Cell synchronization

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Cell synchronization is a process by which cells in a culture at different stages of the cell cycle are brought to the same phase. Cell synchrony is a vital process in the study of cells progressing through the cell cycle as it allows population-wide data to be collected rather than relying solely on single-cell experiments. The types of synchronization are broadly categorized into two groups; physical fractionization and chemical blockade.

Contents

Physical Separation

Physical fractionation is a process by which continuously dividing cells are separated into phase-enriched populations based on characteristics such as the following:

Given that cells take on varying morphologies and surface markers throughout the cell cycle, these traits can be used to separate by phase. There are two commonly used methods.

Centrifugal Elutriation

(Previously called: counter streaming centrifugation) Centrifugal elutriation can be used to separate cells in different phases of the cell cycle based on their size and sedimentation velocity (related to sedimentation coefficient). Because of the consistent growth patterns throughout the cell cycle, centrifugal elutriation can separate cells into G1, S, G2, and M phases by increasing size (and increasing sedimentation coefficients) with diminished resolution between G2 and M phases due to cellular heterogeneity and lack of a distinct size change. [1]

Larger cells sediment faster, so a cell in G2, which has experienced more growth time, will sediment faster than a cell in G1 and can therefore be fractionated out. Cells grown in suspension tend to be easier to elutriate given that they do not adhere to one another and have rounded, uniform shapes. However, some types of adherent cells can be treated with trypsin and resuspended for elutriation as they will assume a more rounded shape in suspension. [2]

Flow Cytometry and Cell Sorting

Flow cytometry allows for detection, counting, and measurement of the physical and chemical properties of cells. Cells are suspended in fluid and put through the flow cytometer. Cells are sent one at a time through a laser beam and the light scatter is measured by a detector. Cells or their components can be labeled with fluorescent markers so that they emit different wavelengths of light in response to the laser, allowing for additional data collection.

For quantitative cell cycle analysis, cells are usually fixed with ethanol and stained with DNA-binding dyes like propidium iodide, Hoechst 33342, DAPI, 7-Aminoactinomysin D, Mithramycin, DRAQ5, or TO-PRO-3, allowing for determination of phase by DNA quantity. [3] However, if these cells have been fixed, they are dead and cannot be maintained for continued growth. Cells can also be resuspended in media and dyed with non-toxic dyes to maintain living cultures. Cells can also be genome edited such that some cellular proteins are made with conjugated fluorescent tags such as GFP, mCherry, and Luciferase that can be used to detect and quantify those components. For example, chimeric histone H2B-GFP constructs can be made and used to measure DNA content and determine replication status as a means of discerning cell phase. [4] Light scatter measurements can be used to determine characteristics like size, allowing for distinction of cell phase without tagging.

Flow cytometers can be used to collect multiparameter cytometry data, but cannot be used to separate or purify cells. Fluorescence-activated cell sorting (FACS) is a technique for sorting out the cells based on the differences that can be detected by light scatter (e.g. cell size) or fluorescence emission (by penetrated DNA, RNA, proteins or antigens). The system works much like flow cytometry, but will also charge each cell droplet after it has been measured based on a defined parameter. The charged droplet will then encounter an electrostatic deflection system that will sort the cell to a different container based on that charge. This allows cells to be separated on the basis of fluorescent content or scatter.

To summarize, flow cytometry alone can be used to gather quantitative data about cell cycle phase distribution, but flow cytometry in coordination with FACS can be used to gather quantitative data and separate cells by phase for further study. Limitations include:

Chemical blockade

The addition of exogenous substrates can be used to block cells in certain phases of the cell cycle and frequently target cell cycle checkpoints. These techniques can be carried out in vitro and do not require removal from the culture environment. The most common type of chemical blockade is arrest-and-release, which involves treatment of a culture with a chemical block and subsequent release by washing or addition of a neutralizing agent for the block. While chemical blockade is typically more effective and precise than physical separation, some methods can be imperfect for various reasons, including:

Arrest in M

Mitotic arrest can be achieved through many methods and at various points within M-phase, including the G2/M transition, the metaphase/anaphase transition, and mitotic exit.

