Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Summary

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Abstract

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.  

Introduction

In the cell cycle, cells undergo a series of highly regulated and temporally controlled events for the accurate duplication of their genome and proliferation. In mammals, the cell cycle consists of interphase and M-phase. In interphase, which consists of three stages- G1, S, and G2, the cell duplicates its genome and undergoes growth that is necessary for normal cell cycle progression^1,2. In the M-phase, which consists of mitosis (prophase, prometaphase, metaphase, anaphase, and telophase) and cytokinesis, a parental cell produces two genetically identical daughter cells. In mitosis, sister chromatids of dupli....

Protocol

1. Double Thymidine Block and Release: Reagent Preparations

1. Make 500 mL Dulbecco's modified Eagle medium (DMEM) medium supplemented with 10% FBS, penicillin, and streptomycin.
2. Make 100 mM stock of thymidine in sterile water and store in aliquots at -80 °C.

2. Protocol for Fixed Cell Imaging of Mitotic Progression (Figure 1A)

1. On day 1, seed ~2 x 10⁵ HeLa cells into the wells of a 6-well plate with a cover slip (sterilized with 70% ethanol and UV irradiation) and 2 mL of DMEM medium. Grow the cells in a humidified incubator for 24 h at 37 °C and 5%....

Results

Study of mitotic progression and microtubule stability in cells fixed after release from double thymidine block
Cdt1 is involved in licensing of DNA replication origins in the G1 phase. It is degraded during S phase but re-accumulates in G2/M phase. To study its role in mitosis, the endogenous Cdt1 needs to be depleted specifically at the G/M phase using the most suitable cell synchronization technique, the double thymidine block¹⁵. Cells were .......

Discussion

The most critical advantage of double thymidine synchronization is that it provides an increased sample size of mitotic cells in a short time window with many of these cells entering mitosis in unison, thus also enabling analyses of chromosome alignment, bipolar spindle formation, and chromosome segregation with much higher efficiency.

Many regulatory protein complexes and signaling pathways are devoted to ensuring normal progression through mitosis and deregulation of this process may led to .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM (1x)	Life Technologies	11965-092	Store at 4 °C
DPBS (1x)	Life Technologies	14190-144	Store at 4 °C
Leibovitz’s (1x) L-15 medium	Life Technologies	21083-027	Store at 4 °C
Serum reduced medium (Opti-MEM)	Life Technologies	319-85-070	Store at 4 °C
Penicillin and streptomycin	Life Technologies	15070-063 (Pen Strep)	1:1,000 dilution
Dharmafect2	GE Dharmacon	T-2002-02	Store at 4 °C
Thymidine	MP Biomedicals LLC	103056	Dissolved in sterile distiled water
Cdt1 siRNA	Life Technologies		Ref 10
Hec1 siRNA	Life Technologies		Ref 13
HeLa cells expressing GFP-H2B
HeLa cells expressing GFP-α-tubulin and mCherry H2B	Generous gift from Dr. Kozo Tanaka of Tohoku university, Japan
Formaldehyde solution	Sigma-Aldrich Corporation	F8775	Toxic, needs caution
DAPI	Sigma-Aldrich Corporation	D9542	Toxic, needs caution
Mouse anti-α-tubulin	Santa Cruz Biotechnology	Sc32293	1:1,000 dilution
Rabbit ant-Zwint1	Bethyl	A300-781A	1:400 dilution
Mouse anti-phospho-γH2AX (Ser139)	Upstate Biotechnology	05-626, clone JBW301	1:300 dilution
Alexa 488	Jackson ImmunoResearch		1:250 dilution
Rodamine Red-X	Jackson ImmunoResearch		1:250 dilution
BioLite 6 well multidish	Thermo Fisher Scientific	130184
35 mm Glass bottom dish	MatTek Corporation	P35GCOL-1.5-14-C
Nikon Eclipse TiE inverted microscope	Nikon Instruments
Spinning disc for confocal	Yokagawa	CSU-X1
Ultra 888 EM-CCD Camera	Andor	iXon Ultra EMCCD
4 wave length laser	Agilent Technologies
Incubation System for Microscopes	Tokai Hit	TIZB
NIS-elements software	Nikon Instruments