Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Summary

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.

Abstract

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.

Introduction

Time-lapse fluorescence microcopy is a valuable tool for studying the dynamics of meiotic chromosome segregation in budding yeast^1,2. Budding yeast cells can be induced to undergo meiosis through starvation of key nutrients³. During meiosis, cells undergo one round of chromosome segregation followed by two divisions to create four meiotic products that are packaged into spores (Figure 1). Individual cells can be visualized throughout each stage of meiosis, which generates spatial and temporal data that can be easily missed by fixed-cell imaging. This protoc....

Protocol

1. Preparation of necessary materials

1. Prepare reagents for the growth and sporulation of yeast cells.
   NOTE: If budding yeast strains are ade2- and trp1-, supplement all media in steps 1.1.1-1.1.3 with a final concentration of 0.01% adenine and 0.01% tryptophan from 1% stocks. If sterilizing media by autoclave, add these amino acids only after the reagents have been autoclaved and allowed to cool to room temperature.
   1. For vegetative growth, prepare 2x synthetic complete + dextrose medium (2XSC) by dissolving 6.7 g of yeast nitrogen base without amino acids, 2 g of complete amino acid mix, and 20 g of dext....

Results

To monitor chromatin segregation, histone protein Htb2 was tagged with mCherry. In prophase I, the chromatin appears as a single Htb2 mass. After homologous chromosomes segregate in the first meiotic division, the chromatin appears as two distinct masses (Figure 3A). After the sister chromatids segregate, the chromatin appears as four masses. If some chromosomes fail to attach to spindle microtubules, additional masses can be seen after meiosis I or meiosis II.

Th.......

Discussion

This protocol combines the NDT80-in system to synchronize cells, the anchor away technique to deplete proteins from the nucleus, and fluorescence time-lapse microscopy to image budding yeast cells during meiosis. The NDT80-in system is a method for meiotic cell cycle synchronization that utilizes a prophase I arrest and release^4,8. Although individual cells will vary slightly in the amount of time spent in each of the subsequent meiotic stages, .......

Materials

Name	Company	Catalog Number	Comments
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