The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.
The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer’s1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 °C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.
1. Transgenic Flies and Maintenance
2. Fly Food Preparation (Lab scale)
3. Small-scale Egg Collection
4. Preparing Coverslips and Slides
5. Dechorionate Embryos
6. Imaging Embryos
7. Provoke the SAC by Microinjecting the Embryos with Appropriate Reagents
8. Representative Results
Figure 1. Required materials: a. fine pen brush, b. small square broken-glasses container, c. 22 x 50 mm coverslip, d. microscope slide, e. double-sided sticky tape, f. dry yeast powder in test tube with holes on the lid, g. yeast granule used for fly maintenance, h. fly food vial, i. a half of a tweezers used for dechorionate embryos, j. a pair tweezers, k. heptane glue liquid container (volumetric flask).
Figure 2. The diagrams show the preparation of the coverslips and slides in step 4, embryo dechorionation in step 5 and needle preparations for microinjection in step 7. A. The top picture shows a coverslip with a strip of heptane glue across its middle and a small square of a broken coverslip stuck to one end. The bottom picture shows a microscope slide with a thin layer of water at its four corners to hold a coverslip. The reason for using a slide to hold the coverslip is to keep roughly the same focal distance as that found when you have double-sided tape on the slides and thus avoiding refocusing the microscope while you are dissecting and transferring the embryos between tape and coverslip. B. A slide with a short length of double-sided Scotch tape applied before peeling off its cover paper used for dechorionating the embryo. C. The picture on the left shows embryos with intact chorion shells with marked anterior and posterior ends; the picture on the right shows the embryos in the broken chorion shells before they were transferred onto the coverslip with the glue strip. D & E. Diagrams showing two ways for transferring, placing and aligning the dechorionated embryos on glue strips. The embryos were covered by 10S oil after an appropriate desiccation period. F & G. Diagrams showing how to open the tip of the needle and position it so as to inject.
Single or time-lapse images obtained from the above described experiments can be directly analyzed using Leica software or saved in .TIF files as an open format available for further quantification or editing analyses using other common image analysis programs such as Image J, Photoshop and MetaMorph etc. Two examples to illustrate this are discussed below:
Example 1: A time-lapse movie (Figure 3) shows the dynamic kinetochore recruitment of GFP-Cdc20 and chromosome motion in living Drosophila syncytial embryos. The region of interest from original time-lapse sequence images was determined/edited and automated-batch converted using Photoshop software. The movie was assembled using QuickTime software.
Figure 3. A time-lapse movie shows the dynamic kinetochore recruitment of GFP-Cdc20 and chromosome motion in living Drosophila syncytial embryos. (A) Time-lapse images were taken from a transgenic syncytial embryo co-expressing GFP-Cdc20 (in green) and RFP-Histone 2B (in red) fusion proteins and were recorded using a Leica TCS SP2 confocal system at 18 °C during nuclear division cycles 7-8. Frames were taken every 10 seconds. The frame with the cells already in prophase is treated as the zero time point10. Click here to view movie for Figure 3A. (B) GFP-Cdc20 can be readily observed on prophase and prometaphase kinetochores (B3 & 4, white arrows) and persists on metaphase and anaphase kinetochores (B5 & 6, white arrows). GFP-Cdc20 is excluded from interphase nucleus (White arrowhead), entering the nucleus by early prophase. Chromatin morphologies were determined using co-expressed His2BmRFP as markers (B8-14). Bars= 5mm.
Example 2: SAC functions can be studied by manipulating the embryos by microinjecting antibodies, fluorescently labeled proteins or chemical compounds of interest that potentially trigger the SAC. For instance injecting colchicine to depolymerize the microtubules so provoking the SAC as indicated in Figure 410.
Figure 4. Mad2 is essential for colchicine-invoked SAC function10. gfp-cdc20; mad2+/+, gfp-cdc20; mad2EY (mad2-/- null mutant) and gfp-mad2; mad2EY embryos were treated with colchicine by microinjection. Time-lapse confocal images were taken before (01, 07 & 13) or after injection (02-06, top panels; 08-12, middle panels; 14-18, bottom panels). GFP-Cdc20 (top and middle panels) or GFP-Mad2 (bottom panel) kinetochore signals were used as cell cycle progression markers. Top panel, the arrows indicate the arrested kinetochores in 02-06. The area marked by an arrow in 01 indicates that before colchicine treatment, GFP-Cdc20 is excluded from the late interphase nucleus. Middle panel, in the absence of endogenous Mad2, GFP-Cdc20 signals continue to oscillate in and out of the nucleus, and on and off the kinetochores, although cytokinesis appears to be defective, as would be expected in embryos lacking microtubules, in the presence of colchicine (indicated by arrows in 07-12) suggesting a failed SAC function in arresting cells in response to colchicine treatment. Separation of daughter nucleus failed in picture 10. Bottom panel, arrowheads in 14-18 indicate arrested kinetochores with accumulated GFP-Mad2 fusion proteins to suggest the functional GFP-Mad2 in rescue the SAC defect phenotype in Mad2 mutant embryo. Arrowhead in 13 indicates GFP-Mad2 accumulation in a late interphase nucleus. Bar=5mm. The embryos were microinjected with ~1% egg volume of a 100mg/ml colchicine stock solution in 1 x PBS.
