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 duplicated genome are condensed (prophase) and are captured at their kinetochores by microtubules of the assembled mitotic spindle (prometaphase), that drives their alignment at the metaphase plate (metaphase) followed by their equal segregation when sister chromatids are split toward and transported to opposite spindle poles (anaphase). The two daughter cells are physically separated by the activity of an actin-based contractile ring (telophase and cytokinesis). The kinetochore is a specialized proteinaceous structure which assembles at the centromeric region of chromatids and serve as attachment sites for spindle microtubules. Its main function is to drive chromosome capture, alignment, and aid in correcting improper spindle microtubule attachment, while mediating the spindle assembly checkpoint to maintain the fidelity of chromosome segregation^3,4.

The technique of cell synchronization serves as an ideal tool for understanding the molecular and structural events involved in cell cycle progression. This approach has been used to enrich cell populations at specific phases for various types of analyses, including profiling of gene expression, analyses of cellular biochemical processes, and detection of subcellular localization of proteins. Synchronized mammalian cells can be used not only for the study of individual gene products, but also for approaches involving analysis of whole genomes including microarray analysis of gene expression⁵, miRNA expression patterns⁶, translational regulation⁷, and proteomic analysis of protein modifications⁸. Synchronization can also be used to study the effects of gene expression or protein knock-down or knock-out, or of chemicals on cell cycle progression.

Cells can be synchronized at the different stages of the cell cycle. Both physical and chemical methods are widely used for cell synchronization. The most important criteria for cell synchronization are that synchronization should be noncytotoxic and reversible. Because of the potential adverse cellular consequences of synchronizing cells by pharmacological agents, chemical-dependent methods can be advantageous for studying key cell cycle events. For example, hydroxyurea, amphidicolin, mimosine, and lovastatin, can be used for cell synchronization at G1/S phase but, because of their effect on the biochemical pathways they inhibit, they activate cell cycle checkpoint mechanisms and kill an important fraction of the cells^9,10. On the other hand, feedback inhibition of DNA replication by adding thymidine to the growth media, known as "thymidine block", can arrest the cell cycle at certain points^11,12,13. Cells can also be synchronized at G2/M phase by treating with nocodazole and RO-3306^9,14.Nocodazole, which prevents microtubule assembly, has a relatively high cytotoxicity. Moreover, nocodazole-arrested cells can return to interphase precociously by mitotic slippage. Double thymidine block arrest cells at G1/S phase and after release from the block, cells are found to proceed synchronously through G2 and into mitosis. The normal progression of the cell cycle for cells released from thymidine block can be observed under high resolution confocal microscopy by either cell fixation or live imaging. The effect of perturbation of mitotic proteins can be studied specifically when cells enter and proceed through mitosis after release from double thymidine block. Cdt1, a multifunctional protein, is involved in DNA replication origin licensing in the G1 phase and is also required for kinetochore microtubule attachments during mitosis¹⁵. To study the function of Cdt1 during mitosis, one needs to adopt a method that avoids the effect of its depletion on replication licensing during G1 phase, while at the same time effecting its depletion specifically during the G2/M phase only. Here, we present detailed protocols based on the double thymidine block to study the mitotic role of proteins performing multiple functions during different stages of the cell cycle by both fixed and live-cell imaging.

