Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg
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Schnerch, D., Follo, M., Felthaus, J., Engelhardt, M., Wäsch, R. Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations. J. Vis. Exp. (67), e4239, doi:10.3791/4239 (2012).
Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.
1. Seeding of U2OS-based Cyclin B-SNAP Reporter Cells (Clone 11 Cells 6) on Microscope Chamber Slides
For seeding of cells onto 8 well microscope chambers at a constant distribution across the entire surface of the microscope chamber, centrifuge 10,000 cells and resuspend in 350 μl of phenol red-free normal growth medium (supplemented with 10% fetal bovine serum, penicillin/streptomycin and sodium pyruvate). Transfer cell suspension to the microscope chamber (Figure 2).
Working with 8 well microscope chambers (maximum cell density in the center).
For seeding of cells at a higher density in the center of the microscope chamber, load the chamber with 300 μl of phenol red-free normal growth medium. Add 5,000 cells carefully to the center of the microscope chamber (Figure 2).
Working with 96 well special optics plates (constant distribution of cells).
For seeding of cells onto 96 well plates at a constant distribution across the entire surface of the well, centrifuge 5,000 cells and resuspend in 300 μl of phenol red-free normal growth medium. Transfer the cell suspension to a 96-well plate. Depending on the total number of cell-containing wells required, adjust the cell number and the total volume of the suspension medium (Figure 2).
Working with 96 well special optics plates (maximum cell density in the center).
For seeding of cells onto 96 well plates, carefully add 1,500 cells in 15 μl of phenol red-free normal growth medium in a small drop to the center of each well to achieve restriction of cell growth to the center of the well (Figure 2).
2. Staining of Reporter Cells with SNAP Substrate
3. Measurement of Fluorescence Intensity
4. Analysis of Proteolytic Profiles using Scan^R
5. Representative Results
Figure 5D and 5E depict cyclin B kinetics, represented by a TMR Star fluorescence intensity curve, of a cell that proceeds through a regular mitosis without signs of chromosomal misalignment (Figure 5E). Upon compaction of the cytoplasm following nuclear envelope breakdown (NEBD, as indicated by the red triangle), TMR Star fluorescence intensity shows an abrupt increase until the isomorphic window (brighter area in the diagram) is reached when the cell enters prometaphase 6. Fluorescence intensity remains at a stable level as long as the cell proceeds through prophase and metaphase and then starts to drop rapidly once all of the chromosomes have established a stable metaphase plate (Figure 5D and 5E). This drop precedes chromosome separation during anaphase (blue dot on the curve). In late mitosis the chromatin starts to decondense (blue bars) and the cell adopts interphase morphology while the fluorescence intensity curve approaches a plateau which is lower than the plateau before mitosis (Figure 5D and 5E).
|Autofocus settings||Histone H2-GFP (main object acquisition settings)||Cyclin B-SNAP labeled with TMR Star
|Acquisition cycle time Repetitions|
|Coarse autofocus +/-39 μm 24 Layers
+/-5.4 μm 14 Layers
|GFP filter set:
Exposure time: 100 msec
Light intensity: 25%
|TRITC filter set:
Exposure time: 150 msec
Light intensity: 33.3%
|2 to 5 min.|
|GFP filter set:
Exposure time: 12 msec
Light intensity: 12.5%
|Up to 48 hr of continued analysis (limited by reduced air humidity of 60%)|
Table 1. Standard settings as used for analysis of cyclin B proteolysis. Histone H2-GFP was used as a reference structure to define the focus plane for measurement of cyclin B-SNAP fluorescence intensity. Light intensities during the focusing procedure are lower as compared to image acquisition settings to avoid cumulative phototoxicity.
Figure 1. Schematic of the measurement of cyclin B proteolysis through expression of a chimeric cyclin B derivative with degradation characteristics similar to the endogenous protein. The decline in fluorescence intensity after pulse-chase labeling is a measure of APC/C-activity.
Figure 2. Analysis of cells on 8 well slides or 96 well plates. Regular distribution of the cells across the surface of the well is achieved by resuspension in a final volume of 300 μl and is recommended, if only a single or few positions are manually defined for analysis. Enrichment of cells in the center region is achieved by addition of cells to the center of prefilled or empty wells. This technique is recommended in case of automated image acquisition in different wells.
Figure 3. Sequence of data acquisition. A) Detection of histone H2-GFP is used for focus plane definition and monitoring of chromosomal alignment during mitosis. B) Fluorescence intensity of the TMR Star fluorescence intensity is a measure for the absolute amount of pulse-chase labeled cyclin B-SNAP.
