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Biology
出芽酵母における細胞周期依存過程の操作と解析
出芽酵母における細胞周期依存過程の操作と解析
JoVE Journal
Biology
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JoVE Journal Biology
Manipulation and Analysis of Cell Cycle-Dependent Processes in Budding Yeast

出芽酵母における細胞周期依存過程の操作と解析

Full Text
651 Views
08:13 min
September 26, 2025

DOI: 10.3791/68887-v

Michael G. Stewart*1,2, Talia C. Scheel*1, Ahmed A. Abouelghar*1, Sara E. Hoppe*1, Matthew P. Miller1

1Department of Biochemistry,University of Utah School of Medicine, 2Department of Molecular Biology and Genetics, Howard Hughes Medical Institute,Johns Hopkins University School of Medicine

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Overview

This study investigates how yeast (S. cerevisiae) cells manage chromosome segregation during mitosis, utilizing synchronized cell cycles to observe cellular dynamics. By employing alpha-factor arrest in BAR1 mutants, researchers can achieve a precise G1 arrest to monitor changes in protein localization and activity throughout the cell cycle.

Key Study Components

Research Area

  • Cell cycle regulation
  • Chromosome segregation
  • Microscopy techniques

Background

  • Synchronized cell cycles unveil molecular processes otherwise hidden in unsynchronized populations.
  • Protein localization changes throughout the cell cycle are crucial for understanding mitosis.
  • Alpha-factor arrest provides a cleaner synchronization method than alternatives.

Methods Used

  • Fluorescence microscopy for imaging protein localization
  • Saccharomyces cerevisiae as the biological model
  • Alpha-factor treatment for G1 synchronization and subsequent release techniques

Main Results

  • Dynamic changes in protein localization were observed, particularly for Stu2-GFP during mitosis.
  • A peak in binucleate cells was noted at approximately 90 minutes post-release, indicating synchronized progress into anaphase.
  • Quantified intensity of protein puncta revealed significant changes correlating with different cell cycle stages.

Conclusions

  • The study effectively demonstrates precise methods for synchronizing yeast cell cycles to investigate mitotic processes.
  • This research has implications for broader biological understanding of cell division and chromosome behavior.

Frequently Asked Questions

What is the significance of studying yeast cells in cell cycle research?
Yeast cells serve as a simple eukaryotic model to study fundamental cell cycle processes that are conserved across species.
How does alpha-factor synchronization improve experiments?
Alpha-factor synchronization provides a precise and reversible means to arrest cells in G1 phase, allowing for controlled studies of the cell cycle.
What techniques are used to visualize protein localization?
Fluorescence microscopy techniques are employed to observe dynamic protein localization changes in real-time during the cell cycle.
What roles do Stu2-GFP and Spc110-mCherry play?
Stu2-GFP is used to track spindle dynamics, while Spc110-mCherry marks spindle pole bodies critical for mitotic spindle formation.
Why is synchronized cell population analysis important?
A synchronized cell population allows researchers to accurately assess changes in cellular behavior and protein dynamics at specific time points during the cell cycle.
What outcomes can one expect from this study?
The study aims to provide insights into the mechanisms underlying chromosome segregation and the dynamics of proteins involved in mitosis.
What potential applications does this research have?
Findings could inform cancer research and the development of therapeutic strategies targeting cell division processes.

このプロトコルは、酵母細胞周期停止とオプションの放出の2つの方法を詳述し、 S. cerevisiaeの細胞周期依存性プロセスを研究するための蛍光顕微鏡の使用について詳しく説明します。

私たちは、分裂する細胞が有糸分裂中に染色体を忠実に伝達する方法を研究し、正確な染色体分離を保証する分子機械やメカニズムに焦点を当てています。細胞を同期させて、細胞周期に伴う分子プロセスの研究を行います。これらの方法がなければ、重要な変化は同期していない細胞集団の中で隠れてしまいます。

他の同期法と比較して、BAR1変異体におけるアルファクター停止はよりクリーンで可逆的なG1停止を提供し、酵母培養全体が周期を同期的に進行している様子を追跡することを可能にします。私たちの研究は、細胞周期を通じて動的なタンパク質の局在化と活性変化を明らかにし、染色体分離やスピンドル維持といった主要な有糸分裂過程に光を当てています。まず、酵母を25ミリリットルのYPAD培地に接種し、一晩培養して600ナノメートルの0.5から2.0の光学密度に到達させます。

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