We thank the Light Microscopy Imaging Center at Indiana University. This work was supported by a grant from the National Institutes of Health (GM105755).

1. Preparation of necessary materials
<ol>
	<li>Prepare reagents for the growth and sporulation of yeast cells. 
	NOTE: If budding yeast strains are ade2- and trp1-, supplement all media in steps 1.1.1-1.1.3 with a final concentration of 0.01% adenine and 0.01% tryptophan from 1% stocks. If sterilizing media by autoclave, add these amino acids only after the reagents have been autoclaved and allowed to cool to room temperature.
	<ol>
		<li>For vegetative growth, prepare 2x synthetic complete + dextrose medium (2XSC) by dissolving 6.7 g of yeast nitrogen base without amino acids, 2 g of complete amino acid mix, and 20 g of dextrose in 500 mL of water. Sterilize the mixture by autoclaving for 20 min at 121 &#176;C or filtering through a 0.2 &#181;m filter.</li>
		<li>For the first step of sporulation, prepare 2x synthetic complete + acetate medium (2XSCA) by dissolving 6.7 g of yeast nitrogen base without amino acids, 2 g of complete amino acid mix, and 20 g of potassium acetate in 500 mL of water. Sterilize the mixture by autoclaving for 20 min at 121 &#176;C or filtering through a 0.2 &#181;m filter.</li>
		<li>For the final step of sporulation, prepare 1% potassium acetate (1% KAc) by dissolving 5 g of KAc in 500 mL of water. Sterilize the mixture by autoclaving for 20 min at 121 &#176;C or filtering through a 0.2 &#181;m filter.</li>
	</ol>
	</li>
	<li>Prepare drugs and reagents for microscopy, synchronization, and anchor away.
	<ol>
		<li>For adhering cells to the coverslip during time-lapse imaging, make 1 mg/mL of concanavalin A (ConA) in 1x PBS. Filter sterilize using a 0.2 &#181;m filter and store small (~10 &#956;L) aliquots at -20 &#176;C.</li>
		<li>To use the NDT80-in system, make 2 mL of 1 mM &#946;-estradiol dissolved in ethanol. Filter sterilize using a 0.2 &#181;m filter and store aliquots (~500 &#956;L) at -20 &#176;C.</li>
		<li>For the anchor away system, make 1 mg/mL of rapamycin dissolved in DMSO. Filter sterilize using a 0.2 &#181;m filter and store small (~10 &#956;L) aliquots at -20 &#176;C. 
		NOTE: Prepare small aliquots of rapamycin to avoid multiple freeze-thaw cycles of the drug.</li>
	</ol>
	</li>
	<li>Generate yeast strains containing the NDT80-in system (PGAL1,10-NDT80/PGAL1,10-NDT80; GAL4-ER/ GAL4-ER) and a target protein tagged with FRB in the anchor away genetic background (tor1-1/tor1-1; fpr1&#916;/ fpr1&#916;; Rpl13A-FKBP12/Rpl13A-FKBP12)4,8. Ctf19-FRB was used for this study. Additionally, one copy of histone protein Htb2 was tagged with mCherry to allow for monitoring of meiotic progression and chromatin segregation.</li>
	<li>Prepare a chamber for time-lapse imaging
	<ol>
		<li>Start preparing the chamber 6 h-24 h prior to imaging (Figure 2). Cut an 18 mm x 18 mm chamber from the pipette tip box insert using a heated scalpel or other sharp blade.</li>
		<li>Use a plastic pipette tip to spread a thin layer of silicone sealant around the bottom edges of the chamber that will adhere to the coverslip. Add enough sealant so that the edges of the chamber are completely covered (see Figure 2).</li>
		<li>Adhere the chamber to a 24 mm x 50 mm coverslip by gently placing the chamber, sealant-side-down, onto the coverslip. Make sure that there are no gaps in the sealant so that the chamber does not leak.</li>
		<li>Spread 8-10 &#181;L of ConA onto the coverslip. Dispense ConA onto the middle of the coverslip and use a pipette tip to spread the ConA into a thin layer such that it covers most of the coverslip that is surrounded by the chamber. 
		&#8203;NOTE: Plastic chambers can be reused indefinitely. After time-lapse imaging is completed, remove the chamber from the coverslip using a razor, clean the silicone sealant from the chamber, and keep the chambers submerged in 95% ethanol for future use.</li>
	</ol>
	</li>
</ol>
2. Sporulation of yeast cells
<ol>
	<li>Perform the following steps for starvation of yeast cells to induce the meiotic program. 
	NOTE: These steps are for the sporulation of the W303 strain of yeast. Other strains may require different protocols15. Sporulation efficiency is highly variable between lab strains, with W303 exhibiting a sporulation efficiency of ~60%16,17,18.
	<ol>
		<li>Take a single colony of the appropriate diploid yeast strain from a plate and inoculate 2 mL of 2XSC and let grow at 30 &#176;C on a roller drum for 12-24 h to saturation.</li>
