Recombinant Probes for Visualizing Endogenous Synaptic Proteins in Living Neurons

Gross, Garrett G.; Junge, Jason A.; Mora, Rudy J.; Kwon, Hyung Bae; Olson, C. Anders; Takahashi, Terry T.; Liman, Emily R.; Ellis‐Davies, Graham C. R.; McGee, Aaron W.; Sabatini, Bernardo L.; Roberts, Richard W.; Arnold, Don W.

doi:10.1016/j.neuron.2013.04.017

Cited by 272 publications

(388 citation statements)

References 56 publications

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“…As expected, we found gephyrin-FingR-GFP clustering at the AIS, which was identified by colabeling with ankyrin-G antibody ( Figure 5A, arrows). GFP-labeled puncta were also seen on the soma and dendrites ( Figure 5A, arrowheads), concordant with the previously described distribution of endogenous gephyrin (22).…”

Section: Discussionsupporting

confidence: 90%

“…To test whether Sptan1-deleted cortical pyramidal cells exhibit a defect in inhibitory input, we first used the FingR (fibronectin intrabodies generated with mRNA display) system to label endogenous postsynaptic gephyrin, a GABA type A receptor (GABA A R) anchoring protein, as a surrogate for GABAergic synapses (22). The gephyrin-FingR-GFP construct incorporates a transcriptional regulation system that ties expression to the level of the target, allowing inhibitory synapses to be visualized without altering localization, morphology, and function, in either fixed or living neurons (22). To test the construct, we cotransfected gephyrin-FingR-GFP and RFP plasmids (the latter to fill cells) via IUE at E13-14 and examined brain sections at P21, a time when the majority of synaptogenesis has occurred.…”

Section: Discussionmentioning

confidence: 99%

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Critical roles of αII spectrin in brain development and epileptic encephalopathy

Wang¹,

Ji²,

Nelson³

et al. 2018

Journal of Clinical Investigation

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Critical roles of αII spectrin in brain development and epileptic encephalopathy

Wang¹,

Ji²,

Nelson³

et al. 2018

Journal of Clinical Investigation

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“…To verify that the photoswitching hot-spots of α1-LiGABAR represent clusters of functional receptor at synapses, we targeted inhibitory synapses using a genetically-encoded fluorescent intrabody for gephyrin (a scaffolding protein that tethers GABA A receptors at synapses; Gross et al, 2013). Neurons expressing the gephyrin intrabody exhibit fluorescent puncta at postsynaptic sites.…”

Section: Resultsmentioning

confidence: 99%

A Comprehensive Optogenetic Pharmacology Toolkit for In Vivo Control of GABA A Receptors and Synaptic Inhibition

Lin

Tsai

Davenport

et al. 2015

Neuron

124

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SUMMARY Exogenously-expressed opsins are valuable tools for optogenetic control of neurons in circuits. A deeper understanding of neural function can be gained by bringing control to endogenous neurotransmitter receptors that mediate synaptic transmission. Here we develop a comprehensive optogenetic toolkit for controlling GABAA receptor-mediated inhibition in the brain. We synthesized a series of photoswitch ligands and the complementary genetically-modified GABAA receptor subunits. By conjugating the two components we generated light-sensitive versions of the entire GABAA receptor family. We validate these light-sensitive receptors for applications across a broad range of spatial scales, from subcellular receptor mapping to in vivo photo-control of visual responses in the cerebral cortex. Finally, we generated a knock-in mouse in which the “photoswitch-ready” version of a GABAA receptor subunit genomically replaces its wild-type counterpart, ensuring normal receptor expression. This optogenetic pharmacology toolkit allows scalable interrogation of endogenous GABAA receptor function with high spatial, temporal, and biochemical precision.

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“…Another strategy would be to reduce the unbound fraction of the fluorescent Cas proteins. For example, in a recent approach, a zinc-finger (ZF) binding domain and transcription repressor KRAB have been fused to Cas13a-FP, and the ZF binding site was inserted to the promoter controlling the expression of the fusion protein (132, 134). In this way, a negative-feedback loop is created, and the unbound Cas13a-FP therefore auto-represses its expression to control the background fluorescence.…”

Section: Emerging Strategies and Approachesmentioning

confidence: 99%

RNA Localization in Bacteria

Fei

Sharma

2018

Microbiol Spectr

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Diverse mechanisms and functions of post-transcriptional regulation by small regulatory RNAs (sRNAs) and RNA binding proteins (RBPs) have been described in bacteria. In contrast, little is known about the spatial organization of RNAs in bacterial cells. In eukaryotes, subcellular localization and transport of RNAs play important roles in diverse physiological processes, such as embryonic patterning, asymmetric cell division, epithelial polarity, and neuronal plasticity. It is now clear that bacterial RNAs also can accumulate at distinct sites in the cell. However, due the small size of bacterial cells, localized RNA and translation are more challenging to study in bacterial cells, and the underlying molecular mechanisms of transcript localization are less understood. Here we review the emerging examples of RNAs localized to specific subcellular locations in bacteria, with indications that subcellular localization of transcripts might be important for regulatory processes. Diverse mechanisms for bacterial RNA localization have been suggested, including close association to their genomic site of transcription, or to the localizations of their protein products in translation-dependent or independent processes. We also provide an overview of the state-of-the-art of technologies to visualize and track bacterial RNAs, ranging from hybridization-based approaches in fixed cells to in vivo imaging approaches using fluorescent protein reporters and/or RNA aptamers in single living bacterial cells. We conclude with a discussion of open questions in the field and ongoing technological developments regarding RNA imaging in eukaryotic systems that might likewise provide novel insights into RNA localization in bacteria.

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Recombinant Probes for Visualizing Endogenous Synaptic Proteins in Living Neurons

Cited by 272 publications

References 56 publications

Critical roles of αII spectrin in brain development and epileptic encephalopathy

Critical roles of αII spectrin in brain development and epileptic encephalopathy

A Comprehensive Optogenetic Pharmacology Toolkit for In Vivo Control of GABA A Receptors and Synaptic Inhibition

RNA Localization in Bacteria

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