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% CO₂.
2. 1^st thymidine block: On day 2, thoroughly mix the required volume of thymidine in fresh DMEM media (100 mM stock, 2 mM final concentration).
3. Add 2 mL of thymidine-containing media to the cells in each well of the 6-well plate and incubate for 18 h.
4. On day 3, aspirate the medium and wash the cells twice with 2 mL of 1x PBS and once with fresh prewarmed DMEM; grow cells in fresh prewarmed DMEM medium for 9 h to release cells from block.
5. 2^nd thymidine block: Again add 2 mL of thymidine-containing media (2 mM final concentration) to the cells in each well of the 6-well plate.
6. Incubate for another 18 h.
7. On day 4, aspirate the medium and wash the cells twice with 2 mL of 1x PBS and once with fresh prewarmed DMEM.
8. Dilute siRNAs (control and Cdt1) and transfection reagent in serum free growth medium separately for 10 min. Mix them together and incubate for 20 min at RT. The final concentration of siRNA used in each transfection was 100 nM. Add the reaction mixture to the cells that have been washed out of Thymidine.
9. Incubate cells at 37 °C for 9-10 h to release from blocking and fix the cells on the cover slips with 4% PFA for 20 min at RT.
   Caution: PFA is toxic, wear appropriate protection.
10. Immunostain cells, after permeabilizing with 0.5% (v/v) detergent for 10 min at RT and washing the cells twice with 1x PBS for 5 min each.
    1. Block cells with 1% BSA (w/v) in 1x PBS for 1 h at RT followed by treating cells with 50 µL of primary antibodies (mouse anti-α-tubulin antibody diluted at 1:1,000, rabbit anti-Zwint1 antibody diluted at 1:400, and mouse anti-phospho-ɣ H2AX diluted at 1:300) in 1% (w/v) BSA in 1x PBS for 1 h at 37 ^oC.
    2. After washing out the cells with 1x PBS thrice, treat cells with 50 µL of secondary antibodies for each cover glass (Alexa 488 and Rhodamine Red at 1:250 dilutions in 1% (w/v) BSA in 1x PBS) for 1 h at RT.
    3. After washing the cells with 1x PBS twice for 5 min each, treat cells with DAPI (0.1µg/mL in 1x PBS) for 5 min at RT.
    4. After washing the cells with 1x PBS twice for 5 min each, place the cover slip facing cells to the appropriate mounting media on a clear microscopic slide.
11. Image the cells for the immunostained proteins with 60X or 100X 1.4 NA Plan-Apochromatic DIC oil immersion objective mounted on an inverted high resolution confocal microscope equipped with an appropriate camera as necessary for the quality of images required.
12. Acquire images at room temperature as z-stacks of 0.2 µm thickness using software appropriate for the microscope.

3. Protocol for Live Cell Imaging of Mitotic Progression (Figure 3A)

1. On day 1, seed approximately 0.5-1 x 10⁵ HeLa cells stably expressing mCherry-H2B and GFP-α-tubulin (slightly variable based on cell type and the proteins they express) into 35 mm glass bottom dishes with 1.5 mL of DMEM medium and grow them in a humidified incubator for 24 h at 37 ^oC and 5% CO₂.
2. 1^st thymidine block: On day 2, add 1.5 mL of thymidine-containing media to the cells in the dish (100 mM thymidine, 2 mM final concentration,).
3. Incubate for 18 h.
4. On day 3, aspirate the medium containing thymidine and wash the cells thrice, twice with 2 mL of 1x PBS and once with fresh prewarmed DMEM.
5. Dilute siRNAs (control and Hec1) and transfection reagent with serum free growth medium separately for 10 min. Mix them together and incubate for 20 min at RT. The final concentration of siRNA used in each transfection was 100 nM. Add the reaction mixture to the cells that have been washed out of Thymidine.
6. Grow cells in 1.5 mL of fresh prewarmed DMEM medium for 8 h to release cells from block.
7. 2^nd thymidine block: Add 1.5 mL of thymidine-containing media (2 mM final concentration) to the cells in the dish.
8. Incubate for another 18 h.
9. On day 4, aspirate the medium and wash the cells thrice, twice with 2 mL of 1x PBS and once with fresh prewarmed DMEM. Then grow cells in fresh prewarmed Leibovitz's (L-15) medium supplemented with 10% FBS and 20 mM HEPES at pH 7.0 for 8 h to release cells from the block.
10. Place the dish in the temperature control chamber set in the high resolution confocal microscope which has already switched on at least 30 min before beginning the imaging to stabilize the on-stage and experimental temperatures.
11. Focus in bright field with 60x objective until cells are visible, then manually sift the on-stage to the region of choice.
12. Set up the light and laser power/exposure, image acquisition parameters, and duration of the experiment using the microscope image acquisition software of choice. Use filters for GFP (488 excitation; 385 nm emission) and mCherry (561 nm excitation and 385 nm emission) to acquire images.
13. Perform the time-lapse experiment by separately acquiring pulsed transmitted light and fluorescence images every 10 min for a period up to 16 h.
14. Acquire images as twelve 1.0 µm-separated z-planes at 9 h after release from the double thymidine block using software appropriate to the microscope.
15. Analyze the images tracking individual cells for mitotic progression and finally assemble the corresponding movie with the source software Fiji/ImageJ.

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 wer...

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

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