		<li>Dilute the saturated culture into 2XSCA by adding 80 &#181;L of the culture from step 2.1.1 to 2 mL of 2XSCA. Let it grow at 30 &#176;C on a roller drum for 12-16 h. Do not leave in 2XSCA for longer than 16 h because cells can become sick or auto-fluorescent.</li>
		<li>Perform two washes by spinning down the culture at 800 x g at room temperature (25 &#176;C) for 1 min, discarding the liquid, and resuspending the pellet in 2 mL of sterile distilled water.</li>
		<li>After the second wash, remove the liquid and resuspend the pellet in 2 mL of 1% KAc and let it grow at 25 &#176;C on a roller drum for 8-12 h. Cells that have entered meiosis will arrest in pachytene of prophase I.</li>
	</ol>
	</li>
	<li>Prophase I release system
	<ol>
		<li>Add &#946;-estradiol directly to the sporulation culture to a final concentration of 1 &#956;M and vortex the culture tube quickly. The &#946;-estradiol will release cells from prophase I.</li>
	</ol>
	</li>
</ol>
3. Depletion of target protein from the nucleus using the anchor away technique
<ol>
	<li>Add rapamycin to the cells to deplete the FRB-tagged protein from the nucleus at a particular stage.
	<ol>
		<li>To deplete proteins from the nucleus at prophase I exit, add rapamycin to a final concentration of 1 &#956;g/mL to the sporulation culture tube at the same time as &#946;-estradiol addition.</li>
		<li>To deplete proteins from the nucleus at a particular stage of meiosis, monitor the cell cycle stage starting at 60 min after &#946;-estradiol addition. Every 20 min, pipette 5 &#181;L of culture onto a 24 mm x 40 mm coverslip and cover the cells with an 18 mm x 18 mm coverslip. Keep cultures spinning on the roller drum at 25 &#176;C between time points.</li>
		<li>Image the cells using the A594/mCherry filter and the 60x objective of a fluorescence microscope. Set the percent transmittance to 2% and the exposure time to 250 ms. The presence of one, two, or four DNA masses will indicate the meiotic stage at which the cells have progressed. Add rapamycin to a final concentration of 1 &#956;g/mL at the meiotic stage of interest.</li>
	</ol>
	</li>
	<li>Keep cells spinning on the roller drum at 25 &#176;C until they are ready for imaging. Nuclear depletion of a target protein occurs 30-45 min after rapamycin addition2,8.</li>
</ol>
4. Time-lapse fluorescence microscopy
<ol>
	<li>Make an agar pad that will be used to create a monolayer of cells for imaging (see step 4.2).
	<ol>
		<li>Cut off and discard the cap and bottom 1/3 of a 1.5 mL microfuge tube to create a cylinder. The cylinder will serve as a mold for the agar pad. Make two cylinders to have an extra in case the agar does not polymerize properly in the first one.</li>
		<li>Place the cut microfuge tube cylinder on a clean glass slide with the top of the tube upside down sitting on the slide.</li>
		<li>Make 6 mL of a 5% agar solution (use 1% KAc as solvent) in a 50 mL beaker and microwave it until the agar is fully dissolved. 
		NOTE: The agar will easily boil over in the microwave; watch the agar and start and stop the microwave when the agar starts to boil and swirl the beaker. Start and stop the microwave several times to fully dissolve the agar. The agar solution is made in excess of the amount needed to ensure that the agar dissolves fully.</li>
		<li>Cut off the tip of the pipet to make a larger opening and pipet ~500 &#181;L of melted agar into each microfuge tube cylinder. Let it sit at room temperature until the agar solidifies (~10-12 min).</li>
	</ol>
	</li>
	<li>Preparing yeast cells for imaging
	<ol>
		<li>Spin down 200 &#181;L of the sporulation culture from step 3.2 at 800 x g for 2 min in 1 mL microcentrifuge tubes. Remove and discard 180 &#181;L of the supernatant. Resuspend the pellet in the remaining supernatant by swirling and flicking the tube.</li>
		<li>Pipet 6 &#181;L of the concentrated cells onto the coverslip in the middle of the chamber made in step 1.4.</li>
		<li>Hold the cylinder with the agar pad made in step 4.1 and carefully slide it off the glass slide. Make sure the agar is completely flat at the bottom and using the bottom of a pipette tip, apply a slight amount of pressure to the microfuge mold such that the agar pad is pushed out slightly above the boundary of the tube.</li>
		<li>Invert the mold such that the agar pad is facing down toward the chamber.</li>
		<li>Using forceps, gently place the agar pad (still in the microfuge tube mold) on top of the cells. Use a pipette tip to gently slide the agar pad around the chamber 10-20 times to create a monolayer of cells on the coverslip.</li>
		<li>Leave the agar pad in the chamber for 12-15 min. This step will allow the cells to adhere to the ConA on the coverslip.</li>