Nocodazole

Nocodazole is a rapidly-reversible inhibitor of microtubule polymerization that can be used to arrest cells before Anaphase at the spindle assembly checkpoint in the metaphase/anaphase transition. The microtubule poison works by blocking the formation of the mitotic spindles that attach to and pull apart sister chromatids in dividing cells. Cells will remain arrested until the nocodazole has been washed out. Nocodazole does not appear to disrupt interphase metabolism, and released cells return to normal cell cycle progression. [5] Because microtubules are vital in other cellular functions, sustained use of nocodazole can result in disruption of those functions, causing cell death.

Inhibition of CDK1

CDK1 is necessary for the transition from G2 to M phase. RO-3306 is a selective CDK1 inhibitor that can reversibly arrest cells at the G2/M border. RO-3306 synchronized >95% of cycling cells (including cancer cells), and released cells rapidly enter mitosis. [6]

Roscovitine

Roscovitine can be used to inhibit the activity of cyclin-dependent kinases (CDKs) by competing with ATP in the ATP-binding region of the kinase. Its effects are potent, arresting cells by knocking down the function of CDKs necessary for cell cycle progression. Roscovitine can be used to arrest cells in G0/G1, G1/S, or G2/M transitions. [7]

Colchicine

Colchicine arrests cells in metaphase and is a microtubule poison preventing mitotic spindle formation, much like nocodazole. It works by depolymerizing tubulin in microtubules, blocking progression to anaphase through sustained arrest at the spindle assembly checkpoint.

Arrest in S (G1/S arrest)

Arrest in S phase typically involves inhibition of DNA synthesis as the genome is being replicated. Most methods are reversible through washing.

Double thymidine block

High concentrations of thymidine interrupt the deoxynucleotide metabolism pathway through competitive inhibition, thus blocking DNA replication. A single treatment with thymidine arrests cells throughout S phase, so a double treatment acts to induce a more uniform block in early S phase. [8] The process begins with a treatment with thymidine, washing of the culture, followed by another thymidine treatment.

Hydroxyurea

Hydroxyurea decreases the production of dNTPs by inhibiting the enzyme ribonucleotide reductase. This serves to halt DNA synthesis by depriving DNA polymerase of dNTPs at replication forks. [9] Hydroxyurea is also used to treat certain types of cancer and blood disorders.

Aphidicolin

Aphidocolin is a fungus-derived tetracyclic diterpenoid that acts as a selective inhibitor for DNA polymerase α. [10] This enzyme is necessary for replicative DNA synthesis, but does not disrupt DNA repair synthesis or mitochondrial DNA replication. [11]

Arrest in G1

A single commonly-used chemical method exists for synchronization of cells in G1. It involves Lovastatin, a reversible competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an enzyme vital in the production of mevalonic acid. Mevalonic acid is a key intermediate in the mevalonate pathway responsible for synthesis of cholesterol. Addition of cholesterol to Lovastatin-treated cells does not undo the arrest affect, so Lovastatin appears to inhibit the formation of some early intermediate in the pathway that is essential for progression through early G1. [12]

Other Methods of Synchronization

Mitotic Selection

Mitotic selection is a drug-free procedure for the selection of mitotic cells from a monolayer undergoing exponential growth. [13] During mitosis, cells undergo changes in morphology, and mitotic selection takes advantage of this in adherent cells grown in a monolayer. The cells become more spherical, decreasing the surface area of cell membrane attached to the culture plate. Mitotic cells can therefore be completely detached by gently shaking and collected from the supernatant. [3]

Nutrient/Serum Deprivation

Elimination of serum from the culture medium for about 24 hours results in the accumulation of cells at the transition between G0 quiescence and early G1. This arrest is easily reversible through addition of serum or the deprived nutrient. Upon release, progression through the cell cycle is variable, as some cells remain quiescent while others proceed through the cell cycle at variable rates. [14]

