Name: examination of mitotic and meiotic fission yeast nuclear dynamics by fluorescence live-cell microscopy
Uploaded: 2019-06-24T14:45:00
Duration: 12 min 4 s
Description: Watch this Scientific Journal Video about Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy at JoVE.com

The authors would like to thank Natalia La Fourcade and Mon LaFerte for making the preparation of this manuscript a more enjoyable endeavor. Thanks to members of the Forsburg Lab who contributed to the completion of this work with their insight, experiment ideas, and moral support. This project was supported by NIGMS R35 GM118109 to SLF.

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The authors have nothing to disclose.

Live-cell imaging during mitosis and meiosis offers the opportunity to examine nuclear dynamics without substantially disrupting fission yeast physiology during data acquisition. However, caution must be exercised to ensure cells grow to the desired densities free of environmental stresses; and for meiosis, that the cells are imaged during the time of terminal differentiation and not too early or late. Excess cellular crowding, starvation, and inappropriate growth temperatures are common factors that contribute to irreproducible observations. In addition, during strain construction, it is crucial to test that fluorescent tags do not interfere with the functions of target genes nor impact overall cell fitness. Failure to closely inspect the phenotypes of strains carrying fluorescent markers may result in inconsistent and incomparable results across similar genotypes.
Though microscope agarose pads are simple to assemble, they can also pose a hurdle in obtaining valuable imaging data. Creating agarose pads is as simple as placing three microscope slides parallel to each other, dispensing molten agarose on the middle slide, and putting a slide that transverses the top of the other slides. It is useful, however, to assemble a slide-maker apparatus that allows for width modifications to manufacture resistant, situation-specific agarose pads. A simple slide-maker that gives pad width flexibility consists of a platform (pipette tips holder or the bench), two microscope slides, and lab tape acting as the pad-width adjusting mechanism (Figure 1A). Exact pad thickness will depend on imaging time requirements, agarose brand, temperature, coverslip sealant, evaporation rate, etc. Thus, it is important to calibrate the imaging system before data acquisition to increase technical reproducibility and to enhance the quality of live-cell imaging1,2,3. Pad surface, rigidity, and composition are essential during prolonged image acquisition. Agarose has low background fluorescence, can be easily molded, and at the appropriate concentration, withstands structural deformation due to evaporation. Moreover, combining fission yeast media with agarose does not compromise image quality and allows for extended observation of multiple mitoses and meiosis cycles. Also, it is important to avoid any air bubbles for time-lapse microscopy. Inside agarose pads and within the sample, air pockets expand over time, causing cell-shifting and disrupting focal planes. Provided cells are efficiently spread apart by coverslip rotation, researchers can follow mitotic processes across several generations and meiotic events from nuclear fusion to sporulation. Making and choosing the right pad for imaging experiments takes time to master, but once achieved, can contribute to highly informative microscope observations.
As is the case with other methods, live-cell microscopy is not free of technical limitations. The use of fluorescently-tagged proteins introduces boundaries to experiments that limit the scope of what can be studied. Photo-bleaching and photo-toxicity are problems typical of prolonged image acquisition. They can be counteracted by not exposing cells to short wavelengths for too long or too frequently. For experiments involving GFP, CFP, YFP, mRFP, and DsRed, typical fluorescent proteins used in fission yeast live-cell imaging, it is common to employ 5-15% excitation light and 100-500 ms exposure time for routine experiments. These parameters change, however, according to the specific experiment requirements of the researcher. In addition, z-stacks comprised of 9-13 sections with 0.5 &#181;m spacing using a 60x objective is enough to resolve the thickness of a fission yeast cell. Moreover, it is important to confirm that fluorescent markers do not disrupt proper regulation or function of target proteins or generate spurious phenotypes. Thus, it is advisable to construct strains containing different fluorescent and affinity tags for the same target gene to corroborate observations by different means. Furthermore, it is the responsibility of researchers to ensure that experimental parameters can be reproduced across experiments and that the collected data is fit for downstream analysis1,3. No technology can replace the time-tested practice of meticulously validating experimental systems by careful observation and rigorous examination of linearity in detecting fluorescent signals.
Finally, it is crucial to analyze microscopy data in formats that do not modify raw images. Fiji provides excellent documentation tools to keep track of raw data measurements and allows researchers to examine different cell parameters in a single session. These features, as well as its wealth of online tutorials and extensive literature coverage, make Fiji an important tool for measuring, analyzing, and presenting live cell imaging data that describe the nuclear dynamics of fission yeast in mitosis and meiosis.

