Measuring Bulk Translation Activity in Single Mammalian Cells During the Integrated Stress Response

Alyssa M English; Stephanie L Moon

doi:10.1007/978-1-0716-1975-9_4

Measuring Bulk Translation Activity in Single Mammalian Cells During the Integrated Stress Response

Methods Mol Biol. 2022:2428:63-73. doi: 10.1007/978-1-0716-1975-9_4.

Authors

Alyssa M English^{1

2}, Stephanie L Moon^{3

4}

Affiliations

¹ Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA.
² Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI, USA.
³ Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA. smslmoon@umich.edu.
⁴ Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI, USA. smslmoon@umich.edu.

PMID: 35171473
DOI: 10.1007/978-1-0716-1975-9_4

Abstract

The attenuation of global translation is a critical outcome of the integrated stress response (ISR). Consequently, it is important to effectively detect and measure protein synthesis in studies seeking to evaluate the ISR. This chapter details two methods, surface sensing of translation (SUnSET) and fluorescent noncanonical amino acid tagging (FUNCAT), to measure global translation activity in individual cells using fluorescence microscopy as a read-out. Detecting bulk translation activity in single cells is advantageous for the concurrent observation of newly synthesized proteins and other cellular structures and to identify differences in translation activity among individuals within a population of cells.

Keywords: Click chemistry; FUNCAT; Fluorescent noncanonical amino acid tagging; Immunofluorescence; Integrated stress response; Puromycin; Puromycylation; SUnSET; Surface sensing of translation; Translation.

MeSH terms

Amino Acids / metabolism
Animals
Humans
Microscopy, Fluorescence
Protein Biosynthesis*
Proteins* / metabolism
Puromycin

Substances

Amino Acids
Proteins
Puromycin