Contact Inhibition

Contact inhibition occurs when cells are allowed to grow to high or full confluence, maximizing cell-to-cell contact. This triggers arrest in early G1 in normal cells. Arrest is reversed by replating cells at a lower density. [14] Because of the proliferation-promoting mutations intrinsic to cancer, tumor cell lines are not usually able to undergo contact inhibition, though there are exceptions. [15]

Related Research Articles

<span class="mw-page-title-main">Cell cycle</span> Series of events and stages that result in cell division

The cell cycle, or cell-division cycle, is the series of events that take place in a cell that causes it to divide into two daughter cells. These events include the duplication of its DNA and some of its organelles, and subsequently the partitioning of its cytoplasm, chromosomes and other components into two daughter cells in a process called cell division.

<span class="mw-page-title-main">Mitosis</span> Process in which chromosomes are replicated and separated into two new identical nuclei

Mitosis is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis, is an equational division which gives rise to genetically identical cells in which the total number of chromosomes is maintained. Mitosis is preceded by the S phase of interphase and is followed by telophase and cytokinesis; which divides the cytoplasm, organelles and cell membrane of one cell into two new cells containing roughly equal shares of these cellular components. The different stages of mitosis altogether define the mitotic phase of a cell cycle—the division of the mother cell into two daughter cells genetically identical to each other.

<span class="mw-page-title-main">Cell division</span> Process by which living cells divide

Cell division is the process by which a parent cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome(s) before dividing. In eukaryotes, there are two distinct types of cell division: a vegetative division (mitosis), producing daughter cells genetically identical to the parent cell, and a cell division that produces haploid gametes for sexual reproduction (meiosis), reducing the number of chromosomes from two of each type in the diploid parent cell to one of each type in the daughter cells. In Mitosis is a part of the cell cycle, in which, replicated chromosomes are separated into two new nuclei. Cell division gives rise to genetically identical cells in which the total number of chromosomes is maintained. In general, mitosis is preceded by the S stage of interphase and is followed by telophase and cytokinesis; which divides the cytoplasm, organelles, and cell membrane of one cell into two new cells containing roughly equal shares of these cellular components. The different stages of mitosis all together define the M phase of an animal cell cycle—the division of the mother cell into two genetically identical daughter cells. Meiosis undergoes two divisions resulting in four haploid daughter cells. Homologous chromosomes are separated in the first division of meiosis, such that each daughter cell has one copy of each chromosome. These chromosomes have already been replicated and have two sister chromatids which are then separated during the second division of meiosis. Both of these cell division cycles are used in the process of sexual reproduction at some point in their life cycle. Both are believed to be present in the last eukaryotic common ancestor.

G<sub>1</sub> phase First growth phase in the eukaryotic cell cycle

The G1 phase, gap 1 phase, or growth 1 phase, is the first of four phases of the cell cycle that takes place in eukaryotic cell division. In this part of interphase, the cell synthesizes mRNA and proteins in preparation for subsequent steps leading to mitosis. G1 phase ends when the cell moves into the S phase of interphase. Around 30 to 40 percent of cell cycle time is spent in the G1 phase.

<span class="mw-page-title-main">Telophase</span> Final stage of a cell division for eukaryotic cells both in mitosis and meiosis

Telophase is the final stage in both meiosis and mitosis in a eukaryotic cell. During telophase, the effects of prophase and prometaphase are reversed. As chromosomes reach the cell poles, a nuclear envelope is re-assembled around each set of chromatids, the nucleoli reappear, and chromosomes begin to decondense back into the expanded chromatin that is present during interphase. The mitotic spindle is disassembled and remaining spindle microtubules are depolymerized. Telophase accounts for approximately 2% of the cell cycle's duration.

<span class="mw-page-title-main">Anaphase-promoting complex</span> Cell-cycle regulatory complex

Anaphase-promoting complex is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. The APC/C is a large complex of 11–13 subunit proteins, including a cullin (Apc2) and RING (Apc11) subunit much like SCF. Other parts of the APC/C have unknown functions but are highly conserved.

