Measuring protein degradation in living organisms

Protein degradation governs many biological processes, but studying degradation can be technically challenging. We developed a degradation assay and a software package that overcome several of these challenges.

HFSP Career Development Award holder Patrick Müller and HFSP Program Grant holder Alexander Schier and colleagues
authored on Mon, 02 November 2015

Neuronal activity, embryonic development, cell division, and cell death are among the diverse biological processes that depend on protein degradation. Insights into these and other processes can be gained by measuring the degradation of key proteins.

Studies aimed at measuring protein degradation are often hampered by technical challenges that can introduce artifacts and cast doubt on the validity of the measurements. Two major drawbacks affect many degradation assays. Firstly, the protein of interest is often studied outside of its normal environment. Since degradation rates depend on environmental context, observing a protein’s behavior outside of its normal environment is potentially problematic. Secondly, degradation rates are often inferred by processing dead tissues or cells. Under these conditions, observation of degradation dynamics over time within the same tissue or cell population is impossible.

To circumvent these problems, we developed an in vivo degradation assay, Fluorescence Decay After Photoconversion (FDAP), and provide a step-by-step video protocol for performing FDAP in zebrafish embryos. Additionally, we created the software package PyFDAP to facilitate the analysis of FDAP data. With FDAP it is possible to dynamically measure the degradation of proteins in their normal environments in living tissues.  

Figure: A zebrafish embryo producing a protein fused to Dendra2 was transiently exposed to blue light and imaged over five hours. The red fluorescence intensity present in the spaces between cells (dark spots) decreases over time as the protein is degraded. The fluorescence intensity data can be used to determine the protein’s half-life.

Our FDAP assay relies on a class of fluorescent proteins known as photoswitchable proteins. We used Dendra2, a photoswitchable protein that normally emits green fluorescence. Upon exposure to blue light, the fluorescence of Dendra2 switches from green to red. This useful property can be exploited to pulse-label a protein of interest by fusing it to Dendra2. When tissues producing the fusion protein are transiently exposed to blue light, the protein of interest switches its fluorescence from green to red, pulse-labeling the protein. As the pulse-labeled protein is degraded, the decrease in red fluorescence intensity over time is monitored using microscopy.   

Data from FDAP experiments can be easily and rapidly analyzed using our new software package, PyFDAP. Our software extracts fluorescence intensity information from FDAP experiments and fits decay models to the resulting data. PyFDAP then determines protein half-lives.

New tools and technologies that directly measure biological processes provide opportunities to gain novel insights into biology. For example, we previously used FDAP to measure the degradation rates of proteins involved in embryonic development, leading to a better understanding of how embryos generate the correct proportions of different cell types. In the future, FDAP and PyFDAP can be employed to investigate diverse biological systems.   

Text by Katherine W. Rogers and Patrick Müller

References

[1] Measuring protein stability in living zebrafish embryos using fluorescence decay after photoconversion (FDAP). Katherine W. Rogers, Alexander Bläßle, Alexander F. Schier and Patrick Müller. Journal of Visualized Experiments. 2015; 95:52266. doi: 10.3791/52266.

[2] PyFDAP: automated analysis of fluorescence decay after photoconversion (FDAP) experiments. Alexander Bläßle and Patrick Müller. Bioinformatics. 2015; 31:972-4. doi: 10.1093/bioinformatics/btu735.

Pubmed link [1]

Pubmed link [2]