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

Summary

This protocol details two methods of yeast cell cycle arrest and optional release, and elaborates on the use of fluorescence microscopy to study cell cycle-dependent processes in S. cerevisiae.

Abstract

Eukaryotic cells follow a conserved cell cycle that regulates diverse processes, including DNA maintenance and organelle homeostasis. Studying cellular processes in a cell cycle-dependent manner is often necessary to properly interpret experimental results. There are chemical and genetic methods available to produce cell cycle synchronization in cultured cells across a wide swath of organisms, including vertebrate models, enabling the study of cell cycle-dependent processes. However, among model organisms, budding yeast remains a powerhouse for cell cycle analysis due to its particularly robust synchronization methods, short generation time, and genetic tractability. Yeast shares core cell cycle machinery with other eukaryotes, which has enabled landmark discoveries in cell cycle regulation. This protocol details methods for cell cycle analysis in yeast, focusing on G1 arrest-release and mitotic arrest-release experiments, including strain construction, culture preparation, and microscopy. PCR tagging methods for producing suitable strains for cell cycle arrests and fluorescence microscopy are presented. A G1 arrest is achieved using the peptide pheromone α-factor, and brief washes result in synchronous release and cell cycle progression. Samples are taken at different time points following release into the cell cycle and fixed for microscopy. A second method arrests yeast cells in mitosis by depleting the cell cycle regulator Cdc20 to achieve a metaphase-arrested population, as well as optional release into anaphase. Samples are fixed and prepared for imaging pre- and post-release, and are imaged and analyzed. Image analysis focuses on cataloging dynamic localization and population abundance changes of proteins in the cell cycle. These synchronization methods are suitable for diverse cell cycle manipulations, and while their use in imaging fixed cells is highlighted here, they can be adapted for many other analyses, including live cell imaging as well as biochemical and molecular assays.

Introduction

Eukaryotic cell division is highly regulated through a program called the cell cycle. The highly conserved and dynamic processes occurring in the cell cycle make it interesting to study in-and-of-itself, but also have wide-spread implications that inform investigations of other cell biological processes-for example, many organelles undergo dramatic remodeling during cell division, and the abundance and localization of many proteins is highly regulated throughout^1,2,3. Although there are some additional layers of complexity present in metazoan systems compared to yeast, includ....

Protocol

1. Construction of strains for cell cycle analysis and imaging

1. Design primers to C-terminally tag the gene of interest using Pringle Tagging Plasmids (pFA6a plasmids)²⁴. In short, design F2 and R1 primers that, when used with a pFA6a-series plasmid, produce a PCR product that can be directly transformed into yeast to generate a 'tagged' version of the gene of interest. Use the primer pairs and plasmids contained in Table 1 to tag the genes of interest in this protocol. Plasmid and primer design strategies for C-terminal tagging of yeast genes are described in detail by Longtine et al.²⁴

Results

Analyzing changes in cell cycle-dependent protein localization by fluorescence microscopy can be readily accomplished using the methods we describe here. Our group has long been interested in the dynamic regulation and function of the mitotic spindle. In yeast, spindle pole bodies (marked by component Spc110) function as microtubule organizing centers from which microtubule filaments emanate to create the structure of the mitotic spindle³⁰. The microtubule binding .......

