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
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

我们感谢印第安纳大学的光学显微镜成像中心。这项工作得到了美国国立卫生研究院（GM105755）的资助。

1. 准备必要的材料
<ol><li>准备酵母细胞生长和孢子形成的试剂。 注意：如果出芽酵母菌株是 ade2 -和 trp1-，则在步骤1.1.1-1.1.3中补充所有培养基，终浓度为0.01%腺嘌呤和0.01%色氨酸。如果通过高压灭菌器灭菌培养基，则仅在试剂高压灭菌并冷却至室温后添加这些氨基酸。<ol><li>对于营养生长，通过将 6.7 g 不含氨基酸的酵母氮碱、2 g 完全氨基酸混合物和 20 g 葡萄糖溶解在...

<ol>
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M., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis+I+is+established+through+division-specific+translational+control+of+a+cyclin.">Meiosis I is established through division-specific translational control of a cyclin.</a> Cell. 133 (2), 280-291 (2008).</li><li>Benjamin, K. R., Zhang, C., Shokat, K. M., Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+landmark+events+in+meiosis+by+the+CDK+Cdc28+and+the+meiosis-specific+kinase+Ime2.">Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2.</a> Genes and Development. 17 (12), 1524-1539 (2003).</li><li>Xu, L., Ajimura, M., Padmore, R., Klein, C., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NDT80,+a+meiosis-specific+gene+required+for+exit+from+pachytene+in+Saccharomyces+cerevisiae.">NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae.</a> Molecular and Cellular Biology. 15 (12), 6572-6581 (1995).</li><li>Chu, S., Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gametogenesis+in+yeast+is+regulated+by+a+transcriptional+cascade+dependent+on+Ndt80.">Gametogenesis in yeast is regulated by a transcriptional cascade dependent on Ndt80.</a> Molecular Cell. 1 (5), 685-696 (1998).</li><li>Haruki, H., Nishikawa, J., Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anchor-away+technique:+rapid,+conditional+establishment+of+yeast+mutant+phenotypes.">The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes.</a> Molecular Cell. 31 (6), 925-932 (2008).</li><li>Borek, W. E., 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=The+proteomic+landscape+of+centromeric+chromatin+reveals+an+essential+role+for+the+Ctf19(CCAN)+complex+in+meiotic+kinetochore+assembly.">The proteomic landscape of centromeric chromatin reveals an essential role for the Ctf19(CCAN) complex in meiotic kinetochore assembly.</a> Current Biology. 31 (2), 283-296 (2021).</li><li>Mehta, G. D., Agarwal, M., Ghosh, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+kinetochore+protein,+Ctf19+in+meiosis+I:+an+implication+of+differential+impact+of+Ctf19+on+the+assembly+of+mitotic+and+meiotic+kinetochores+in+Saccharomyces+cerevisiae.">Functional characterization of kinetochore protein, Ctf19 in meiosis I: an implication of differential impact of Ctf19 on the assembly of mitotic and meiotic kinetochores in Saccharomyces cerevisiae.</a> Molecular Microbiology. 91 (6), 1179-1199 (2014).</li><li>Ortiz, J., Stemmann, O., Rank, S., Lechner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+putative+protein+complex+consisting+of+Ctf19,+Mcm21,+and+Okp1+represents+a+missing+link+in+the+budding+yeast+kinetochore.">A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore.</a> Genes and Development. 13 (9), 1140-1155 (1999).