Figure 4. Representative Scan^R Analysis software-based depiction of mean TMR Star fluorescence intensities (total TMRstar intensity divided by area) over time. Selection of a representative cell trace (blue). The corresponding images (shown on the right) allow the simultaneous visualization of histone H2-GFP (green) and cyclin B-SNAP (red). Click here to view larger figure.
Figure 5. A+B) Example for gating of dots representing a cell of interest on the XY-dot plot. C) Visualization of the mean TMRstar fluorescence intensity (total TMRstar intensity divided by area) over time using the dot plot. D+E) Representative fluorescence intensity curve of cyclin B-SNAP with isomorphic window (brigher field on the diagram) between NEBD (nuclear envelope breakdown) and chromatin decondensation (blue bar) as generated in Microsoft Excel. Anaphase is indicated by the blue dot on the curve. Click here to view larger figure.
Movie 1. Click here to view supplemental movie.
We present here a live-cell imaging-based approach facilitating the simultaneous monitoring of cyclin B proteolysis and chromosome alignment. This approach allows the study of mitotic control in unperturbed cell populations at the single cell level. Cyclin B degradation curves facilitate direct insights into the activity of the APC/C and thus indirectly reflect the status of the SAC 6.
This approach, even though established based on the Scan^R software, can easily be reproduced on any comparable microscope station. Moreover, using our retroviral cyclin B-SNAP expression construct, a reporter cell line can easily be established by stable retroviral integration into any desired cell line. To make the system even more flexible, the chimeric SNAP proteins can be stained with various fluorochromes.
The implementation of histone H2-GFP, besides its requirement in monitoring chromosome alignment, facilitates fast and easy autofocusing. Autofocus and intensity settings also depend on the kind of cell and have to be established for each cell line. The acquisition of proteolytic profiles of large numbers of cell divisions using Scan^R technology furthermore allowed us to select for single mitoses, which showed chromosome alignment errors. This enabled us to define differences between the proteolytic profiles of aberrant and regular divisions 6.
Mitoses displaying chromosomal misalignment can also be selected by analysis of raw image stacks, although this is less time-efficient. Cyclin B-SNAP fluorescence intensities can be quantified using freeware programs for image analysis such as ImageJ. Fluorescence intensity curves can be calculated based on the mean pixel intensity over time inside an area/gate defining the mitotic cell (technique described by Wolthuis et al. 11). Within an isovolumetric window, which has been described previously 6, the shape and the volume of the cell remain nearly constant. This provides a rationale for using the same area/gate between nuclear envelope breakdown until early anaphase for measuring mean pixel intensities to manually estimate cyclin B proteolysis during prometaphase, metaphase and early anaphase.
Using the Scan^R-based approach, further mathematical analyses of degradation curves using specific software, such as Microsoft Excel, requires gating at the single cell level. Innovative gating strategies to allow automated data export of all analyzed cells might facilitate a more time-efficient analysis of whole cell populations. A fully automated approach covering all cell traces might lead to more representative results, further enhance precision of this technique and hence facilitate deeper insights into the regulation of mitotic exit.
Fast and efficient automated image acquisition enables our model to be used in large-scale screening analyses. Here, screens using shRNA libraries may lead to the identification of novel mitotic regulators. Moreover, the system should be useful in screening small molecules with regard to their potency in interfering with APC/C-dependent proteolysis in order to identify novel drugs for antimitotic therapy.
Cyclin B kinetics have been widely addressed by monitoring cyclin B-GFP fluorescence intensities. Here, we present a system that facilitates the delineation of cyclin B proteolysis from whole protein expression and therefore constitutes a valuable amendment to widely distributed techniques in the mitosis field6.
No conflicts of interest declared.
We are grateful to S. Taylor for providing the pLPCX-Histone H2-GFP plasmid. We thank R. Mertelsmann for continuous support. This work was supported by the Deutsche Forschungsgemeinschaft.
|Reporter cell line||generated in-house as described 6||clone 11 reporter cells
(U2Os-based cyclin B-SNAP expressing cells)
|Retroviral cyclin B-SNAP expression vector||generated in-house as described 6||pLNCX2-cyclin B mut5-SNAP|
|Phenolred-free DMEM||Gibco||21063-029||Supplementation with FCS, sodium pyruvate, penicillin/strepto-mycin required|
|SNAP-Cell TMR-Star||New England Biolabs||S9105S||Stock solution 400 μM in DMSO|
|Special Opstics Plate, 96 well||Costar||3720|
|μ-Slide 8 well, ibiTreat||Ibidi||80826|
|Microscopy unit||Olympus||IX-81 inverse microscope with climate chamber|
|Objective||Olympus||UPLSAPO 20x objective (N.A. 0.75)|
|Acquisition software||Olympus||Scan^R Acquisition software (v.2.2.09)|
|Analysis software||Olympus||Scan^R Analysis software (v.188.8.131.52)|