Abstract
Spatial and temporal regulation of protein expression plays important roles in many cellular functions, particularly for highly polarized cell types. While the subcellular proteome can be altered by relocalizing proteins from other domains of the cell, transporting mRNAs to subcellular domains provides a means to locally synthesize new proteins in response to different stimuli. Localized protein synthesis is a critical mechanism in neurons that extend dendrites and axons long distances from their cell bodies. Here, we discuss methodologies that have been developed to study localized protein synthesis using axonal protein synthesis as an example. We provide an in-depth method using dual fluorescence recovery after photobleaching to visualize sites of protein synthesis using reporter cDNAs that encode two different localizing mRNAs along with diffusion-limited fluorescent reporter proteins. We show how this method can be used to determine how extracellular stimuli and different physiological states can alter the specificity of local mRNA translation in real time.
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Acknowledgments
This work was supported by grants from the National Institutes of Health (R01NS089633 and R01NS117821 to JLT), National Science Foundation (MCB-1020970 to JLT), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to JLT), and South Carolina Spinal Cord Injury Research Fund (2019-PD-02 to PKS). JLT is the incumbent SmartState Chair in Childhood Neurotherapeutics at the University of South Carolina.
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Sahoo, P.K., Twiss, J.L. (2023). Profiling Locally Translated mRNAs in Regenerating Axons. In: Udvadia, A.J., Antczak, J.B. (eds) Axon Regeneration. Methods in Molecular Biology, vol 2636. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3012-9_8
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DOI: https://doi.org/10.1007/978-1-0716-3012-9_8
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