<span class="mw-page-title-main">Nocodazole</span> Chemical compound

Nocodazole is an antineoplastic agent which exerts its effect in cells by interfering with the polymerization of microtubules. Microtubules are one type of fibre which constitutes the cytoskeleton, and the dynamic microtubule network has several important roles in the cell, including vesicular transport, forming the mitotic spindle and in cytokinesis. Several drugs including vincristine and colcemid are similar to nocodazole in that they interfere with microtubule polymerization.

<span class="mw-page-title-main">Cyclin</span> Group of proteins

Cyclin is a family of proteins that controls the progression of a cell through the cell cycle by activating cyclin-dependent kinase (CDK) enzymes or group of enzymes required for synthesis of cell cycle.

Maturation-promoting factor (abbreviated MPF, also called mitosis-promoting factor or M-Phase-promoting factor) is the cyclin-Cdk complex that was discovered first in frog eggs. It stimulates the mitotic and meiotic phases of the cell cycle. MPF promotes the entrance into mitosis (the M phase) from the G2 phase by phosphorylating multiple proteins needed during mitosis. MPF is activated at the end of G2 by a phosphatase, which removes an inhibitory phosphate group added earlier.

<span class="mw-page-title-main">Spindle checkpoint</span> Cell cycle checkpoint

The spindle checkpoint, also known as the metaphase-to-anaphase transition, the spindle assembly checkpoint (SAC), the metaphase checkpoint, or the mitotic checkpoint, is a cell cycle checkpoint during metaphase of mitosis or meiosis that prevents the separation of the duplicated chromosomes (anaphase) until each chromosome is properly attached to the spindle. To achieve proper segregation, the two kinetochores on the sister chromatids must be attached to opposite spindle poles. Only this pattern of attachment will ensure that each daughter cell receives one copy of the chromosome. The defining biochemical feature of this checkpoint is the stimulation of the anaphase-promoting complex by M-phase cyclin-CDK complexes, which in turn causes the proteolytic destruction of cyclins and proteins that hold the sister chromatids together.

G<sub>2</sub> phase Second growth phase in the eukaryotic cell cycle, prior to mitosis

G2 phase, Gap 2 phase, or Growth 2 phase, is the third subphase of interphase in the cell cycle directly preceding mitosis. It follows the successful completion of S phase, during which the cell’s DNA is replicated. G2 phase ends with the onset of prophase, the first phase of mitosis in which the cell’s chromatin condenses into chromosomes.

Endoreduplication is replication of the nuclear genome in the absence of mitosis, which leads to elevated nuclear gene content and polyploidy. Endoreplication can be understood simply as a variant form of the mitotic cell cycle (G1-S-G2-M) in which mitosis is circumvented entirely, due to modulation of cyclin-dependent kinase (CDK) activity. Examples of endoreplication characterized in arthropod, mammalian, and plant species suggest that it is a universal developmental mechanism responsible for the differentiation and morphogenesis of cell types that fulfill an array of biological functions. While endoreplication is often limited to specific cell types in animals, it is considerably more widespread in plants, such that polyploidy can be detected in the majority of plant tissues.

A spindle poison, also known as a spindle toxin, is a poison that disrupts cell division by affecting the protein threads that connect the centromere regions of chromosomes, known as spindles. Spindle poisons effectively cease the production of new cells by interrupting the mitosis phase of cell division at the spindle assembly checkpoint (SAC). However, as numerous and varied as they are, spindle poisons are not yet 100% effective at ending the formation of tumors (neoplasms). Although not 100% effective, substantive therapeutic efficacy has been found in these types of chemotherapeutic treatments. The mitotic spindle is composed of microtubules that aid, along with regulatory proteins, each other in the activity of appropriately segregating replicated chromosomes. Certain compounds affecting the mitotic spindle have proven highly effective against solid tumors and hematological malignancies.

