Tracking Individual Chromosomes with Integrated Arrays of lacop Sites and GFP-laci Repressor: Analyzing Position and Dynamics of Chromosomal Loci in Saccharomyces cerevisiae
The visualisation of specific DNA sequences in living cells, achieved through the integration of lac operator arrays (lacop) and expression of a GFP-lac repressor fusion, has provided new tools to examine how the nucleus is organised and how basic events such as sister chromatid separation occur (Straight et al., 1996; Belmont, 2001). In contrast to other methods, such as fluorescence in situ hybridisation, the lacop GFP-lac repressor (GFP-laci) technique is noninvasive and therefore interferes minimally with nuclear structure and function. In addition, it facilitates analysis of the rapid dynamics of specific DNA loci (Gasser, 2002). Although this technique has been adapted to organisms from bacteria to humans, the ease with which GFP fusions can be targeted to specific chromosomal sites depends on the ability of the organism to carry out homologous recombination. This process is very efficient in budding yeast, allowing pairs of chromosomal loci to be analysed at the same time through the use of two bacterial repressors (laci and tetR) fused to different GFP variants. Given the relatively advanced state of the art in budding yeast, this article presents protocols optimised for this organism. These provide a starting point for adapting multilocus tagging to other species. Moreover, the techniques described here for the quantitative analyses of locus dynamics are universally applicable.
II. MATERIALS AND INSTRUMENTATION
Yeast minimal and rich media (SD, YPD) are described in Guthrie et al. (1991). Cells can be mounted on a depression slide (Milian SA, Cat. No. CAV-1, Fig. 2A) upon 1.4% agarose (Eurobio Cat. No. 018645) containing SD medium with 4% glucose (Fluka). Aliquots of this can be kept at 4 °C for several months. Alternatively, cells can be immobilised on a 18-mm coverslip treated with concanavalin A (Con A, Sigma, Cat. No. C-0412) in a cell observation chamber (Ludin chamber, Life Imaging Services, Fig. 2B). Con A dissolved to 1mg/ml in H2O is stable at -20°C for months. Widefield microscopy is performed on a Metamorph-driven Olympus IX 70 inverted microscope with Olympus Planapo 60×/NA = 1.4 or Zeiss Planapo 100×/NA = 1.4 objectives on a piezoelectric translator (PIFOC; Physik Instrumente), illuminating with a PolychromeII monochromator (T.I.L.L. Photonics). Also needed is a CoolSNAP-HQ digital camera (Roper Scientific) or equivalent, and both the FITC filter set for detecting GFP (Chroma, Ref. 41001) and the CFP/YFP filter set (e.g., Chroma, Ref. 51017). Confocal microscopy can be performed on a Zeiss LSM510 Axiovert 200M, equipped with a Zeiss Plan-Apochromat 100×/NA = 1.4 oil immersion or a Plan-Fluar 100×/NA = 1.45 oil immersion objective. The stage is equipped with a hyperfine motor HRZ 200. Temperature is stabilised using a temperature-regulated box surrounding the microscope (The Box, Life Imaging Services). Software used for analysis is (a) Excel (Microsoft), (b) ImageJ public domain software (Rasband), (c) Imaris v 3.3 (Bitplane), (d) Mathematica 4.1 (Wolfram Research), and (e) Metamorph v 4.6r6 (Universal Imaging Corp.).
1. Plasmids and Strains
Yeast transformation and growth are as described (Guthrie et al., 1991). The lacop/GFP-laci system for site recognition exploits the high affinity and specificity of the bacterial lac repressor for its recognition sequence (lacop All procedures are performed analogously for the tetR/tetop system (Michaelis et al., 1997).
2. Growth and Cell Preparation
3. Temperature Control
In order to have a stable condition for microscopic observation, the temperature of the microscope and room should be controlled carefully (±2°C). Two mechanisms are used standardly. The first is to enclose the entire imaging part of the microscope in a commercially available temperature-regulated box (e.g., Life Imaging Services or Zeiss). A second, less precise method is to regulate the temperature of the slide through a heated stage.