</li><li>Hyland, K. M., Kingsbury, J., Koshland, D., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ctf19p:+A+novel+kinetochore+protein+in+Saccharomyces+cerevisiae+and+a+potential+link+between+the+kinetochore+and+mitotic+spindle.">Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle.</a> Journal of Cell Biology. 145 (1), 15-28 (1999).</li><li>Fernius, J., Marston, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+cohesion+at+the+pericentromere+by+the+Ctf19+kinetochore+subcomplex+and+the+replication+fork-associated+factor,+Csm3.">Establishment of cohesion at the pericentromere by the Ctf19 kinetochore subcomplex and the replication fork-associated factor, Csm3.</a> PLoS Genetics. 5 (9), 1000629 (2009).</li><li>Meyer, R. E., 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=Ipl1/Aurora-B+is+necessary+for+kinetochore+restructuring+in+meiosis+I+in+Saccharomyces+cerevisiae.">Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae.</a> Molecular Biology of the Cell. 26 (17), 2986-3000 (2015).</li><li>Elrod, S. L., Chen, S. M., Schwartz, K., Shuster, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+sporulation+conditions+for+different+Saccharomyces+cerevisiae+strain+backgrounds.">Optimizing sporulation conditions for different Saccharomyces cerevisiae strain backgrounds.</a> Methods in Molecular Biology. 557, 21-26 (2009).</li><li>Deutschbauer, A. M., Davis, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+mapped+to+single-nucleotide+resolution+in+yeast.">Quantitative trait loci mapped to single-nucleotide resolution in yeast.</a> Nature Genetics. 37 (12), 1333-1340 (2005).</li><li>Ben-Ari, G., 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=Four+linked+genes+participate+in+controlling+sporulation+efficiency+in+budding+yeast.">Four linked genes participate in controlling sporulation efficiency in budding yeast.</a> PLoS Genetics. 2 (11), 195 (2006).</li><li>Primig, M., 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=The+core+meiotic+transcriptome+in+budding+yeasts.">The core meiotic transcriptome in budding yeasts.</a> Nature Genetics. 26 (4), 415-423 (2000).</li><li>Spencer, F., Gerring, S. L., Connelly, C., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+chromosome+transmission+fidelity+mutants+in+Saccharomyces+cerevisiae.">Mitotic chromosome transmission fidelity mutants in Saccharomyces cerevisiae.</a> Genetics. 124 (2), 237-249 (1990).</li><li>Weinhandl, K., Winkler, M., Glieder, A., Camattari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+source+dependent+promoters+in+yeasts.">Carbon source dependent promoters in yeasts.</a> Microbial Cell Factories. 13, 5 (2014).</li><li>Byers, B., Goetsch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+pachytene+arrest+of+Saccharomyces+cerevisiae+at+elevated+temperature.">Reversible pachytene arrest of Saccharomyces cerevisiae at elevated temperature.</a> Molecular and General Genetics. 187 (1), 47-53 (1982).</li><li>Winter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sum1/Ndt80+transcriptional+switch+and+commitment+to+meiosis+in+Saccharomyces+cerevisiae.">The Sum1/Ndt80 transcriptional switch and commitment to meiosis in Saccharomyces cerevisiae.</a> Microbiology and Molecular Biology Reviews. 76 (1), 1-15 (2012).</li><li>Neiman, A. 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>