		<li>Transfer 2 mL of sporulation culture from step 3.2 to two microcentrifuge tubes and spin at 15,700 x g for 2 min. Transfer the supernatant into clean microfuge tubes and spin again at 15,700 x g for 2 min. Transfer the supernatant to clean microcentrifuge tubes to be used in the next step. 
		NOTE: It is important to use the pre-conditioned KAc supernatant with the &#946;-estradiol and rapamycin to ensure efficient sporulation and continued depletion of the protein of interest.</li>
		<li>After the agar pad has sat in the chamber for 12-15 min, float and remove the agar pad before imaging. To do so, add 2 mL of the supernatant from step 4.2.7 dropwise to the chamber. Once the liquid has reached the top of the chamber, the agar pad will most likely float. 
		NOTE: If the agar pad does not float automatically, wait 1-2 min. If the agar pad still does not float, remove it gently with forceps. Ideally, the agar pad would float on its own because removing it prior to floating could lead to removal of the cells.</li>
		<li>After the agar pad has floated, remove it gently with forceps and discard it. Place a 24 mm x 50 mm coverslip on the top of the chamber to prevent evaporation during imaging.</li>
	</ol>
	</li>
	<li>Setting up a movie on the microscope 
	NOTE: The instructions below are for an inverted microscope fitted with a slide holder that accommodates the 24 mm x 50 mm coverslip (See Table of Materials for microscope, camera, and software details). A 60x oil-immersion objective is used for image acquisition. This protocol may need to be altered when using other microscopes or other slide holders. The exact steps for executing step 4.3.2 through step 4.3.16 will vary depending on the microscope and imaging software used. See section 4.4 for instructions for a different microscope.
	<ol>
		<li>Fit the coverslip inside the slide holder. Adhere the molding clay to the side of the coverslip to keep it secure in the slide holder.</li>
		<li>Open the image acquisition software. Use course and fine adjustment knobs to focus the cells using DIC or brightfield.</li>
		<li>On the main menu of the image acquisition software, click on File &#62; Acquire (Resolve 3D). Three windows will pop-up.</li>
		<li>In the window named Resolve 3D, click on the Erlenmeyer Flask icon. This will open a window titled Design/Run Experiment, which contains the controls for setting up an experiment to set up a time-lapse movie.</li>
		<li>Under the Design tab, navigate to the tab labeled Sectioning. Select the box next to Z Sectioning. Set the z stacks as follows: Optical section spacing = 1 &#181;m, Number of optical sections = 5, Sample thickness = 5.0 &#181;m.</li>
		<li>Under the Channels tab, click on the + icon such that one channel option appears. Select the appropriate channel. For this experiment, we select A594, which is used to image mCherry. Select the box next to Reference Image and set the Z position to the middle of the sample from the drop-down menu.</li>
		<li>Select a value from the dropdown menu adjacent to %T and Exp. to set percent transmittance and exposure time, respectively. For this experiment, 2% transmittance and 250 ms exposure time are used in the A594 channel, and 10% transmittance and 500 ms exposure time are used for brightfield. 
		NOTE: The exposure time and percent transmittance will vary with different microscopes. To avoid overexposure, use the lowest percent transmittance and shortest exposure time possible that allows for proper visualization of the protein of interest.</li>
		<li>Under the Time-lapse tab, select the box next to Time-lapse. In the table that appears, enter 10 under the min column in the time-lapse row and 10 under the hours column in the total time row. This will run a time course taking images every 10 min for 10 h.</li>
		<li>Select the box next to Maintain Focus with Ultimate Focus to prevent stage drift during the movie. Under the Points tab, select the box next to Visit Point List.</li>
		<li>In the main menu, click on View &#62; Point List. A window called point list will pop up. Move the stage to an area of the chamber that shows a monolayer of cells. Click on Point Mark in the point list window.</li>
		<li>Move the stage to select 25-30 points without any overlap to avoid overexposing the cells. Each field will be imaged during each time course.</li>
		<li>In the point list window, select Calibrate All to set ultimate focus for each point. Under the Design tab in the Desgin/Run Experiment window, enter the range of point values (obtained from the point list window) in the box next to Visit Point List. Under the Run tab, save the file to the appropriate destination on the computer. On the Design/Run Experiment window, select the Play button (green triangle icon) to start the movie.</li>