B. Image Acquisition
The choice of imaging technique depends on the question being asked. To derive quantitative information on the position of a given locus relative to a fixed structure (e.g., the spindle pole body, nucleolus, or nuclear envelope), three-dimensional (3D) stacks and detection of different wavelengths may be necessary. An analysis of fine movement and chromatin dynamics, however, requires the rapid and extended capture of one or more fluorochromes. Bleaching of the signal is often a major limiting factor in time-lapse imaging. One should note that chromatin movement is very fast [movements >0.5µm in less than 10s (Heun et al., 2001a)], making it necessary to have rapid image acquisition with a minimal interval between sequential images. To optimise acquisition, parameters such as image resolution, the number of z frames, intervals between frames, light intensity, and exposure time can be varied. In all cases, it is of utmost importance to minimise and monitor laser- or light-induced damage to the organism during imaging, in part by comparing the time required for one division cycle in imaged and nonimaged cells.
Cell Cycle Determination
As position and mobility of a chromosomal locus can vary with stages of the cell cycle, it is crucial to determine precisely what stage each imaged cell is in. This is done by monitoring bud presence and bud size, as well as the shape and position of the nucleus, as visualised by the Nup49-GFP fusion and a transmission or phase image. Figure 3 summarises the morphologies that characterise each stage of the cell cycle.
2. Wide-Field Microscopy and Deconvolution
For the imaging of large fields of cells, best results are obtained with a wide-field microscope equipped with a PIFOC, Xenon light source, and monochromator that allows a broad and continuous range of incident light wavelengths, as well as rapid switching between these values. Images are acquired by a highspeed monochrome CCD camera run by a rapid imaging software, such as Metamorph. The limiting step is often the speed of signal transfer from the CCD chip to the RAM and/or hard disk of your computer.
Wide-field microscopy is well adapted to experiments in which a large number of cells (200-300) need to be scored, e.g., when determining the subnuclear position of a given locus relative to the nuclear envelope or another tagged locus or landmark (e.g., spindle pole body or nucleolus). The reference point should optimally be tagged with a different fluorescent protein. If two loci bind the same fluorescent fusion proteins, then their intensities should be significantly different. Rapid through-focus stacks of images using the full chip capacity of the camera are taken of cells growing on agar or in a Ludin chamber (such that 20-30 individual cells are resolved per field). Optimal parameters for GFP are as follows: exposure time, 100-200ms; z spacing of 200nm for 18 focal planes, excitation wavelength 475 nm. For dual-wavelength capture, images of both wavelengths (CFP: 432nm, ~300ms; YFP: 514nm, ~150ms) must be acquired before the focal plane changes. A phase image is taken after every stack of fluorescence images. Wide-field images have out-offocus haze and deconvolution of the z stack is often necessary to reassign blurred intensities back to their original source. Use Metamorph software or other available deconvolution packages.
Three-Dimensional Time Lapse
The conditions for capturing 3D time-lapse series are as follows: 5-11 optical z slices taken every 1 to 4min, z sections are 200 to 400nm in depth, and the exposure time is ~50 ms. Using these settings, up to 300 stacks of five sections each (1500 frames) at 1-min intervals can be captured without affecting cell cycle progression. More rapid sampling with this system, however, leads to bleaching and potential cellular damage. Until this can be remedied by more rapid and more sensitive CCD cameras, wide-field microscopy is recommended for less rapid time-lapse imaging (intervals ≥60s) on larger fields and confocal microscopy (see later) for very rapid time-lapse imaging (intervals ≤2s) on small regions of interest (typically one yeast nucleus).
For very long imaging times (>1h), stray light should be suppressed by inserting an additional shutter. Deconvolution is performed using the Metamorph fast algorithm with five iterations, a sigma parameter of 0.7, and a frequency of 4.
3. Confocal Microscopy
To follow chromatin dynamics in individual cells with rapid time-lapse microscopy, the Zeiss LSM510 scanning confocal microscope is particularly well adapted, although the laser and acousto-optic tuneable filter (AOTF) system is limited in activation wavelengths. Its positive attributes are an ability to limit scanhead motion to a minimal region of interest (ROI), rapid and well-regulated scanning speeds, and the possibility to adjust pinhole aperture and laser intensities to very low levels, while maintaining maximal sensitivity.
To reduce the risk of damage by illumination, the laser transmission is kept as low as possible, and the cells are imaged as rapidly as possible within a minimal ROI. Useful settings for the Zeiss LSM510 are as follows.
Laser: argon/2 458, 488, or 514nm tube current 4,7 amp. Output 25%.
