Manipulation and Analysis of Cell Cycle-Dependent Processes in Budding Yeast

Michael G. Stewart; Talia C. Scheel; Ahmed A. Abouelghar; Sara E. Hoppe; Matthew P. Miller

doi:10.3791/68887

Manipulation et analyse des processus dépendants du cycle cellulaire chez les levures bourgeonnantes

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Manipulation and Analysis of Cell Cycle-Dependent Processes in Budding Yeast

Manipulation et analyse des processus dépendants du cycle cellulaire chez les levures bourgeonnantes

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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.

Ce protocole détaille deux méthodes d’arrêt du cycle cellulaire de levure et de libération facultative, et développe l’utilisation de la microscopie à fluorescence pour étudier les processus dépendants du cycle cellulaire chez S. cerevisiae.

Nous étudions comment les cellules en division transmettent fidèlement leurs chromosomes lors de la mitose, en nous concentrant sur les machines moléculaires et les mécanismes qui assurent une ségrégation chromosomique précise. Nous synchronisons les cellules pour étudier les processus moléculaires qui évoluent avec le cycle cellulaire. Sans ces méthodes, les changements clés seraient cachés dans une population cellulaire non synchronisée.

Comparé à d’autres méthodes de synchronisation, l’arrêt par facteur alpha chez les mutants BAR1 offre un arrêt G1 plus propre et réversible, nous permettant de suivre l’évolution synchrone d’une culture de levure dans le cycle. Nos travaux révèlent la localisation dynamique des protéines et les changements d’activité tout au long du cycle cellulaire, éclairant des processus mitotiques clés, tels que la ségrégation des chromosomes et le maintien du fuseau. Pour commencer, inoculez la levure dans 25 millilitres de milieu YPAD, puis incubez toute la nuit pour atteindre une densité optique de 600 nanomètres comprise entre 0,5 et 2,0.

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