	</ol>
	</li>
	<li>Optional method: Setting up a movie on a microscope 
	NOTE: These instructions are for an inverted microscope fitted with a slide holder that can accommodate a 24 mm x 50 mm coverslip. See Table of Materials for specifications about the microscope, camera, and imaging software used. A 60x oil-immersion objective is used for image acquisition.
	<ol>
		<li>Fit the coverslip inside the slide holder. Open the image acquisition software. Use course and fine adjustment knobs to focus cells using DIC or brightfield. Right click in the open window of the software. A drop-down menu will appear.</li>
		<li>Click on Acquisition Controls &#62; Acquisition. Click on Application &#62; Define/Run Experiment. This will open a window labeled ND Acquisition.</li>
		<li>In the window that opens, check the box next to XY. Move the stage to the desired location and click the box under Point Name to select that location as a point. Repeat this until 25-30 points are selected without any overlap to avoid overexposing the cells. Each field will be imaged during each time course.</li>
		<li>Set percent transmittance for each fluorescent channel. Because the strains produce Htb2-mCherry, the mCherry filter is used here. Under the Acquisition window that opened in step 4.3.3, navigate to the 555 nm button. Set the percent transmittance to 5% by adjusting the sliding scale under the 555 nm button until it reads 5%.</li>
		<li>Set the exposure time for each fluorescent channel. Under the Acquisition window, select 200 ms from the Exposure drop-down menu. 
		NOTE: The exposure time and percent transmittance will vary with different microscopes. To avoid overexposure, use the lowest percent transmittance and shortest exposure time possible that allows for proper visualization of the protein of interest.</li>
		<li>Click on the box next to Z in the ND Acquisition window. Set five z-stacks of the yeast cells that are 1.2 &#181;M apart.</li>
		<li>Click on the box next to &#955;. Select the appropriate channels to be used for the experiment. For DIC, ensure that Home is selected from the drop-down menu under Z pos. For mCherry, ensure that All is selected from the drop-down menu under Z pos. This ensures that, only a single section (in the middle of the z-stack) is taken for DIC.</li>
		<li>Select the box next to Time in the ND Acquisition window. Select the box under Phase. In the drop-down menu under Interval, select 10 min. Under Duration, select 10&#160;hour (s). This will run time course such that images are taken every 10 min for 10 h.</li>
	</ol>
	</li>
</ol>
5. Analysis of chromatin segregation
<ol>
	<li>Open the Fiji software. Open one field of view at a time: for each field, open the DIC and mCherry channels.</li>
	<li>Take the maximum intensity projection of the mCherry channel to obtain a single image: click on Image &#62; Stacks &#62; Z Project, select Max Intensity from the dropdown menu.</li>
	<li>Merge the DIC and mCherry channels in one image: click on Image &#62; Color &#62; Merge Channels.</li>
	<li>Follow a single cell through meiosis. After meiosis II completion, record the number of DNA masses.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+in+the+budding+yeast+Saccharomyces+cerevisiae.">Sporulation in the budding yeast Saccharomyces cerevisiae.</a> Genetics. 189 (3), 737-765 (2011).</li><li>Robinett, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+al.+In+vivo+localization+of+DNA+sequences+and+visualization+of+large-scale+chromatin+organization+using+lac+operator/repressor+recognition.">C. al. In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.</a> Journal of Cell Biology. 135, 1685-1700 (1996).</li><li>Straight, A. F., Belmont, A. S., Robinett, C. C., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+tagging+of+budding+yeast+chromosomes+reveals+that+protein-protein+interactions+can+mediate+sister+chromatid+cohesion.">GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion.</a> Current Biology. 6 (12), 1599-1608 (1996).</li><li>Sym, M., Engebrecht, J. A., Roeder, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZIP1+is+a+synaptonemal+complex+protein+required+for+meiotic+chromosome+synapsis.">ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis.</a> Cell. 72 (3), 365-378 (1993).</li><li>Scherthan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+mobility+during+meiotic+prophase+in+Saccharomyces+cerevisiae.">Chromosome mobility during meiotic prophase in Saccharomyces cerevisiae.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (43), 16934-16939 (2007).</li></ol>