The protocol described here is a generic method for imaging fly syncytial embryos using a Leica TCS SP2 confocal laser scanning microscope and can be modified to suit other microscope systems and can also be adapted to study other gene functions using Drosophila syncytial embryos. We have used this protocol to study many aspects of the spindle assembly checkpoint, protein dynamics and protein proteolysis using transgenic flies or polyclonal antibodies to visualize the protein localisation in living or fixed samples4,6,12-14 .
It is simplest to keep the flies in fresh fly food vials when collecting small numbers (~10 – 100) of eggs. This reduces the hassle of making and preparing additional apple juice agar plates and egg collection chambers as described in other publications15,16. The addition of a small square of broken coverslip glass at one end of the glue strip on the coverslip makes it a lot easier to open the needle and determine the injection volume by adjusting the microinjection system settings while the needle is immersing in the 10S oil. The degree of desiccation of the embryo is important for the success of the injection in avoiding leakages and maintaining the embryos viability especially for those experiments which require a long period of time or when raising transformants after injection. However, there is no golden rule for exactly how long desiccation is required. This varies from experiment to experiment and depends on the amount of the solution injected and the humidity of the working environment.
The functions of a specific protein of interest can be manipulated by microinjecting specific protein inhibitors, mono- or poly-clonal antibodies against a specific protein, messenger RNA or fluorescently labeled peptides under wild type or genetic mutation backgrounds. Many of these mutant lines or GFP-tagged transgenic lines are publicly available from Drosophila stock centers and most of them are listed on Flybase (http://flybase.org/) and can be searched for online.
The authors have nothing to disclose.
This protocol was developed under a Wellcome Trust grant. We thank Ms. Maureen Sinclair for maintaining the fly stocks and preparing the fly food over the years. We would also like to thank Mr. Michael Aitchison for his help and technical support in developing of this protocol.
Essential equipment and reagents:
Confocal imaging system: The imaging system described in this protocol is a Leica TCS SP2 laser scanning confocal inverted microscope system. The method described is also suitable perhaps with some minor modifications for other imaging systems.
Dissecting microscope: We use an Olympus SZX7 with DF PLAPO 1X-4 lens.
Needle puller: Flaming/brown micropipette puller (from Sutter Instrument, Model: P-97).
Microinjection system: An Eppendorf microinjection system is described but any other appropriate system can be used.
Fly food distributor: Jencons Scientific Ltd peristaltic pump.
Reagents:
Ingredients | Weights | Ressourcen | Lot No. |
Maize meal | 100.0g | SUMA, UK | |
Brown sugar | 50.0g | Billington’s, UK | |
Dry yeast | 25.0g | DCL YEAST Ltd. UK | |
Agar | 12.5g | Fisher Scientific | 106556 |
Sorbic acid | 0.4g | BDH, VWR International Ltd. UK | 8829310 |
Benzoic acid | 2.9g | Fisher Scientific | 1019599 |
Nipagin (Methyl -4-Hydro xybenzoate) |
0.9g | BDH, VWR International Ltd. UK | K35969015 |
H2O up to 1L |
Table 1. Fly food ingredients.
Holocarbon 700 and 27 oils purchased from Sigma.
Dry yeast: Thomas Allison, UK.
Preparing the heptane glue: Take about 1.5 meters of double-sided Scotch tape, put it into a 15ml Falcon tube with 5ml of heptane and rotate for 3-5 hours or overnight. After that time split into 4 equal aliquots in 1.5 ml eppendorf centrifuge tubes and spin for 5 min at a fast speed in a bench-top centrifuge to remove any debris. Store the heptane glue solution in a 10ml volumetric flask and keep for use. The glue strength will vary according to the concentration and the amounts of the glue used. Normally, the thinner glue produces lesser noisy background when scanned by confocal microscope.