该协议结合了 NDT80-in系统以同步细胞，锚移技术以从细胞核中去除蛋白质，以及荧光延时显微镜以在减数分裂期间对出芽酵母细胞进行成像。 NDT80-in系统是一种用于减数分裂细胞周期同步的方法，它利用前期I阻滞和释放4，8。尽管单个细胞在随后的每个减数分裂阶段所花费的时间略有不同，但大多数细胞将在整个减数分裂过程中保持高度?...

延时荧光显微镜是研究出芽酵母1，2中减数分裂染色体分离动力学的宝贵工具。通过饥饿关键营养素，可以诱导出芽酵母细胞发生减数分裂3。在减数分裂过程中，细胞经历一轮染色体分离，然后进行两次分裂，产生四种减数分裂产物，这些产物被包装成孢子（图1）。可以在减数分裂的每个阶段可视化单个细胞，从而生成固定细胞成像很容易错过的空间和时间数据。该协议显示了如何将延时荧光显微镜与两种先前建立的方法（诱导NDT80系统（NDT80-in）和锚移技术相结合，可用于研究不同减数分裂阶段特定蛋白质的功能。
NDT80-in系统是减数分裂细胞周期同步的强大工具，它依赖于中间减数分裂转录因子NDT80 4，5的诱导表达。第一阶段出口6，7需要NDT80表达。使用NDT80-in系统，NDT80在表达与雌激素受体（ Gal4-ER）融合的Gal4转录因子的细胞中的GAL1-10启动子的控制下4，5。由于Gal4-ER仅在与β-雌二醇结合时才进入细胞核，因此NDT80-in细胞在没有β-雌二醇的情况下在前期I中停滞，这允许前期I中的细胞同步（图1）。β-雌二醇加成促进Gal4-ER转录因子易位到细胞核中，在那里它结合GAL1-10以驱动NDT80的表达，导致同步进入减数分裂。虽然延时显微镜可以在没有同步的情况下进行，但使用同步的优点是能够在细胞处于减数分裂的特定阶段时添加抑制剂或药物。
锚移技术是一种可诱导系统，通过该系统，可以通过添加雷帕霉素8从细胞核中去除蛋白质。该技术非常适合在出芽酵母的细胞分裂过程中研究核蛋白，因为酵母细胞经历闭合的有丝分裂和减数分裂，其中核包膜不会分解。此外，该技术对于在整个减数分裂中具有多种功能的蛋白质非常有用。与缺失、突变等位基因或减数分裂无效等位基因不同，在特定阶段从细胞核中去除靶蛋白不会损害早期靶蛋白活性，从而可以更准确地解释结果。锚移系统利用核糖体成熟时核和细胞质之间核糖体亚基的穿梭8。为了从细胞核中去除靶蛋白，在核糖体亚基Rpl13A用FKBP12标记的菌株中用FKBP12标记靶蛋白12-雷帕霉素结合结构域（FRB）标记靶蛋白。没有雷帕霉素，FRB和FKBP12不会相互作用，FRB标记的蛋白质保留在细胞核中。雷帕霉素加成后，雷帕霉素与FKBP12和FRB形成稳定的复合物，并且由于与Rpl13A的相互作用，复合物从细胞核中穿梭出来（图1）。为了防止雷帕霉素加成后的细胞死亡，细胞含有TOR1基因的tor1-1突变。此外，这些细胞含有 fpr1Δ，这是酿酒酵母 FKBP12 蛋白的空等位基因，可防止内源性 Fpr1 在 FRB 和雷帕霉素结合方面竞争 Rpl13A-FKBP12。锚定背景突变，tor1-1和fpr1Δ，不影响减数分裂时间或染色体分离2。
为了证明该技术的有效性，动粒蛋白Ctf19在整个减数分裂过程中的不同时间点耗尽。Ctf19是动粒的一种成分，在有丝分裂中是可有可无的，但在减数分裂9，10，11，12，13中适当的染色体分离是必需的。在减数分裂中，动粒在前期I脱落，Ctf19对动粒重新组装很重要9，14。对于该协议，具有NDT80-in系统的细胞是同步的，并且使用锚移技术在前期I释放之前和之后以及减数分裂I染色体分离之后从细胞核中去除靶蛋白Ctf19（图1）。该协议可以适应在减数分裂和有丝分裂的任何阶段消耗其他感兴趣的蛋白质。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#946;-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&#160;</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>&#160;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.&#160;</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&#160; Imaging Software</td>
			<td>Applied Precision</td>
			<td></td>
			<td>Used in Section 4.3</td>
		</tr>
		<tr>
			<td>Synthetic Complete Mixture (Kaiser)&#160;</td>
			<td>Formedium</td>
			<td>DSCK2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Type N immersion oil&#160;</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