The authors declare no competing financial interests.

This protocol combines the NDT80-in system to synchronize cells, the anchor away technique to deplete proteins from the nucleus, and fluorescence time-lapse microscopy to image budding yeast cells during meiosis. The NDT80-in system is a method for meiotic cell cycle synchronization that utilizes a prophase I arrest and release4,8. Although individual cells will vary slightly in the amount of time spent in each of the subsequent meiotic stages, most cells will maintain a high degree of synchrony throughout the meiotic divisions.
The anchor away technique is an adaptable tool for mitotic and meiotic studies. By altering the anchor away target protein and the time of rapamycin addition, this method can be adapted to study various proteins during any stage of budding yeast meiosis. Common methods for making conditional mutants to study budding yeast mitosis are not always suitable for meiotic studies. For example, use of a repressible promoter often requires a change in carbon source to alter the expression of the target protein, which may disrupt meiosis20. Temperature-sensitive mutants rely on a change in temperature to reduce or abolish the activity of the target protein. Many of these conditional mutants cannot be used in meiotic studies because shifting nutrient conditions or temperature can disrupt meiosis21,22,23. Furthermore, deletions or mutants may limit studies of meiosis because expressing a mutant protein early in meiosis might block or adversely affect later stages. The anchor away technique can circumvent these challenges because it does not rely on changes in nutrition or temperature8. Moreover, depletion of the protein can be temporally regulated such that the time of rapamycin addition determines when the protein will be anchored away.
One limitation of the anchor away technique is that it may not be suitable for non-nuclear proteins because it relies on the Rpl13A&#160;ribosomal subunit as an anchor to transport the target protein out of the nucleus. However, there are other applications of the anchor away technique by changing the protein-protein interactions that may also be useful during meiosis, such as anchoring proteins to complexes or tethering them to membranes8. Previous studies show that nuclear depletion of target proteins occurs within 30 min of rapamycin addition2,8. However, it is possible that some proteins need a longer or shorter period to be shuttled out of the nucleus. To ensure that the target protein is being shuttled out of the nucleus, the target protein can be tagged with FRB fused to GFP (FRB-GFP) to monitor the length of time between rapamycin addition and nuclear depletion. Another limitation of the anchor away system is that some proteins are sensitive to the FRB and FRB-GFP tags at the C-terminus. As such, N-terminal tagging of the target protein with FRB is an alternative strategy for anchoring away a target protein.
To monitor chromatin segregation, budding yeast cells expressed Htb2-mCherry. In this strain, the Htb2 protein is tagged at its endogenous locus, which ensures that Htb2 is expressed normally. Other fluorescently tagged proteins could also be used to monitor different aspects of meiosis. When constructing a strain for time-lapse imaging, it is important to consider that some proteins are sensitive to C-terminal fluorescent tags. Growth and sporulation assays of the strain harboring the fluorescently tagged protein ensures that the tag is not altering the function of the protein. One challenge of time-lapse fluorescence microscopy is exposing cells to enough fluorescence so that the fluorescent proteins are detected while avoiding cell death or slowing of meiosis from excess exposure. An important troubleshooting step is to find the appropriate microscope and camera settings to achieve this balance. The required exposure time will vary slightly between proteins, so multiple iterations of an experiment should be performed to determine the appropriate exposure time. When using the chamber method described here for time-lapse imaging experiments, an especially sensitive step is achieving a monolayer of cells to be imaged. Without a monolayer, cells accumulate on top of one another, which presents a challenge for proper imaging and data analysis. Using an agar pad, which helps cells adhere to the ConA and spreads cells around the chamber, is an inexpensive and efficient way to obtain monolayers. Moving the agar pad to spread cells around the chamber such that a monolayer is achieved can be challenging. Ensuring that the agar pad is making very small movements during the moving process can assist in creating a monolayer. Making a reusable chamber allows for the inexpensive generation of time-lapse imaging data. With proper cleaning and disinfecting, the plastic chambers described here can be reused indefinitely. It is best practice that chambers are removed from the coverslip immediately following time-lapse imaging and stored in 95% ethanol until the next use.
Time-lapse imaging provides information about the cell cycle that can be missed from imaging fixed cells1,2. For example, when monitoring the duration of the meiotic stages, variations between single cells can be more accurately examined when imaging live cells compared to populations of fixed cells. Because yeast proteins can be easily tagged with fluorescent proteins, coupling fluorescence time-lapse imaging with the NDT80-in system and the anchor away technique can be adapted for additional studies, such as monitoring individual chromosome segregation and the timing of specific meiotic stages. When monitoring cell cycle progression following prophase I release in NDT80-in cells, it is crucial to have a protein tagged with a fluorescent molecule that will mark the stages of meiosis. Individual chromosomes can be monitored throughout meiosis by integrating repeats of the lac operon (LacO) near the centromere of a chromosome in cells expressing LacI-GFP. LacI-GFP binds tightly to LacO and is visualized as a bright focus, which can be followed throughout the meiotic divisions by time-lapse microscopy24,25. Various proteins with fluorescent tags have been used to monitor the duration of meiotic stages. These include Tub1, the &#945;-tubulin subunit that incorporates into microtubules; Zip1, a component of the synaptonemal complex; and Spc42, a spindle pole body component1,2,26,27.
In conclusion, this method incorporates meiotic cell cycle synchronization, conditional depletion of a target protein, and time-lapse fluorescence microscopy to study meiotic chromosome segregation. By imaging budding yeast cells during meiosis and using conditional mutants that affect only the intended stages of meiosis, this method can provide accurate data about the meiotic cell cycle.

