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>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;0,</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>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;0,</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>FeCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</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>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;0</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>MnSO&lt;sub&gt;4&lt;/sub&gt;,</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>Na&lt;sub&gt;2&lt;/sub&gt;S0&lt;sub&gt;4&lt;/sub&gt;</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>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</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 ...

examination-mitotic-meiotic-fission-yeast-nuclear-dynamics

Nanyang Technological University

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JoVE Journal

Biology

9.5K Views.  University of Southern California. Live celmicroscopie stelt onderzoekers in staat om splijtingsgist nucleaire dynamiek in mitose en meiose in real time te bestuderen. De kracht van deze methode komt van het bestuderen van nucleaire processen in levende cellen onder normale fysiologische omstandigheden die de noodzaak van toxische fixatiemiddelen en vlekken elimineert. Deze techniek behandelt vragen met betrekking tot eiwittiming, stabiliteit en mobiliteit die mogelijk niet vatbaar zijn voor genetische of biomechanische manipu...

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

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Genetics

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...

Identifying Drugs that Affect Polymerization of Bacterial Cytoskeletal Proteins

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB ...

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

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