Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Summary

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Abstract

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Introduction

The levels of proteins in cells and organisms are determined by their rates of production and clearance. Protein half-lives can range from minutes to days^1-4. In many biological systems, the stabilization or clearance of key proteins has important effects on cellular activity. Modulation of intracellular protein stability is required for cell cycle progression^5,6, developmental signaling^7-9, apoptosis¹⁰, and normal function and maintenance of neurons^11,12. Extracellular protein stability affects the distribution and availability of secreted proteins, such as morphogens^13,14, within a tissue.

Protocol

1. Generating a Photoconvertible Fusion Construct and Injecting Dechorionated Zebrafish Embryos

1. Generate a functional construct containing the protein of interest fused to a green-to-red photoconvertible protein (see Discussion), then use in vitro transcription to generate capped mRNA encoding the fusion protein as in Müller et al., 2012¹⁹.
2. Use pronase to remove the chorions from about 30 zebrafish embryos at the one-cell stage. Alternatively, manually dechorionate embryos using forceps²⁸.
   Note: Embryos must be dechorionated for subsequent imaging. If desired, embryos can be injected t....

Results

FDAP has been used to determine the half-lives of extracellular signaling proteins in zebrafish embryos¹⁹. One of these proteins, Squint, induces expression of mesendodermal genes during embryogenesis³⁴. Squint-Dendra2 activates expression of mesendodermal genes at levels similar to untagged Squint, as demonstrated by qRT-PCR and in situ hybridization assays¹⁹. Embryos were co-injected with Alexa488-dextran and mRNA encoding Squint-Dendra2 and subjected to the FDAP assay. A decre.......

Discussion

The success of an FDAP experiment relies on the generation of a functional photoconvertible fusion protein. Tagging a protein can affect its biological activity and/or biophysical properties, including its localization, solubility, and stability^36-41. Be prepared to test the activity of several different fusion constructs in order to find one that is active. We have found that changing the position of the photoconvertible protein relative to the protein of interest or using longer linkers (e.g., using.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PyFDAP (download from the following website: http://people.tuebingen.mpg.de/mueller-lab)			Install and operate using the instructions provided on the PyFDAP website; PyFDAP is compatible with Linux, Mac, and Windows operating systems.
mMessage mMachine Sp6 Transcription Kit	Life Technologies	AM1340	To generate capped mRNA for injection into embryos
Alexa488-dextran conjugate, 3 kDa	Life Technologies	D34682	Co-inject with mRNA to create intracellular and extracellular masks
6-well plastic dish	BD Falcon		Incubate embryos in agarose-coated wells until ready for mounting
Embryo medium			250 mg/L Instant Ocean salt, 1 mg/L methylene blue in reverse osmosis water adjusted to pH 7 with NaHCO3
Protease from Streptomyces griseus	Sigma	P5147	Make a 5 mg/ml stock and use at 1 mg/ml to dechorionate embryos at the one-cell stage
5 cm diameter glass Petri dish			For embryo dechorionation
200 ml glass beaker			For embryo dechorionation
Microinjection apparatus			For injection of mRNA and dye into embryos at the one-cell stage
Stereomicroscope			For injecting and mounting embryos
1x Danieau's medium			Dilute low melting point agarose and perform imaging in this medium; recipe: 0.2 mm filtered solution of 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.3 mM CaCl2, 5 mM HEPES pH 7.2
UltraPure low melting point agarose	Invitrogen	16520-100	For mounting embryos; use at a concentration of 1% in Danieau's medium: add 200 mg to 20 ml Danieau's medium, microwave until dissolved, then aliquot 1 ml into microcentrifuge tubes; aliquots can be stored at 4 °C, re-melted at 70 °C, and cooled to 40–42 °C when ready to use
Glass Pasteur pipette	Kimble Chase (via Fisher)	63A53WT	For mounting embryos; flame the tip to prevent jagged edges from injuring embryos
Metal probe			For positioning embryos during mounting
Glass bottom dishes	MatTek	P35G-1.5-14-C	Use the appropriate cover glass thickness for your objective; part number listed here is for cover glass No. 1.5
15 ml tube filled with ~5 ml embryo medium	BD Falcon		For rinsing residual agarose from the Pasteur pipette
Inverted laser scanning confocal microscope			A mercury arc lamp, 488 nm laser, 543 nm laser, and the appropriate filter sets are required
Heated stage			To maintain embryos at the optimal temperature of 28 °C during the experiment
Confocal software capable of time-lapse imaging			Must be able to define multiple positions and automatically image them at defined intervals
25X or 40X water objective			Objective for imaging
10X air objective			Objective for photoconversion
Immersion oil			Immersion oil with the same refractive index as water