<span class="mw-page-title-main">Cell cycle checkpoint</span> Control mechanism in the eukaryotic cell cycle

Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring only when favorable conditions are met. There are many checkpoints in the cell cycle, but the three major ones are: the G1 checkpoint, also known as the Start or restriction checkpoint or Major Checkpoint; the G2/M checkpoint; and the metaphase-to-anaphase transition, also known as the spindle checkpoint. Progression through these checkpoints is largely determined by the activation of cyclin-dependent kinases by regulatory protein subunits called cyclins, different forms of which are produced at each stage of the cell cycle to control the specific events that occur therein.

<span class="mw-page-title-main">G1/S transition</span> Stage in cell cycle

The G1/S transition is a stage in the cell cycle at the boundary between the G1 phase, in which the cell grows, and the S phase, during which DNA is replicated. It is governed by cell cycle checkpoints to ensure cell cycle integrity and the subsequent S phase can pause in response to improperly or partially replicated DNA. During this transition the cell makes decisions to become quiescent, differentiate, make DNA repairs, or proliferate based on environmental cues and molecular signaling inputs. The G1/S transition occurs late in G1 and the absence or improper application of this highly regulated checkpoint can lead to cellular transformation and disease states such as cancer.

Cyclin A is a member of the cyclin family, a group of proteins that function in regulating progression through the cell cycle. The stages that a cell passes through that culminate in its division and replication are collectively known as the cell cycle Since the successful division and replication of a cell is essential for its survival, the cell cycle is tightly regulated by several components to ensure the efficient and error-free progression through the cell cycle. One such regulatory component is cyclin A which plays a role in the regulation of two different cell cycle stages.

A series of biochemical switches control transitions between and within the various phases of the cell cycle. The cell cycle is a series of complex, ordered, sequential events that control how a single cell divides into two cells, and involves several different phases. The phases include the G1 and G2 phases, DNA replication or S phase, and the actual process of cell division, mitosis or M phase. During the M phase, the chromosomes separate and cytokinesis occurs.

Cell cycle analysis by DNA content measurement is a method that most frequently employs flow cytometry to distinguish cells in different phases of the cell cycle. Before analysis, the cells are usually permeabilised and treated with a fluorescent dye that stains DNA quantitatively, such as propidium iodide (PI) or 4,6-diamidino-2-phenylindole (DAPI). The fluorescence intensity of the stained cells correlates with the amount of DNA they contain. As the DNA content doubles during the S phase, the DNA content (and thereby intensity of fluorescence) of cells in the G0 phase and G1 phase (before S), in the S phase, and in the G2 phase and M phase (after S) identifies the cell cycle phase position in the major phases (G0/G1 versus S versus G2/M phase) of the cell cycle. The cellular DNA content of individual cells is often plotted as their frequency histogram to provide information about relative frequency (percentage) of cells in the major phases of the cell cycle.

Mitotic exit is an important transition point that signifies the end of mitosis and the onset of new G1 phase for a cell, and the cell needs to rely on specific control mechanisms to ensure that once it exits mitosis, it never returns to mitosis until it has gone through G1, S, and G2 phases and passed all the necessary checkpoints. Many factors including cyclins, cyclin-dependent kinases (CDKs), ubiquitin ligases, inhibitors of cyclin-dependent kinases, and reversible phosphorylations regulate mitotic exit to ensure that cell cycle events occur in correct order with fewest errors. The end of mitosis is characterized by spindle breakdown, shortened kinetochore microtubules, and pronounced outgrowth of astral (non-kinetochore) microtubules. For a normal eukaryotic cell, mitotic exit is irreversible.

Induced cell cycle arrest is the use of a chemical or genetic manipulation to artificially halt progression through the cell cycle. Cellular processes like genome duplication and cell division stop. It can be temporary or permanent. It is an artificial activation of naturally occurring cell cycle checkpoints, induced by exogenous stimuli controlled by an experimenter.

