Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. Cerevisiae.
Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. Cerevisiae.
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DOI：
10.3791/64580
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发表时间：
2022-10-11
期刊：
影响因子：
--
通讯作者：
Lacefield S
中科院分区：
文献类型：
--
作者：
Cairo G;MacKenzie A;Tsuchiya D;Lacefield S
延时荧光显微镜通过提供固定细胞成像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。芽殖酵母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高度保守。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上起作用。因此，使用功能丧失或减数分裂无效突变体可以破坏早期过程，阻断或干扰后期过程，使其难以确定与每个个体角色相关的表型。为了规避这一挑战，该方案描述了蛋白质如何在减数分裂的特定阶段有条件地从细胞核中耗尽，同时使用定时显微镜监测减数分裂事件。具体来说，该方案描述了细胞如何在前期I同步，锚移技术如何用于在特定减数分裂阶段从细胞核中消耗蛋白质，以及如何使用定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，着丝点蛋白Ctf19在减数分裂期间的不同时间点从细胞核中消失，并在减数分裂II结束时分析染色质团块的数量。总的来说，该方案可以适应从细胞核中消耗不同的核蛋白，同时监测减数分裂。延时显微镜是研究出芽酵母减数分裂的重要工具。该方案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，以演示如何研究减数分裂染色体分离过程中特定蛋白的功能。
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. 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.
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影响因子：
10.5
作者：
Benjamin, KR;Zhang, C;Herskowitz, I
通讯作者：
Herskowitz, I
影响因子：
3.3
作者：
Neiman, Aaron M.
通讯作者：
Neiman, Aaron M.
DOI：
10.1007/bf00384382
发表时间：
1982-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
BYERS, B;GOETSCH, L
通讯作者：
GOETSCH, L
影响因子：
3.3
作者：
Tsuchiya D;Gonzalez C;Lacefield S
通讯作者：
Lacefield S
DOI：
10.1083/jcb.145.1.15
发表时间：
1999-04-05
期刊：
The Journal of cell biology
影响因子：
--
作者：
Hyland KM;Kingsbury J;Koshland D;Hieter P
通讯作者：
Hieter P