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

The overall goal of the following experiment is to measure intracellular and extracellular protein stability in living zebra fish embryos. This is achieved by tagging a protein of interest with a photo convertible fluorescent protein, and by injecting mRNA, encoding the fusion as well as a fluorescent dye into zebrafish embryos. Next, the photo convertible fusion protein is photo converted, which pulse labels the protein.

In this example, the fusion protein is secreted then the decay in intra and extracellular photo converted fluorescence intensity is monitored over time and fitted with an exponentially decreasing function in order to determine the intracellular and extracellular half lives of the fusion protein. The results show the in vivo stability of photo convertible fusion proteins based on the decay in fluorescent signal over time. This method can help answer key questions in the fields of cell and developmental biology.

For example, how stability influences protein distribution and availability. We are using our software EP for the analysis of FD DP data. EP is freely available on our webpage After generating capped mRNA in coding a encoding a green to red photo convertible fusion protein of interest transfer approximately 31 cell stage zebrafish embryos to a five centimeter diameter glass or agar rose coated plastic Petri dish containing approximately eight milliliters of embryo medium.

Add two milliliters of previously prepared and thawed pro nay stock solution to the dish and incubate at room temperature for five to 10 minutes. Fill a glass speaker with embryo medium and while avoiding exposing the embryos to air or plastic, transfer the embryos to the beaker by tilting the Petri dish while submerging it in the medium. After the embryos have settled to the bottom of the beaker, pour out most of the medium and use fresh medium to carefully replace it.

The mild swirling of the fresh medium will cause the embryos to lose their weakened corion. After repeating the wash, use a glass PEs pipette with a flamed tip to transfer the coated embryos to an agarose coated injection dish. Then co inject the mRNA and a three kilodalton Alexa 4 88 DExT Strand conjugate directly into the center of the cell to ensure even distribution of mRNA and dye.

Once cleavage begins, transfer the injected embryos to a one to 2%agarose coated well of a six well plastic dish filled with embryo, medium, and incubate in the dark at 28 degrees Celsius until the embryos reach late sphere stage with a stereo microscope, identify one to five healthy embryos and use a glass PEs pipette with a flamed tip to remove them from the dish. Gently eject the embryos into a micro centrifuge tube containing approximately one milliliter of melted 1%low melting point aros in one x danios embryo medium. Next, draw the embryos back into the pipette along with some aros.

Then gently eject the aros and embryos onto the cover. Glass of a glass bottom dish. To reuse the glass pipette, clean out the residual aros to prevent clogging by quickly pipetting embryo medium up and down, working quickly before the aros solidifies.

Use a metal probe to position the embryos so that the animal pole faces the cover glass under the stereo microscope. Monitor the embryos positions and readjust as necessary until the agarro hardens. When the agarro has solidified, use one x danios embryo medium to fill the glass bottom dish.

To prepare for photo conversion, set the incubator to 28 degrees Celsius and place a large drop of immersion oil on the objective to ensure that the oil film between the objective and cover glass will not break as the stage moves to different embryo positions. During imaging securely, place the glass bottom dish onto the stage so that the dish will not shift when the stage moves. Next in the confocal microscope's software package, define each embryo's position.

Adjust the Z depth for each embryo and attempt to target roughly the same plane in each embryo using both the 4 88 and 5 43 nanometer lasers. Acquire pre photo conversion images. Then configure the confocal microscope software to image each of the previously defined positions with the appropriate imaging conditions.

Every 10 or 20 minutes or a five hour time course to photo convert the fusion protein switch to a 10 x objective and expose groups of embryos to UV light from the mercury lamp with an approximately 300 to 400 nanometer filter at 100%output for two minutes. Shift the focus along the Z axis to promote uniform photo conversion after photo conversion. Switch back to the 25 x or 40 x objective immediately and to confirm that the previously defined positions are still accurate.

If the dish shifted during photo conversion, redefine the positions of the embryos. Start the imaging time course and allow it to run for five hours. Note the time elapsed between photo conversion and the start of imaging for each embryo.

Occasionally check on the experiment and monitor the level of Danielle's medium, adding more if necessary. Begin by visually inspecting the time course data sets from each embryo. Discard data sets from embryos that died during imaging that shifted significantly, that have very low levels of photo converted signal, or that contain regions of cells that look unusual and that have stopped moving and dividing.

Using the Python based software package, PI F DAP create a mask using the Alexa 4 88 signal, which is strictly intracellular. In order to separate intracellular and extracellular photo converted signal, apply the mask to the corresponding red channel image to prevent intracellular pixels from being considered. When calculating average extracellular intensity to measure average intracellular intensity, invert the mask, display the generated masked images, and visually inspect them and discard data sets in which masks do not accurately distinguish intracellular from extracellular space.

Use PI F DAP to calculate average extracellular and intracellular fluorescence intensities for each image. Then fit the fluorescence data as shown here according to the guidelines in the text protocol, and calculate the extracellular and intracellular protein. Half lives tau from the clearance rate.

Constance K squint is a secreted protein that induces expression of mis endodermal genes during zebrafish embryogenesis. In this experiment, embryos were co injected with Alexa 4 88 dextran and mRNA encoding squint DENDRA two and subjected to the F DAP assay. As shown here, the extracellular photo converted signal intensity decreases over time.

Using PI F dap, extracellular intensity profiles from 23 embryos were generated and the data was fitted with a first order clearance model. The results indicate an average half-life tau of 116 minutes. Similar intensity profiles and protein.

Half-lives were obtained when the intervals between imaging were 10 or 20 minutes. Suggesting that photo bleaching or inadvertent photo conversion did not contribute significantly to intensity changes. After watching this video, you should have a good understanding of how to use fluorescence decay after photo conversion.

To measure in vivo. Half-lifes of photo convertible fusion proteins in living zebrafish embryos.