References

  1. ↑ Keng, PC (September 1980). "Synchronization of 9L rat brain tumor cells by centrifugal elutriation". Cell Biophysics. 2 (3): 191–206. doi:10.1007/BF02790449. PMID   6159093. S2CID   7679022.
  2. ↑ Krek, Wilhelm (1995). Methods in Enzymology. Elsevier Inc. pp. 114–124.
  3. 1 2 3 Banfalvi G. (2011) Overview of Cell Synchronization. In: Banfalvi G. (eds) Cell Cycle Synchronization. Methods in Molecular Biology (Methods and Protocols), vol 761. Humana Press.
  4. ↑ Coquelle, A; et al. (30 November 2006). "Enrichment of non-synchronized cells in the G1, S and G2 phases of the cell cycle for the study of apoptosis". Biochemical Pharmacology. 72 (11): 1396–1404. doi:10.1016/j.bcp.2006.04.014. PMID   16765323.
  5. ↑ Zieve, Gary W.; Turnbull, Deborah; Mullins, J.Michael; McIntosh, J.Richard (April 1980). "Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor Nocodazole: Nocodazole accumulated mitotic cells". Experimental Cell Research. 126 (2): 397–405. doi:10.1016/0014-4827(80)90279-7. PMID   6153987.
  6. ↑ Vassilev, Lyubomir T (8 November 2006). "Cell Cycle Synchronization at the G2/M Phase Border by Reversible Inhibition of CDK1". Cell Cycle. 5 (22): 2555–2556. doi: 10.4161/cc.5.22.3463 . PMID   17172841 – via Taylor & Francis Online.
  7. ↑ Azevedo, W. F.; Leclerc, S.; Meijer, L.; Havlicek, I.; Strnad, M.; Kim, S. H. (1997). "Inhibition of cyclin-dependent kinases by a purine analogs: crystal structure of human cdk2 complexed with roscovitine". Eur. J. Biochem. 243 (1–2): 518–526. doi: 10.1111/j.1432-1033.1997.0518a.x . PMID   9030780.
  8. ↑ G. Banfalvi (ed.), Cell Cycle Synchronization, Methods in Molecular Biology 761, DOI 10.1007/978-1-61779-182-6_10, © Springer Science+Business Media, LLC 2011
  9. ↑ Koc, Ahmet; Wheeler, Linda J.; Mathews, Christopher K.; Merrill, Gary F. (21 October 2003). "Hydroxyurea Arrests DNA Replication by a Mechanism That Preserves Basal dNTP Pools". Journal of Biological Chemistry. 279 (1): 223–230. doi: 10.1074/jbc.m303952200 . PMID   14573610.
  10. ↑ Nagano, H; Ikegami, S (November 1980). "Aphidicolin: a specific inhibitor of eukaryotic DNA polymerase alpha". Seikagaku: The Journal of Japanese Biomedical Society. 52 (11): 1208–1216. PMID   6790638.
  11. ↑ Sala, F.; Galli, M. G.; Levi, M.; Burroni, D.; Parisi, B.; Pedrali-Noy, G.; Spadari, S. (1981). "Functional roles of the plant alpha-like and gamma-like DNA polymerases". FEBS Lett. 124 (1): 112–118. doi:10.1016/0014-5793(81)80064-6. PMID   6783441. S2CID   85153639.
  12. ↑ Keyomarsi, Khandan; Sandoval, Larue; Band, Vilma; Pardee, Arthur B. (1 July 1991). "Synchronization of Tumor and Normal Cells from G1 to Multiple Cell Cycles by Lovastatin". Cancer Research. 51 (13): 3602–3609. PMID   1711413.
  13. ↑ "Mitotic cell selection". Biology Online Dictionary. 3 October 2005. Archived from the original on 2 April 2019. Retrieved 15 December 2018.
  14. 1 2 Davis, Penny K; Ho, Alan; Dowdy, Steven F (June 2001). "Biological Methods for Cell-Cycle Synchronization of Mammalian Cells". BioTechniques. 30 (3): 1322–1331. doi: 10.2144/01306rv01 . PMID   11414226.
  15. ↑ Zeng, Qi; Hong, Wanjin (11 March 2008). "The Emerging Role of the Hippo Pathway in Cell Contact Inhibition, Organ Size Control, and Cancer Development in Mammals". Cancer Cell. 13 (3): 188–192. doi: 10.1016/j.ccr.2008.02.011 . PMID   18328423.
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