Filters: Channel 1: Lp 505 for GFP alone; channel 1 Lp 530, channel 3 Bp 470-500 for YFP/CFP single track acquisition.
Channel setting: Pinhole 1-1.2 airy unit (corresponding to optical slice of 700 to 900nm); detector gain: 930 to 999; amplifier gain: 1-1.5; amplifier offset: 0.2-0.1 V; laser transmission AOTF = 0.1-1% for GFP alone, 1-15% for YFP, and 10-50% for CFP in single track acquisition. In order to use minimal laser transmission the pinhole must be aligned regularly.
Scan setting: Speed 10 (0.88µs/pixel); 8 bits one scan direction; 4 average/mean/line; zoom 1.8 (pixel size: 100 x 100 nm)
Imaging intervals: 1.5 s
Note: If CFP and YFP signals are very weak, images can be acquired sequentially using the more sensitive LSM 510 channel 1 in multitrack mode. This allows the use of broader filters: long-pass filter Lp 475 for CFP and Lp 530 for YFP. Alternatively, and to avoid any cross talk, recover the YFP signal as before and use Bp 470-500 on channel 3 for CFP. These latter parameters will slow the imaging process.
Two- or Three-Dimensional Time Lapse
If maximal capture speed is desired, only one image per time point can be taken, as long as the GFP spot stays in the imaged plane of focus (called 2D time lapse). Often the plane of focus has to be changed manually to follow the spot. Image acquisition in 3D has two main advantages. (1) The GFP spot does not have to be followed manually as it is always present in one of the focal planes. A subsequent maximal projection along the z axis produces a complete 2D time sequence without loss of focus on the GFP spot. (2) After image reconstruction, one can visualise the nucleus and calculate distances in 3D. Such measurements are nonetheless compromised by the reduced optical resolution in z (≥0.5 µm for 488-nm light).
Specific 3D time-lapse settings are as follows: six to eight optical slices in z, 300- to 450-nm spacing in z with Hyperfine HRZ 200 motor using a ROI of 3 × 3 to 4 × 4 µm and time intervals of 1.5 s. A 12-min time-lapse series at 0.2% laser transmission did not influence cell cycle progression.
C. Image Analysis
1. z Stacks
Determination of the subnuclear position of a GFPtagged locus is monitored relative to the centre of the Nup49-GFP ring. Nuclei in which the tagged locus is at the very top or bottom of the nucleus are not scored because the pore signal no longer forms a ring but a surface and a peripheral spot will appear internal.
2. Three-Dimensional Time Lapse
A prerequisite for the precise description of chromatin movement is the knowledge of the coordinates of the locus and of the nuclear centre for each frame of a time-lapse movie. In collaboration with D. Sage and M. Unser (Swiss Federal Institute of Technology, Lausanne), a best-fit algorithm has been developed that reliably tracks a moving spot in 2D time-lapse movies or in maximal projections of z stacks in 3D time lapse using nuclei carrying Nup49-GFP or expressing tetR-GFP to detect the nucleoplasmic signal. This system is complete and dramatically improves reproducibility and the speed of analysis, while allowing user intervention at several stages. The algorithm has been implemented as a Java plug in for the public domain ImageJ software (Rasband; Sage et al., 2003). The spatiotemporal trajectory is exported as x,y coordinates for each time point in a spreadsheet. An implementation for 3D image stacks over time is also available (Sage et al, 2005). Automated image analysis requires three steps.
Characterisation of Movement
Because each time-lapse series represents a single cell, it is indispensable to average 8-10 movies over a total time >40min for a given strain or condition. Subtle differences require a larger data source. Useful parameters for quantitative analysis include the following.
It is very difficult to accurately quantify the intensity of a small, mobile GFP-laci focus. Even in deconvolved images it can differ by twofold in sequential images.
This protocol shows the optimal method for the described microscope setups. For different microscopes, the values and methods of this protocol are simply a starting point for further optimisation. As improvements in technology (e.g., more sensitive and rapid CCD cameras) and reagents (e.g., more stable or more intense GFP variants) evolve, future adjustments of this protocol will be indispensable.
The method described here can also be applied to Schizosaccharomyces pombe with a few changes, one being immobilisation on a coverslip with isolectin B (1 mg/ml) (Williams et al., 2002) or lectin from Bandeiraea simplicifolia (lyophilized powder, Sigma Cat. No. L2380).
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