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Biol Open. 2018 Dec 13;7(12). pii: bio039362. doi: 10.1242/bio.039362.

An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos.

Author information

1
Sorbonne Université, CNRS UMR7622, Inserm U1156, Institut de Biologie Paris-Seine (IBPS), Laboratoire de Biologie du développement (LBD), F-75005 Paris, France.
2
Sorbonne Université, CNRS UMR7622, Inserm U1156, Institut de Biologie Paris-Seine (IBPS), Laboratoire de Biologie du développement (LBD), F-75005 Paris, France francois.giudicelli@inserm.fr.

Abstract

Control of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive and reliable procedures to visualize sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique ( Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. The addition of a cell-permeable protease inhibitor instantly stabilizes newly produced tagged protein allowing us to distinguish recently synthesized proteins from pre-existing ones. After a brief protease inhibitor treatment, the ratio of tagged versus non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged proteins it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development.

KEYWORDS:

Local translation; Neuron; SPoT; TimeStamp; Zebrafish

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

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