Time-lapse fluorescence microcopy is a valuable tool for studying the dynamics of meiotic chromosome segregation in budding yeast1,2. Budding yeast cells can be induced to undergo meiosis through starvation of key nutrients3. During meiosis, cells undergo one round of chromosome segregation followed by two divisions to create four meiotic products that are packaged into spores (Figure 1). Individual cells can be visualized throughout each stage of meiosis, which generates spatial and temporal data that can be easily missed by fixed-cell imaging. This protocol shows how combining time-lapse fluorescence microscopy with two previously established methods, the inducible NDT80 system (NDT80-in) and the anchor away technique, can be used to study the function of specific proteins in distinct meiotic stages.
The NDT80-in system is a powerful tool for meiotic cell cycle synchronization that relies on the inducible expression of the middle meiosis transcription factor NDT804,5. NDT80 expression is required for prophase I exit6,7. With the NDT80-in system, NDT80 is under the control of the GAL1-10 promoter in cells expressing the Gal4 transcription factor fused to an estrogen receptor (Gal4-ER)4,5. Because Gal4-ER only enters the nucleus when bound to &#946;-estradiol, NDT80-in cells arrest in prophase I in the absence of &#946;-estradiol, which allows the synchronization of cells in prophase I (Figure 1). &#946;-estradiol addition promotes the translocation of the Gal4-ER transcription factor into the nucleus, where it binds GAL1-10 to drive expression of NDT80, leading to synchronous entry into the meiotic divisions. Although time-lapse microscopy can be performed without synchronization, the advantage of using synchronization is the ability to add an inhibitor or a drug while cells are at a specific stage of meiosis.
The anchor away technique is an inducible system by which a protein can be depleted from the nucleus with the addition of rapamycin8. This technique is ideal for studying nuclear proteins during cell division in budding yeast because yeast cells undergo closed mitosis and meiosis, in which the nuclear envelope does not break down. Furthermore, this technique is very useful for proteins that have multiple functions throughout meiosis. Unlike for deletions, mutant alleles, or meiotic null alleles, the removal of a target protein from the nucleus at a specific stage does not compromise target protein activity at earlier stages, allowing for a more accurate interpretation of results. The anchor away system utilizes the shuttling of ribosomal subunits between the nucleus and cytoplasm that occurs upon ribosomal maturation8. To deplete the target protein from the nucleus, the target protein is tagged with the FKBP12-rapamycin-binding domain (FRB) in a strain in which the ribosomal subunit Rpl13A is tagged with FKBP12. Without rapamycin, FRB and FKBP12 do not interact, and the FRB-tagged protein remains in the nucleus. Upon rapamycin addition, the rapamycin forms a stable complex with FKBP12 and FRB, and the complex is shuttled out of the nucleus due to the interaction with Rpl13A (Figure 1). To prevent cell death upon rapamycin addition, cells harbor the tor1-1 mutation of the TOR1 gene. Additionally, these cells contain fpr1&#916;, a null allele of the S. cerevisiae FKBP12 protein, which prevents endogenous Fpr1 from out-competing Rpl13A-FKBP12 for FRB and rapamycin binding. The anchor away background mutations, tor1-1 and fpr1&#916;, do not affect meiotic timings or chromosome segregation2.
To demonstrate the usefulness of this technique, the kinetochore protein Ctf19 was depleted at different timepoints throughout meiosis. Ctf19 is a component of the kinetochore that is dispensable in mitosis but required for proper chromosome segregation in meiosis9,10,11,12,13. In meiosis, the kinetochore is shed in prophase I, and Ctf19 is important for kinetochore re-assembly9,14. For this protocol, cells with the NDT80-in system were synchronized, and the anchor away technique was used to deplete the target protein Ctf19 from the nucleus before and after the release from prophase I, and after meiosis I chromosome segregation (Figure 1). This protocol can be adapted to deplete other proteins of interest at any stage of meiosis and mitosis.