Discussion

Utilizing cell cycle synchronization in budding yeast enables studying important mechanisms for a variety of cellular processes. The use of G1 arrest-releases with α-factor treatment allows synchronous progression of a population of cells through the stages of the cell cycle, and as we showed, can reveal dynamic localization patterns of cellular regulators like Stu2³⁶. Cell cycle arrests can also be accomplished using genetic means via depletion of cell cycle regulators like Cdc20, w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
?-factor	University of Utah Core Synthesis Facility		Sequence: WHWLQLKPGQPMY
1.5 mL Eppendorf Tubes	Axygen	MCT-175-C
10mM dNTP mix	Thermo Scientific	R0193
50 mL conical tubes	greiner bio-one	227 261
5x Phusion HF reaction buffer	New England BioLabs	B0518S
Acetic Acid, Glacial	Fisher Chemical	BP2401C-212
adenine hemisulfate salt	Sigma-Aldrich	A9126-100G
Agar, Granulated	Apex Chemicals and Reagents	20-275
agarose	Apex Bioresearch Products	20-102GP
Autoclave Amsco Century Steam Sterilizer	Steris	SV-1262
autoclaved DI water
Auxin	Sigma-Aldrich	Cat#I3750-5G-A; CAS: 87-51-4
Cargille Laser Liquid	Cargille Laboratories	20130
D-Sorbitol	Sigma-Aldrich	S1876-500G
DAPI (40 ,6-Diamidino-2-Phenylindole, Dihydrochloride)	Molecular Probes	Cat#D1306
Deoxyribonucleic acid sodium salt from salmon testes	Sigma-Aldrich	D1626
Dextrose	Fisher Chemical	D16-10
Disodium Ethylenediamine Tetraacetate	Fisher Chemical	S811-10
DMSO	Thermo Scientific	20688
FIJI/ImageJ2 vs 2.14.0/1.54f	ImageJ2	https://imagej.net/software/fiji/
Fixed Speed Vortex Mixer	VWR	https://dabos.com/product/vortex-mixers-vwr-fixed-speed-vortex-mixer-00001-24763?srsltid=AfmBOoo5TH0aoExvrrrphDaFt8XAsDqLvkjxtEUj1QWlFbWh7_gwzMObLT4&gQT=2
Fluorescent microscope DV Ultra	Leica	https://www.leica-microsystems.com/c/am/lsr-w/fluorescence-microscope-wf/?nlc=20250214-SFDC-022570&utm_source=google&utm_medium=cpc&utm_campaign=25-AM-LSR-L3-LSPO-LSWF-SE-Google-Ads-WF-Thunder-Search&utm_content=text_ad&utm_term=fluorescence%20microscopes&gad_source=1&gad_campaignid=170130111&gbraid=0AAAAADrbsAF-dGDbxzgT8m_cvXSlf4BB0&gclid=CjwKCAjwmenCBhA4EiwAtVjzmkMJUGFksaHezZvlBUlbbS1tR8RqXP24dbSRzcRgTT8RmJy7nyeThBoC3yQQAvD_BwE	Serial #: NV01063. No longer supported
Formaldehyde	Fisher Chemical	Cat#F79-500
gel apparatus	Thermo Scientific	Owl EasyCast B1
GeneRuler DNA Ladder Mix	Fermentas	SM0333
glass beads	Fisher Scientific	11312A
Glass Slides	VWR	48300-026
Innova 2300 Platform Shaker	New Brunswick	NB-2300
Kimwipes	Kimtech	06-666
Laboratory centrifuge for 1.5 mL tubes	Eppendorf	2525
Laboratory centrifuge for 50 mL tubes	Eppendorf	5804
Lithium acetate dihydrate	Sigma-Aldrich	L4158-250G
Master cycler nexus X2	eppendorf	https://www.eppendorf.com/us-en/Products/PCR/Thermocyclers/Mastercycler-nexus-X2-p-PF-82586
Micro-pipettes p2, p20, p200 and p1000 and corresponding tips	Rainin	L-2XLS+R, L-20XLS-R, L-200XLS-R, L-1000XLS-R
Microscope Cover Glass	Fisher Scientific	12541014
Nocodazole	Calbiochem	Cat#487928; CAS: 31430-18-9; Lot#B35705
Orange G	Sigma-Aldrich	O7252
PEG	Hampton Research	HR2-591
Peptone granulated	Fisher Bioreagents	BP9725-5
Phusion HF DNA Polymerase	New England BioLabs	M0530L
Pipet-X	Rainin	PX-100R
Potassium phosphate, dibasic	Thermo Scientific	424195000
Potassium phosphate, monobasic	Thermo Scientific	424200025
power source	Bio-Rad	23786
Start Acquire Ultra 1.2.2	softWoRx Cytiva	Obtain with DV Ultra
Tris Base	Fisher Bioreagents	BP152-10
Triton X-100	Sigma-Aldrich	9002-93-1
tube rotator	VWR	10136-084
water bath	VWR	WBE10A11B
Water, Ultra Pure	Apex Bioresearch Products	18-194
Yeast extract Granulated	Fisher Bioreagents	BP9727-5