延时荧光显微镜通过提供成像固定细胞通常看不到的时间和空间数据，彻底改变了对减数分裂细胞周期事件的理解。芽酵母已被证明是研究减数分裂染色体分离的重要模式生物，因为许多减数分裂基因是高度保守的。出芽酵母减数分裂的延时显微镜可以监测不同的减数分裂突变体，以显示突变如何破坏减数分裂过程。然而，许多蛋白质在减数分裂的多个点起作用。因此，使用功能丧失或减数分裂零突变体会破坏早期过程，阻断或干扰后期过程，并且难以确定与每个个体角色相关的表型。为了规避这一挑战，该协议描述了如何在减数分裂的特定阶段有条件地从细胞核中耗尽蛋白质，同时使用延时显微镜监测减数分裂事件。具体来说，该协议描述了细胞如何在前期I中同步，如何使用锚移技术在特定减数分裂阶段从细胞核中去除蛋白质，以及如何使用延时成像来监测减数分裂染色体分离。作为该技术实用性的一个例子，在减数分裂期间的不同时间点从细胞核中去除了动粒蛋白Ctf19，并在减数分裂II结束时分析染色质质量的数量。总体而言，该方案可以适应于从细胞核中消耗不同的核蛋白，同时监测减数分裂。

为了监测染色质分离，用mCherry标记组蛋白Htb2。在前期I中，染色质显示为单个Htb2肿块。同源染色体在第一次减数分裂中分离后，染色质显示为两个不同的肿块（图3A）。在姐妹染色单体分离后，染色质显示为四个肿块。如果某些染色体未能附着在纺锤体微管上，则在减数分裂I或减数分裂II后可以看到额外的肿块。
上述方法用于研究动粒成分 Ctf19 在确保?...

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

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

延时显微镜是研究出芽酵母减数分裂的宝贵工具。该协议描述了一种结合细胞周期同步，延时显微镜和目标蛋白的条件耗竭的方法，以证明如何在减数分裂染色体分离期间研究特定蛋白质的功能。