Live-cell microscopy allows researchers to examine nuclear dynamics without killing fission yeast cells and eliminates the need for lethal fixatives and stains. It is possible to observe the stability, movement, localization, and timing of fluorescently tagged proteins that are involved in important events such as chromosome replication, recombination, and segregation, in addition to the membrane and cytoskeletal movements. By maintaining cell viability and non-toxic signal detection, there is minimal physiological intrusion. When performed properly, this microscopy method can reduce confounding effects that arise from the extended technical handling or from spurious chemical reactivity. Furthermore, during an experiment, researchers can make pertinent observations of fission yeast nutritional and stress states, proliferative efficiency, and apoptotic status that indicate inappropriate imaging parameters or abnormal cell physiology1,2,3.
Mitosis and meiosis are characterized by nuclear and cellular division. In meiosis, contrary to mitosis, genetic content is halved as parent cells give rise to daughter cells4. Because live-cell imaging provides a temporal dimension in addition to the spatial relationship, large numbers of cells can be examined in real-time. Live-cell microscopy has enabled researchers to examine the dynamics of nuclear division using fluorescent tags for histones5, cohesin subunits6, microtubules7, centromeres8, kinetochores9, components of the spindle pole body (SPB)10, and those of the chromosome passenger complex (CPC)11. Outside of the chromosome segregation apparatus, live-cell imaging has also captured the behavior of proteins necessary, but not directly involved in chromosome segregation. The function of these proteins has been shown to influence replication, chromosome recombination, nuclear movement, and chromosome attachment to microtubules12,13,14. These observations have contributed to a better understanding of cellular events whose functional components were not amenable to biochemical or genetic examination. With new advances in microscopy technology, limitations such as poor resolution, photobleaching, phototoxicity, and focus stability will lessen, thereby facilitating better real-time observations of mitotic and meiotic nuclear dynamics1,2,3. Meiosis is particularly challenging as it is a terminal differentiation pathway that requires close attention to timing.
The goal of this protocol is to present a relatively simple method for examining real-time fission yeast nuclear dynamics in mitosis and meiosis. To accomplish this, it is necessary to use cells that have not been exposed to environmental stress, prepare agarose pads that can withstand prolonged imaging, and mount cells at densities that facilitate visualization of single-cell dynamics. Moreover, this protocol makes use of cells carrying fluorescently tagged proteins that serve as useful markers for nuclear kinetics (Hht1-mRFP or Hht1-GFP), chromosome segregation (Sad1-DsRed), cytoskeleton dynamics (Atb2-mRFP), transcriptional activation in G1/S (Tos4-GFP), and cohesion stability in meiosis (Rec8-GFP). This method also introduces additional nuclear and cytosolic markers (Table I) that can be used in different combinations to address questions about specific cellular processes. Furthermore, basic Fiji tools are featured to help researchers process live-cell images and analyze different types of data. The strength of this approach stems from the use of real-time observations of protein dynamics, timing, and stability to describe processes that are integral for the proper execution of mitosis and meiosis.

<table>
	<tbody>
		<tr>
			<td>60x Plan Apochromat objective lens</td>
			<td>Olympus</td>
			<td>AMEP4694</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A-8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>7558B</td>
			<td>IB4917-10 kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Belly Dancer rotator</td>
			<td>Stovall</td>
			<td>US Patent #4.702.610</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma</td>
			<td>B-4501</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma</td>
			<td>B-6768-5kg</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H20,</td>
			<td>Sigma</td>
			<td>C3306-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma</td>
			<td>C-0759</td>
			<td></td>
		</tr>
		<tr>
			<td>Convolve 3D software plug-in</td>
			<td>OptiNav, Inc.</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSnap HQ CCD camera</td>
			<td>Roper</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slip</td>
			<td>VWR</td>
			<td>16004-302</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4&#183;5H20,</td>
			<td>END</td>
			<td>CX-2185-1</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision deconvolution fluorescence microscope</td>
			<td>GE Healthcare/Applied Precision</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Diffraction PSF 3D software plug-in</td>
			<td>OptiNav, Inc.</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Edinburgh minimal medium (EMM) Notrogen</td>
			<td>Sunrise</td>
			<td>2023;1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl2&#183;6H2O</td>
			<td>END</td>
			<td>FX9259-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G-7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat plate</td>
			<td>Barnstead</td>
			<td>Thermo Lyne</td>
			<td></td>
		</tr>
		<tr>
			<td>Histidine</td>
			<td>Sigma</td>
			<td>H8125-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Huygens deconvolution software</td>
			<td>SVI</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris deconvolution software</td>
			<td>Bitplane</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Shell lab</td>
			<td>#3015</td>
			<td></td>
		</tr>
		<tr>
			<td>Inositol</td>
			<td>Sigma</td>
			<td>I-5125</td>
			<td></td>
		</tr>
		<tr>
			<td>Iterative Deconvolve 3D software plug-in</td>
			<td>OptiNav, Inc.</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Mallinckvadt</td>
			<td>6858-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Lanolin</td>
			<td>Sigma</td>
			<td>L7387-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>Sigma</td>
			<td>L8912-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>Sigma</td>
			<td>L5626-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Malt extract (ME)</td>
			<td>MP</td>
			<td>4103-032</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H20</td>
			<td>AMRESCO</td>
			<td>0288-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph</td>
			<td>Molecular Devices</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>VWR</td>
			<td>16004-422</td>
			<td></td>
		</tr>
		<tr>
			<td>MnSO4,</td>
			<td>Mallinckvadt</td>
			<td>6192-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Molybdic acid</td>
			<td>Sigma</td>
			<td>M-0878</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2S04</td>
			<td>Mallinckvadt</td>
			<td>8024-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinic acid,</td>
			<td>Sigma</td>
			<td>N-4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Pantothenic acid</td>
			<td>Sigma</td>
			<td>P-5161</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>Fisher</td>
			<td>s80119WX</td>
			<td></td>
		</tr>
		<tr>
			<td>Pombe glutamate medium (PMG)</td>
			<td>Sunrise</td>
			<td>2060-250</td>
			<td></td>
		</tr>
		<tr>
			<td>softWorx v3.3 image processing software</td>
			<td>GE Healthcare</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Sporulation Medium powder</td>
			<td>Sunrise</td>
			<td>1821-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controlled centrifuge</td>
			<td>Beckman</td>
			<td>Allwgra 6KR xentrifuge</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>AMRESCO</td>
			<td>0847-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td>Equaline</td>
			<td>F79658</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>EMD</td>
			<td>1.03753.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract plus supplements (YES)</td>
			<td>Sunrise</td>
			<td>2011-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4&#183;7H2O</td>
			<td>J.T.Baker</td>
			<td>4382-04</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of mitotic and meiotic fission yeast nuclear dynamics by fluorescence live-cell microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Watch this Scientific Journal Video about Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy at JoVE.com

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Research

JoVE Journal

Biology

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