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 progression1,2. 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 segregation3,4.
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 cells9,10. 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 points11,12,13. Cells can also be synchronized at G2/M phase by treating with nocodazole and RO-33069,14.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
Representative Results
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 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful to Dr. Kozo Tanaka of Tohoku University, Japan for sharing HeLa cells stably expressing mCherry-Histone H2B and GFP-α-tubulin. This work was supported by an NCI grant to DV (R00CA178188) and by start-up funds from Northwestern University.
Materials
| DMEM (1X) | Life Technologies | 11965-092 | Store at 4 degree C |
| DPBS (1X) | Life Technologies | 14190-144 | Store at 4 degree C |
| Leibovitz’s (1X) L-15 medium | Life Technologies | 21083-027 | Store at 4 degree C |
| Serum reduced medium (Opti-MEM) | Life Technologies | 319-85-070 | Store at 4 degree C |
| Penicillin and streptomycin | Life Technologies | 15070-063 (Pen Strep) | 1:1000 dilution |
| Dharmafect2 | GE Dharmacon | T-2002-02 | Store at 4 degree 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 | 1:1000 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 | |
| 35MM 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 |
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