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Ribosome Profiling

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...

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In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

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Soma-Targeted Imaging of Neural Circuits by Ribosome Tethering.

Yiming Chen¹, Heeun Jang², Perry W E Spratt¹

¹Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.

|June 24, 2020

Summary

Researchers developed novel ribosome-tethered reporters to visualize neuronal cell bodies and calcium dynamics. These tools enhance imaging specificity in neuroscience by confining fluorescent proteins to the soma, overcoming neuropil interference.

Keywords:

C. elegans GCaMP calcium imaging ribo-GCaMP ribosomal tagging soma-targeting whole-brain imaging

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Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

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Published on: July 6, 2021

Area of Science:

Neuroscience
Molecular Biology
Biotechnology

Background:

Neuronal imaging is crucial for understanding neural circuits.
Overlapping fluorescence from axons and dendrites often obscures neuronal cell bodies in neuropil.
Existing imaging techniques struggle with specificity in dense neural tissues.

Purpose of the Study:

To develop strategies for restricting fluorescent protein expression to neuronal somata.
To enable clearer visualization of neuronal cell bodies and their dynamics.
To improve the specificity of calcium imaging in neuroscience.

Main Methods:

Utilized ribosome-tethered nanobodies to trap fluorescent proteins (GFP) in the neuronal soma.
Engineered ribosome-tethered GCaMP for imaging somatic calcium dynamics.
Tested reporter efficacy in mouse brain and C. elegans models.

Main Results:

Ribosome-tethered nanobodies enabled direct visualization of previously obscured GFP fluorescence in neuronal cell bodies.
Ribosome-tethered GCaMP accurately tracked somatic calcium dynamics in mice, eliminating neuropil cross-talk.
In worms, the reporter facilitated whole-brain imaging with improved kinetics and brightness compared to nuclear GCaMPs.

Conclusions:

Developed two generalizable strategies using ribosome-tethering to enhance imaging specificity in neurobiology.
These methods allow for precise visualization of neuronal somata and their activity.
The engineered reporters offer significant advantages for studying neural circuits in various model organisms.