Name: use of time-lapse microscopy and stage-specific nuclear depletion of proteins to study meiosis in s. cerevisiae
Uploaded: 2022-10-11T14:40:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae at JoVE.com

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, ...

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.
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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. 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.

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

Szybkie Technika Wizualizacja żywo unieruchomionych komórek drożdży

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

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

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 ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

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 ...

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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4D Microscopy of Yeast