The aim of this experiment is to demonstrate the visualization of in vivo protein dynamics and localization patterns in live drosophila embryos by time-lapse confocal microscopy. This is accomplished by first harvesting embryos from maintained drosophila stocks. Next, transfer the embryos onto a slide and then manually remove the coons from the embryos to coated embryos.
A subsequently mounted in an organized fashion onto previously prepared slides, and depending on the experiment, may be micro injected with appropriate spindle checkpoint inducing reagents. The final step in this procedure is to perform time-lapse imaging of embryos using confocal microscopy. Ultimately, time-lapse and focal microscopy is used to show spindle checkpoint protein, colocalization chromosome behaviors, and the effect of checkpoint inducing agents in checkpoint related transgenic oph lines.
The method can help answer the key questions in the ME regulation field, such as the effect of the mutations or depletions of spindle checkpoint protein through the use of the genic lines on the colocalization of the other checkpoint components, and the consequence of this on the ability of the spindle assembly checkpoint to function efficiently. Transgenic flies are maintained at 25 degrees Celsius in plastic vials containing fly food, and with additional dry yeast powder on the top. The vial is routinely replaced every two to three weeks, depending on growing conditions.
To prepare fly food, heat an appropriate amount of the fly food mix with constant stirring to dissolve the components using a peristaltic pump, distribute eight to 10 milliliters of this medium as slurry into each plastic vial. When the food slurry has set and called drun temperature, plug each vial with a cotton foam plug. Place these food vials in a tray, seal in a plastic bag, and store at four degrees Celsius for a small scale egg collection.
Transfer about 50 pairs of two to three day old adult flies to a fresh fly food vial supplied with additional dry yeast powder on its surface for laying embryos. Incubate at 25 degrees Celsius, which in this case is the temperature maintained by air conditioning. For the room every hour, transfer the flies to a fresh file and leave the embryos for 30 minutes.
To ensure that some of the collected embryos raged around nuclear division cycle eight to 10. The first hour collection is normally discarded as it often contains aged embryos that were retained in the female bodies when conditions were not suitable for laying. Therefore, only embryos from the transfer at the second hour onward will be used in these experiments.
Microscope slides and 50 by 22 millimeter cover slips are used for this procedure. Begin by taking a cover slip and with a moist fine pen brush slightly wet the four corners on one side with a very small amount of water. Place the cover slip on a microscope slide.
The capillary surface tension caused by the thin liquid film should prevent the cover slip from moving. Apply a thin strip of heptane glue across the middle of the cover slip. The heptane should evaporate in seconds leaving the glue on the cover slip.
Next, use a diamond pen to cut another cover slip into small squares of about 1.5 millimeter square. Pick up one square and place it on one end of the glue strip. This will be used to open the needle tip when microinjection is required.
Take another microscope slide stick a two centimeter long piece of double-sided sticky tape to it and peel off the cover paper to begin the procedure for coating embryos. Transfer the flies to a fresh food vial with a moist and fine pen brush. Transfer about 50 eggs onto the double-sided sticky tape on the prepared slide.
Use an appropriate amount of liquid to allow the eggs to be spread out. When the liquid has evaporated, the eggs will stick to the tape under a dissecting microscope. Touch the eggs gently several times along their long axis with the outer side of one half of a pair of tweezers until the Corian is broken and open.
Leave the embryo in the Corian shell to prevent rapid dehydration. Continue this process until 10 to 20 of the eggs have been treated. Next, pick up the embryos and remove them from the Corian shell one by one and gently place the embryos in a vertical strip.
On the glue strip. Align the embryos with the long side anti-parallel to the long side of the glue strip. Place the embryos in a desiccation chamber for three to eight minutes.
Finally, cover the embryos with an appropriate amount of volter left tennis oil to prevent over dehydration prior to microinjection. The micro ingestion is the most difficult aspects of this procedure. In order to make the micro ingestion as easy and efficient as possible, the specific protocol for slight preparation should be followed.
