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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| β-estradiol | Millipore Sigma | E8875 | Make 1mM stocks in 95% EtOH |
| 0.22 uM Threaded Bottle-top Filter | Millipore Sigma | S2GPT02RE | |
| 100% EtOH | Fisher Scientific | 22-032-601 | |
| 10X PBS | Fisher Scientific | BP399500 | Dilute 1:10 to use as solvent for ConA |
| 24 mm x 50 mm coverslip No. 1.5 | VWR North American | 48393241 | |
| 25 mm x 75 mm microscope slides | VWR North American | 48300-026 | |
| Adenine hemisulfate salt | Millipore Sigma | A9126 | To supplement SC, SCA, and 1% Kac |
| Bacto Agar | BD | 214030 | |
| Concanavialin A | Mllipore Sigma | C2010 | Make as 1mg/mL in 1X PBS |
| CoolSNAP HQ2 CCD camera | Photometrics | Used in Section 4.3 | |
| D-glucose | Fisher Scientific | D16-10 | |
| Difco Yeast Nitrogen Base w/o Amino Acids | BD | 291920 | |
| Dimethyl sulfoxide (DMSO) | Millipore Sigma | D5879 | |
| Eclipse Ti2 inverted-objective micrscope | Nikon | Used in Section 4.4 | |
| Fiji | NIH | Download from https://fiji.sc/ | |
| GE Personal DeltaVision Microscope | Applied Precision | Used in Section 4.3 | |
| L-Tryptophan | Millipore Sigma | T0254 | To supplement SC, SCA, and 1% Kac |
| Modeling Clay | Crayola | 2302880000 | To secure coverslip in slide holder |
| NIS-Elements AR 5.30.04 Imaging Software | Nikon | Used in Section 4.4 | |
| ORCA-Fustion BT Camera | Hamamatsu | C15440-20UP | Used in Section 4.4 |
| Plastic pipette tip holder | Dot Scientific | LTS1000-HR | Cut a 4 square x 4 square section of the rack portion of this product. |
| Pottassium Acetate | Fisher Scientific | BP264 | |
| Rapamycin | Fisher Scientific | BP29631 | Make 1mg/mL stocks in DMSO |
| Silicone Sealant | Aqueon | 100165001 | Also known as aquarium glue. |
| SoftWorx7.0.0 Imaging Software | Applied Precision | Used in Section 4.3 | |
| Synthetic Complete Mixture (Kaiser) | Formedium | DSCK2500 | |
| Type N immersion oil | Nikon | MXA22166 |
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