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Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules
Published on: May 12, 2017
Establishment of a stress granule reporter system for evaluating in vitro colon toxicity
Namjoon Cho1, Da-Min Jung1, Eun-Mi Kim2
1Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, Republic of Korea.
Abstract:
Exposure to toxic molecules from food or oral medications induces toxicity in colon cells that cause various human diseases; however, in vitro monitoring systems for colon cell toxicity are not well established. Stress granules are nonmembranous foci that form in cells exposed to cellular stress. When cells sense toxic environments, they acutely and systemically promote stress granule formation, with Ras GTPase-activating protein-binding protein 1 (G3BP1) acting as a core component to protect their mRNA from abnormal degradation. Here, we knocked in green fluorescent protein (GFP)-coding sequences into the C-terminal region of the G3BP1 gene in a human colon cell line through CRISPR-Cas9-mediated homologous recombination and confirmed the formation of stress granules with the G3BP1-GFP protein in these cells under cellular stress exposure. We demonstrated the formation and dissociation of stress granules in G3BP1-GFP expressing colon cells through real-time monitoring using a fluorescence microscope. Furthermore, we validated the toxicity monitoring system in the established colon cell line by observing stress granule formation following exposure to dihydrocapsaicin, bisphenol A, and sorbitol. Taken together, we established a stress granule reporter system in a colon cell line, providing a novel assessment for the real-time monitoring of colon toxicity in response to various chemicals.
Insights
Scientists developed a new method to monitor colon cell toxicity using stress granules. This real-time system tracks stress granule formation in G3BP1-GFP colon cells, offering a novel way to assess chemical toxicity.
Area of Science:
- Cell Biology
- Toxicology
- Biotechnology
Background:
- Colon cell toxicity from environmental toxins is a significant health concern.
- Existing in vitro methods for monitoring colon cell toxicity are limited.
- Stress granules, including those involving Ras GTPase-activating protein-binding protein 1 (G3BP1), form in response to cellular stress.
Purpose of the Study:
- To establish a novel in vitro system for real-time monitoring of colon cell toxicity.
- To develop a reporter system based on stress granule formation in human colon cells.
Main Methods:
- Utilized CRISPR-Cas9 gene editing to create a human colon cell line expressing G3BP1 fused with green fluorescent protein (GFP).
- Observed stress granule formation and dissociation in real-time using fluorescence microscopy.
- Validated the system by exposing cells to known toxicants like dihydrocapsaicin, bisphenol A, and sorbitol.
Main Results:
- Successfully generated a human colon cell line that forms stress granules labeled with G3BP1-GFP under stress.
- Demonstrated real-time visualization of stress granule dynamics (formation and dissociation) in response to cellular stress.
- Confirmed that stress granule formation accurately reflects exposure to specific toxic chemicals.
Conclusions:
- A novel stress granule reporter system has been established in a human colon cell line.
- This system enables real-time monitoring of colon cell toxicity induced by various chemicals.
- Provides a valuable new tool for assessing the toxicological impact of compounds on colon cells.
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