The embryos must be carefully prepared so that they are in optimal condition for micro ingestion. The needles for micro injecting embryos are prepared beforehand. Using a fine loading tip, backfill a needle with one to two microliters of an appropriate concentration of the reagent in injection buffer.
Mount the needle in the needle holder on a Leica TCSP two inverted confocal microscope with the pressure pipe connected to the injection control system. Find an embryo under the 40 times objective. Next, find the needle shadow without changing the focal plane.
This is accomplished by moving the needle into the center of the field of view and gradually lowering it to the right focal plane. Move the microscope stage very gently to find the small square of cover slip prem mounted at one end of the glue strip. Very gently move the needle tip until it hits the edge of the small square cover slip and gently breaks open.
Move the stage so that the embryos come back into view and select an embryo at the right age for injection. Carefully move the embryo close to the needle and readjust the focal plane for both the embryo and the needle. Move the embryo into the needle.
Inject a drop of reagent into the side of the embryo and then move the embryo away. Time lapse or single images of living embryos are collected using a Leica TCSS SP two inverted confocal microscopy system with a 40 times oil immersion objective. When imaging multiple fluorophores that have overlapping spectra and are co-expressed in the same embryo, each fluorescent protein emission wavelength is collected sequentially.
The images emerged later when setting up the timeframes for time-lapse imaging. These factors must be considered. A single image is commonly obtained as an average of two scan lines.
With an average of two frame scans, it takes a minimum of 6.3 seconds to scan a five 12 by five 12 pixel image in a simultaneous scan mode and 15.44 seconds for a sequential scan mode and to produce a good signal to noise ratio. The pinhole is normally set to one to two au or airy unit and the laser power is adjusted to the lowest level possible to avoid any significant photo bleaching. This representative time-lapse movie shows the dynamic kinetic core recruitment of CDC 20 and chromosome motion in living ophs in cial embryos.
Time-lapse images were taken from a transgenic inital embryo coex expressing G-F-P-C-D-C 20, shown in green and RFP his stone two B shown in red. During nuclear division cycles, seven to eight frames were taken every 10 seconds and the frame with the cells already in prophase is treated as a zero time point. This figure shows the frames from the time-lapse movie G fp CDC 20 can be readily observed on prophase and prometaphase kinetico as indicated by the white arrows in images three and four respectively and persists on metaphase and anaphase kinetta course as observed in images five and six respectively.
G-F-P-C-D-C 20 is excluded from the interface nucleus indicated by the white arrow head in image one and enters the nucleus by early prophase as shown in image two. In images eight to 14 chromatin morphologies were determined using co-expressed RFP histone two B as markers merged. Images are shown in the bottom row.
Using this protocol spindle assembly checkpoint or SAC functions can be studied by manipulating the embryos by micro injecting antibodies, fluorescently labeled proteins or chemical compounds of interest that potentially trigger the SAC. In this example, embryos are with colchicine to depolymerize the microtubules, thus provoking the SACG FP CDC 20, or GFP MAD. Two kinetic core signals were used as cell cycle progression markers.
In the top panel, the ARES indicate the arrested esci in images two to six. The area marked by an arrow in image one indicates the before colchicine treatment, G-F-P-C-D-C 20 is excluded from the late interface nucleus. The images in the middle panel show that in the absence of endogenous MAD two G-F-P-C-D-C 20 indicated by arrows in seven to 12 continue to oscillate in and outta the nucleus and on and off the kinetic course.
Although cytokinesis appears to be defective, this suggests a failed SAC function in arresting cells in response to Colchicine treatment. An example of a failed separation of daughter nucleus is observed in image 10. Finally, in the bottom panel arrow heads in images 14 to 18 indicator rested kinetic cores with accumulated GFP MA two fusion proteins, which suggests the functional GFP MA two rescues the sac defect phenotype in the MA two mutant embryo.
The arrowhead in image 13 indicates GFP MAD two accumulation in a late interface nucleus. After watching these videos, you should have a good understanding how to harvest the two software embryos from maintain the stalks, manually de coordinator embryos before preparing slides for micro ingestion performing in vivo time, left imaging using confocal microscopy.
