In Vivo Quantification of Protein Turnover in Aging <em>C. Elegans</em> using Photoconvertible Dendra2

Maria  Lucia Pigazzini; Janine Kirstein

doi:10.3791/61196

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible De...

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In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2

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09:45 min

June 13, 2020

DOI: 10.3791/61196-v

Maria Lucia Pigazzini^1,2, Janine Kirstein^1,3

¹Leibniz Research Institute for Molecular Pharmacology im Forschungsverbund, ²NeuroCure Cluster of Excellence,Charité - Universitätsmedizin Berlin, ³Department of Biology and Chemistry,University of Bremen

Overview

This study presents a protocol for monitoring the degradation of the huntingtin protein fused to Dendra2 in live C. elegans, providing insights into protein turnover within the proteostasis network as well as transport and trafficking. The technique is applicable to both mammalian systems and zebrafish, allowing for non-invasive tracking of protein dynamics.

Key Study Components

Research Area

Protein degradation monitoring
Live imaging techniques
Cellular dynamics in nematodes

Background

Understanding protein degradation is crucial for elucidating cellular processes
The huntingtin protein is associated with neurodegenerative diseases
Quantitative analysis of protein turnover aids in the study of proteostasis

Methods Used

Live imaging of protein dynamics using Dendra2 fluorophore
C. elegans as a model organism
Confocal microscopy to analyze Dendra2 conversion and degradation

Main Results

Successful tracking of Dendra2 fluorescence to assess huntingtin degradation
Fluorescent signal conversion demonstrated specific dynamics over time
Quantitative metrics obtained for protein degradation using image analysis

Conclusions

The method effectively reveals protein degradation patterns in vivo
This protocol enhances understanding of proteostasis and associated mechanisms in various biological systems

Frequently Asked Questions

What is Dendra2?

Dendra2 is a photoconvertible fluorophore used for live-cell imaging to track protein dynamics.

Why is C. elegans used as a model organism?

C. elegans is a well-characterized model organism that allows for in vivo studies of cellular processes.

How does the conversion of Dendra2 work?

Dendra2 can be switched from green to red fluorescence using a specific wavelength of light, enabling tracking of protein movement and degradation.

What are the key advantages of this imaging method?

The method is non-invasive, adaptable to different models, and allows live tracking of protein behavior over time.

What measurements are important in this study?

Area and integrated density of the fluorescence signal are key measurements for analyzing protein degradation.

Can this method be used in mammalian systems?

Yes, the protocol is adaptable to mammalian systems and can provide insights into similar processes in eukaryotic cells.

What implications does this research have?

Understanding protein degradation has significant implications for studying neurodegenerative diseases and cellular health.

Presented here is a protocol to monitor degradation of the protein huntingtin fused to the photoconvertible fluorophore Dendra2.

This protocol allows for tracking and quantification of the degradation of a protein of interest in live and aging C.elegans. This in vivo and noninvasive technique requires only little microscopy set up to reliably convert Dendra2 into a bright and stable fluorophore. The method is adaptable to mammalian systems, as well as zebrafish.

To study, put in turnover within the proteostatis network as well as transport and trafficking. Take care not to provoke unwanted conversion of Dendra2 while scanning with the blue light laser, and to test and optimize the acquisition settings and conversion settings for each system and fusion protein. Begin by growing age-matched nematodes at 20 degrees Celsius to the desired age for the experiment.

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