutamate-induced RNA localization and translation in neurons

Yoon

Buxbaum

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

166

164

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Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapsespecific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single β-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcodebinding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of β-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.single molecule | glutamate uncaging | β-actin | RNA localization | HaloTag S ubcellular localization of mRNA allows control of protein synthesis with respect to space and time (1). By sorting mRNAs to their respective compartments, neurons can regulate translation in response to extracellular signal at the place of protein function (2). Many mRNAs have been shown to be present in dendrites and axons (3), and efforts to characterize mRNA transport revealed that depolarization can lead to detectable increases in alpha calcium calmodulin kinase II (αCaMKII), BDNF, or β-actin mRNAs in dendrites (4-6). Likewise, studies in local translation have shown that dendrites can synthesize the necessary complement of proteins for synaptic plasticity (7). Furthermore, electron microscopy observations of polyribosomes within dendrites and synaptic spines have confirmed that translation occurs readily in neuronal subdomains far from the soma (8, 9). The development and use of fluorescent protein-based translation reporters were pivotal in visualizing local translational output within dendrites (10-12). However, missing from these findings was the high-resolution detection of spatial and kinetic events that result in dynamic repositioning of individual mRNAs and translated proteins within dendrites in response to locally defined input.Actin is the major cytoskeletal component of dendritic spines where filamentous actin (F-actin) dynamics confer motility and structural plasticity (13). Interestingly, the mRNA that encodes for the most abundant actin isoform in neurons, β-actin, is also present in dendrites in relatively large numbers (3). Similar to β-actin, the mRNAs for PSD-95 and αCaMKII (among many) also are found at high levels, with the latter considered one of the most abundant mRNAs in dendrites. Why such abundant synaptic proteins need ...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Glutamate-induced RNA localization and translation in neurons

Yoon

Buxbaum

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

166

164

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“…However, mRNAs also can be sequestered in stress granules or P-bodies (see Decker and Parker 2012) or localized in specific regions of a cell's cytoplasm (see Chao et al 2012;Lasko 2012), indicating that mRNA accessibility can influence the efficiency of translation.…”

Section: Principles Of Translational Controlmentioning

confidence: 99%

Principles of Translational Control: An Overview

Hershey

Sonenberg²,

Mathews

2012

Cold Spring Harbor Perspectives in Biology

348

305

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“…At the same time, literature reports show extensive results about evolution and behavior of single cells obtained by studying their fluorescence emitted at excitation with noncoherent and/or laser radiation. [15][16][17] Some organic dyes, such as Rhodamine 6G (Rh6G), are promising candidates to build biocompatible fluorescent systems, due to their good acceptance by biological entities. This is why they are used in a wide range of applications in biology as markers for in vitro or in vivo imaging, 3 as well as sensitizers and in particular photosensitizers.…”

Section: Introductionmentioning

confidence: 99%

Enhanced fluorescence emitted by microdroplets containing organic dye emulsions

et al. 2015

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In this paper, laser beam resonant interaction with pendant microdroplets that are seeded with a laser dye (Rhodamine 6G (Rh6G)) water solution or oily Vitamin A emulsion with Rhodamine 6G solution in water is investigated through fluorescence spectra analysis. The excitation is made with the second harmonic generated beam emitted by a pulsed Nd:YAG laser system at 532 nm. The pendant microdroplets containing emulsion exhibit an enhanced fluorescence signal. This effect can be explained as being due to the scattering of light by the sub-micrometric drops of oily Vitamin A in emulsion and by the spherical geometry of the pendant droplet. The droplet acts as an optical resonator amplifying the fluorescence signal with the possibility of producing lasing effect. Here, we also investigate how Rhodamine 6G concentration, pumping laser beam energies and number of pumping laser pulses influence the fluorescence behavior. The results can be useful in optical imaging, since they can lead to the use of smaller quantities of fluorescent dyes to obtain results with the same quality.

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Imaging Translation in Single Cells Using Fluorescent Microscopy

Cited by 35 publications

References 69 publications

Glutamate-induced RNA localization and translation in neurons

Glutamate-induced RNA localization and translation in neurons

Principles of Translational Control: An Overview

Enhanced fluorescence emitted by microdroplets containing organic dye emulsions

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