<table><tbody><tr><td>&amp;beta;-estradiol</td><td>Millipore Sigma</td><td>E8875</td><td>Make 1mM stocks in 95% EtOH</td></tr><tr><td>0.22 uM Threaded Bottle-top Filter</td><td>Millipore Sigma</td><td>S2GPT02RE</td><td></td></tr><tr><td>100% EtOH</td><td>Fisher Scientific</td><td>22-032-601</td><td></td></tr><tr><td>10X PBS</td><td>Fisher Scientific</td><td>BP399500</td><td>Dilute 1:10 to use as solvent for ConA</td></tr><tr><td>24 mm x 50 mm coverslip No. 1.5</td><td>VWR North American</td><td>48393241</td><td></td></tr><tr><td>25 mm x 75 mm microscope slides</td><td>VWR North American</td><td>48300-026</td><td></td></tr><tr><td>Adenine hemisulfate salt</td><td>Millipore Sigma</td><td>A9126</td><td>To supplement SC, SCA, and 1% Kac</td></tr><tr><td>Bacto Agar</td><td>BD</td><td>214030</td><td></td></tr><tr><td>Concanavialin A</td><td>Mllipore Sigma</td><td>C2010</td><td>Make as 1mg/mL in 1X PBS</td></tr><tr><td>CoolSNAP HQ2 CCD camera</td><td>Photometrics</td><td></td><td>Used in Section 4.3</td></tr><tr><td>D-glucose</td><td>Fisher Scientific</td><td>D16-10</td><td></td></tr><tr><td>Difco Yeast Nitrogen Base w/o Amino Acids</td><td>BD</td><td>291920</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Millipore Sigma</td><td>D5879</td><td></td></tr><tr><td>Eclipse Ti2 inverted-objective micrscope</td><td>Nikon</td><td></td><td>Used in Section 4.4</td></tr><tr><td>Fiji</td><td>NIH</td><td></td><td>Download from https://fiji.sc/</td></tr><tr><td>GE Personal DeltaVision Microscope</td><td>Applied Precision</td><td></td><td>Used in Section 4.3</td></tr><tr><td>L-Tryptophan&amp;nbsp;</td><td>Millipore Sigma</td><td>T0254</td><td>To supplement SC, SCA, and 1% Kac</td></tr><tr><td>Modeling Clay</td><td>Crayola</td><td>&amp;nbsp;2302880000</td><td>To secure coverslip in slide holder</td></tr><tr><td>NIS-Elements AR 5.30.04 Imaging Software</td><td>Nikon</td><td></td><td>Used in Section 4.4</td></tr><tr><td>ORCA-Fustion BT Camera</td><td>Hamamatsu</td><td>C15440-20UP</td><td>Used in Section 4.4</td></tr><tr><td>Plastic pipette tip holder</td><td>Dot Scientific</td><td>LTS1000-HR</td><td>Cut a 4 square x 4 square section of the rack portion of this product.&amp;nbsp;</td></tr><tr><td>Pottassium Acetate</td><td>Fisher Scientific</td><td>BP264</td><td></td></tr><tr><td>Rapamycin</td><td>Fisher Scientific</td><td>BP29631</td><td>Make 1mg/mL stocks in DMSO</td></tr><tr><td>Silicone Sealant</td><td>Aqueon</td><td>100165001</td><td>Also known as aquarium glue.</td></tr><tr><td>SoftWorx7.0.0&amp;nbsp; Imaging Software</td><td>Applied Precision</td><td></td><td>Used in Section 4.3</td></tr><tr><td>Synthetic Complete Mixture (Kaiser)&amp;nbsp;</td><td>Formedium</td><td>DSCK2500</td><td></td></tr><tr><td>Type N immersion oil&amp;nbsp;</td><td>Nikon</td><td>MXA22166</td><td></td></tr></tbody></table>