使用延时显微镜和蛋白质的阶段特异性核耗竭来研究酿酒酵母的减数分裂

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

通过在前期一同步细胞，然后在特定阶段消耗目标蛋白，该方法可以识别特定蛋白质的暂时不同功能。在肌病的特定阶段有条件地消耗蛋白质可以防止传统肌突变体在后期阶段产生的残留效应。该方法可用于研究出芽酵母有丝分裂和其他不经历核包膜分解的生物。此外，该方法可用于其他生物体，以研究核包膜分解之前的细胞周期事件。首先，制作一个琼脂垫，用于创建用于成像的单层细胞。切下并丢弃1.5毫升微量离心管的盖子和底部三分之一，以创建一个圆柱体，该圆筒将用作琼脂垫的模具。将切割好的微量离心管筒放在干净的载玻片上，将管的顶部倒置，坐在载玻片上。在 50 毫升烧杯中制作 6 毫升 5% 琼脂溶液。切掉移液器的尖端以形成更大的开口，并将大约500微升融化的琼脂移液到微量离心管中。让它在室温下静置，直到琼脂凝固。为了制备酵母细胞，在一毫升微量离心管中以800G旋转200微升孢子培养物两分钟。弃去180微升上清液。通过旋转和轻弹试管将沉淀重悬于剩余的上清液中。用ConA将六微升浓缩细胞移液到腔室中间的盖玻片上。用琼脂垫握住圆筒，然后小心地将其从载玻片上滑落。确保琼脂底部完全平坦，并使用移液器尖端的底部，对微量离心机模具施加轻微的压力，使琼脂垫略高于管的边界。接下来，倒置模具，使琼脂垫朝下朝向腔室。使用镊子轻轻地将琼脂垫放在细胞顶部，用移液器尖端将琼脂垫在腔室周围轻轻滑动 10 到 20 次，以在盖玻片上形成单层细胞。将琼脂垫在腔室中保持 12 至 15 分钟。接下来，将两毫升孢子培养物转移到两个微量离心管中，并以15， 700 G离心两分钟。将上清液转移到干净的微量离心管中后，在相同条件下再次旋转并收集上清液以清洁微量离心管。将两毫升上清液滴加到含有琼脂垫的腔室中。一旦液体到达腔室顶部，琼脂垫很可能会漂浮。用镊子轻轻取出琼脂垫并丢弃。在腔室顶部放置一个 24 x 50 毫米的盖玻片，以防止成像过程中蒸发。要在显微镜上设置视频，请将盖玻片安装在载玻片支架内。将成型粘土粘附在盖玻片的侧面，以将其牢固地固定在载玻片支架中。打开图像采集软件。使用粗调和微调旋钮使用 DIC 或明场聚焦细胞。在图像采集软件的主菜单上，单击文件，选择采集，将弹出四个窗口。在名为 Resolve 3D 的窗口中，单击锥形瓶图标。这将打开一个名为“设计运行实验”的窗口，其中包含用于设置实验以设置延时摄影影片的控件。在“设计”选项卡下，导航到标有“切片”的选项卡。选中 Z 切片旁边的框，并将光学切片间距设置为 1 微米，将光学切片数量设置为 5。接下来，在“频道”选项卡下，单击加号图标并选择适当的频道。然后，选择参考图像旁边的框，并从下拉菜单中将 Z 位置设置为样品的中间。从下拉菜单中选择透射率和曝光时间的值。在“延时摄影”选项卡下，选中“延时摄影”旁边的框，然后输入分钟和小时值以标识图像采集的时间间隔。选中“使用终极对焦保持对焦”旁边的框，以防止影片期间出现舞台漂移。在“点”选项卡下，选中“访问点列表”旁边的框。在主菜单中，单击“查看”并选择“点列表”。将载物台移动到显示单层细胞的腔室区域。单击“点列表”窗口中的标记点。移动载物台以选择 25 到 30 个点，没有任何重叠，以避免在每个时间过程中过度曝光单元格并成像每个场。在“点列表”窗口中，选择“全部校准”以为每个点设置最终焦点。在“运行”选项卡下，将文件保存到计算机上的相应目标。在“运行实验”窗口中，选择“播放”按钮以启动影片。打开斐济软件。打开 DIC 和 mCherry 通道。通过单击图像，然后单击堆栈和Z投影，然后从下拉菜单中选择最大强度，获得mCherry通道的单个最大强度投影。要将DIC和mCherry通道合并到一个图像中，请单击图像，颜色，然后选择合并通道。跟随单个细胞通过减数分裂。减数分裂II完成后，记录DNA质量数。在具有锚点背景的野生型细胞中，减数分裂II末端通常有四个DNA团块，代表减数分裂的四种产物。当Ctf19-FRB在前期I释放时锚定时，大约47%的细胞在减数分裂完成后显示超过四个DNA质量，表明动粒和微管的附着存在缺陷。随着 Ctf19-FRB 的锚定，无论是在动粒组装之后但在减数分裂 I 之前，还是在减数分裂 II 之后，大约 16% 的细胞显示出额外的 DNA 质量。该方案最重要的部分之一是将上清液滴加到您的腔室中，以避免破坏您的单层。此外，请注意准备成像所需的时间，以便在分析中考虑它。通过改变荧光包装的蛋白质，该程序可用于回答有关细胞周期持续时间，染色体分离，蛋白质丰度和蛋白质定位的问题。

Research

JoVE Journal

Biology

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

Assay for Adhesion and Agar Invasion in S. cerevisiae

化验粘附和酿酒酵母琼脂入侵

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

科研

生物学

Monitoring Actin Disassembly with Time-lapse Microscopy

拆装时间推移显微镜监测肌动蛋白

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

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

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

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

Identification of protein complexes with quantitative proteomics in S. cerevisiae

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Time-lapse Imaging of Mitosis After siRNA Transfection

有丝分裂的siRNA转染后的时间推移成像

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...