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.

To monitor chromatin segregation, histone protein Htb2 was tagged with mCherry. In prophase I, the chromatin appears as a single Htb2 mass. After homologous chromosomes segregate in the first meiotic division, the chromatin appears as two distinct masses (Figure 3A). After the sister chromatids segregate, the chromatin appears as four masses. If some chromosomes fail to attach to spindle microtubules, additional masses can be seen after meiosis I or meiosis II.
The method described above was used to study the role of the kinetochore component Ctf19 in ensuring proper chromosome segregation in budding yeast meiosis. Anchor away yeast strains expressing Ctf19 tagged with FRB were synchronized in prophase I using the NDT80-in system (tor1-1; fpr1Δ; RPL13A-FKBP12; CTF19-FRB; GAL1-10 promoted NDT80; GAL4-ER). As a control, an NDT80-in strain with the anchor away strain background but without the FRB-tagged protein was used (tor1-1; fpr1Δ; RPL13A-FKBP12; GAL1-10 promoted NDT80; GAL4-ER). Cells were released from the prophase I arrest by the addition of β-estradiol. Rapamycin was added to deplete Ctf19-FRB from the nucleus either at release (before kinetochore reassembly), 45 min after release (after kinetochore reassembly but before meiosis I), or 3 h after release (just before meiosis II) (Figure 3A-F). Following the addition of rapamycin, time-lapse fluorescence microscopy was performed, and images were acquired every 10 min for the remaining duration of meiosis.
After imaging, the open-access software Fiji was used to open the images and perform analysis for figures showing time progression (Figure 3A-F). The number of DNA masses after meiosis II were counted in at least 100 cells that were undergoing meiosis per condition. In wildtype cells with the anchor away background, there are typically four DNA masses at the end of meiosis II, representing the four products of meiosis (Figure 3A). In a small fraction of the cells, only three masses are visible after meiosis II (Figure 3B,G). However, it is likely that three masses are the result of a cell that has four products of meiosis but two of these masses appear together after meiosis II.
When Ctf19-FRB is anchored away at the time of release of prophase I exit (t = 0 h), approximately 47% of cells display more than four DNA masses upon the completion of meiosis, suggesting a defect in the attachment of kinetochores and microtubules (Figure 3C,G). With anchoring away of Ctf19-FRB either after kinetochore assembly but before meiosis I (t = 45 min) or before meiosis II (t = 3 h), approximately 16% of cells display additional DNA masses. These results support the hypothesis that Ctf19 is important for kinetochore re-assembly at prophase I release but is less important in chromosome segregation once the kinetochore has been reassembled.
The results obtained here show that time-lapse microscopy combined with cell cycle synchronization and conditional depletion of a target protein allow for the study of a protein in a specific meiotic stage. Although Ctf19 is not essential for mitosis, the meiotic kinetochore is extensively reorganized, and Ctf19 has a crucial role in kinetochore re-assembly after prophase I exit9,11,12. The results in Figure 3 show that when Ctf19 is depleted prior to kinetochore re-assembly, a large fraction of cells display additional DNA masses after meiosis II. When Ctf19 is depleted from the nucleus after kinetochore reassembly but before either meiosis I or meiosis II, there are fewer cells that display additional DNA masses. These results suggest that the most important role for Ctf19 is at the end of prophase I. This difference in chromosome segregation fidelity with depletion of Ctf19 at various stages highlights the importance of using stage-specific conditional alleles to study the function of proteins specifically in meiosis I or meiosis II. A meiotic null mutant would have displayed a severe defect and would not have provided information about the role of Ctf19 at each stage.
Additionally, although CTF19 is not an essential gene, there is a chromosome transmission fidelity (ctf) phenotype during vegetative growth of CTF19 mutants19. Use of a null allele or mutant CTF19 allele could create a population of aneuploid cells that may not undergo meiosis properly. Use of the anchor away technique and NDT80-in synchronization system circumvent this problem by precisely depleting Ctf19 from the nucleus just prior to meiosis I or meiosis II, reducing the concern that the analyzed cells were aneuploid.
<img alt="Meiosis phases diagram; spindle structure; β-estradiol and RAP effects on target protein in yeast." class="xfigimg" src="/files/ftp_upload/64580/64580fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Overview of the experiment. Cartoon of yeast cell showing a single pair of homologous chromosomes. NDT80-in cells enter meiosis and arrest at prophase I. After addition of β-estradiol, cells enter the meiotic divisions synchronously. Addition of rapamycin determines when the target protein (marked with a star) is anchored away. In this experiment, the target protein Ctf19 was anchored away at three different time points by adding rapamycin (RAP) at the same time as prophase I (RAP addition at t = 0 min), after prophase I release (RAP addition at t = 45 min), and after meiosis I chromosome segregation (RAP addition at t = 3 h). The thick black arrows indicate cell cycle stage of rapamycin addition and, therefore, target protein depletion. Abbreviations: RAP = rapamycin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64580/64580fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chamber setup diagram with labeled sections for cell culture and silicone sealant application." class="xfigimg" src="/files/ftp_upload/64580/64580fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Chamber set-up for time-lapse imaging experiments. (A) Reusable chamber (right) cut from a pipette tip holder insert (left). The pipette tip holder insert is cut with a red-hot blade to remove a 4 square x 4 square portion of the insert. The dashed lines indicate a sample section of the tip holder that can be cut to make a chamber. The remaining plastic dividers within the 4 square x 4 square cut-out are trimmed away and a hollow chamber remains (right). (B) To create the final chamber, silicone sealant is added to edges on one side of the chamber, and then placed on top of a cover slip. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64580/64580fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Wildtype and Ctf19-FRB NDT80-in cells; time-lapse microscope images; DNA masses post-MII; graph results." class="xfigimg" src="/files/ftp_upload/64580/64580fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Time at which Ctf19 is anchored away affects chromatin segregation. (A-F) Representative time lapse images of cells expressing Htb2-mCherry during meiosis. Images were taken every 10 min, immediately following the addition of β-estradiol. Scale bars= 5 µm. Numbers in the upper-right corner of each frame label the time that has elapsed between each frame, and time 0 indicates the frame in which chromatin segregates in meiosis I. Only cells that completed the meiotic divisions were counted. (A) Wildtype cell (no protein tagged with FRB) in which β-estradiol was added 12 h after KAc transfer. Cell contains four DNA masses after completion of meiosis II. (B) Ctf19-FRB cell in which rapamycin was added at t = 0 (simultaneously with β-estradiol addition). This cell shows three DNA masses after meiosis II. (C) Ctf19-FRB cell in which rapamycin was added at t = 0 (simultaneously with β-estradiol addition). This cell shows five DNA masses after meiosis II. (D) Ctf19-FRB cell in which rapamycin was added at t = 0 (simultaneously with β-estradiol addition). This cell dies after completing meiosis II. (E) Ctf19-FRB cell in which rapamycin was added 45 min after β-estradiol addition. This cell shows five DNA masses after meiosis II. (F) Ctf19-FRB cell in which rapamycin was added 45 min after β-estradiol addition. After meiosis II, six DNA masses are present. (G) Quantification of the number of DNA masses in at least 100 cells for each condition (two movies analyzed for each condition). t = under each bar indicates the time at which rapamycin was added relative to β-estradiol addition. Abbreviations: MII = meiosis II. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64580/64580fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.

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 ...

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1.8K Views.  Indiana University. Door cellen in profase één te synchroniseren en vervolgens het doeleiwit in een bepaald stadium uit te putten, kan deze methode tijdelijk verschillende functies van specifieke eiwitten identificeren. Voorwaardelijk uitputten van eiwitten in specifieke stadia van myose voorkomt de resterende effecten die een traditionele myotische mutant in latere stadia kan hebben. Deze methode kan worden gebruikt om ontluikende gistmitose en andere organismen te bestuderen die geen nucleaire enveloppe-afbr...

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

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Developmental Biology

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

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 ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. 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 ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

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

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