Cell synchronization: Difference between revisions

Revision as of 03:16, 15 December 2018

Revision as of 04:53, 15 December 2018

Line 1:

'''Cell Synchronizationsynchronization''' is a process by which cells in a culture at different stages of the [[cell cycle]] are brought to the same phase. “CellCell synchrony”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:; “Physicalphysical Fractionation”fractionization and “Chemicalchemical blockade.”

==Physical Separation==

Line 15:

'''(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 phase|G1]], [[S phase|S]], [[G2 phase|G2]], and [[M-phase|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<ref>{{Cite journal|last=Keng|first=PC|date=September 1980|title=Synchronization of 9L rat brain tumor cells by centrifugal elutriation.|url=https://www-ncbi-nlm-nih-gov.stanford.idm.oclc.org/pubmed/6159093?dopt=Abstract|journal=Cell Biophysics|volume=2(3)|pages=191-206|via=PubMed}}</ref>.

Essentially, largerLarger 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<ref>{{Cite book|title=Methods in Enzymology|last=Krek|first=Wilhelm|publisher=Elsevier Inc.|year=1995|isbn=|___location=|pages=114-124}}</ref>.

===[[Flow cytometry|Flow Cytometry]] and [[Cell sorting|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]], [[Bisbenzimide|Hoechst 33342]], [[DAPI]], [[7-Aminoactinomycin D|7-Aminoactinomysin D]], [[Plicamycin|Mithramycin]], [[Anthraquinone|DRAQ5]], or TO-PRO-3, allowing for determination of phase by DNA quantity<ref name=":0">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.</ref>. However, if these cells have been fixed, they are dead and can no longercannot 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 editing|genome edited]] such that some cellular proteins are made with conjugated fluorescent tags (likesuch as [[Green fluorescent protein|GFP]], [[mCherry]], and [[Luciferase]], etc.) 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<ref>{{Cite journal|last=Coquelle|first=A|last2=et al.|date=30 November 2006|title=Enrichment of non-synchronized cells in the G1, S and G2 phases of the cell cycle for the study of apoptosis.|url=|journal=Biochemical Pharmacology|volume=72(11)|pages=1396-1404|doi=10.1016/j.bcp.2006.04.014|via=PubMed}}</ref>. 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. LimitationLimitations include:

* for light scatter measurements, poor resolution between G2 and M (likeas with centrifugal elutriation)

* for fixed cells, unable to maintain living cultures

* for unfixed but dyed cells, possible disruption or [[mutagenesis]] of cells because of dye treatment

* some population heterogeneity may be maintained, as size separation may not always be accurate for measuring phase and not all cells may be at the same point within each phase (early G1 vs late G1)

* for DNA -edited cells, extensivethe timeprocess mustmay betake putan intoextended thisperiod processof time

==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 checkpoint|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:

* the proportion of synchronized cells is not sufficiently highinsufficient

* chemical manipulations may disrupt cellular function and/or kill a portion of cells

* the treatment is toxic and not applicable ''in vivo'' (only relevant if considering clinical application)<ref name=":0" />

=== Arrest in M ===

Mitotic arrest can be 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 checkpoint|spindle assembly checkpoint]] in the metaphase/anaphase transition. The microtubule poison works by blocking the formation of the [[Spindle apparatus|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.<ref>{{Cite journal|last=Zieve|first=Gary W.|last2=Turnbull|first2=Deborah|last3=Mullins|first3=J.Michael|last4=McIntosh|first4=J.Richard|date=April 1980|title=Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor Nocodazole: Nocodazole accumulated mitotic cells|url=|journal=Experimental Cell Research|volume=126(2)|pages=397-405|doi=10.1016/0014-4827(80)90279-7|via=Elsevier Science Direct}}</ref> Because microtubules are vital in other cellular functions, sustained use of nocodazole can result in disruption of those functions, andcausing cell death.

==== Inhibition of [[Cyclin-dependent kinase 1|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 synchronziedsynchronized >95% orof cycling cells (including cancer cells), and released cells rapidly enter mitosis.<ref>{{Cite journal|last=Vassilev|first=Lyubomir T|date=8 November 2006|title=Cell Cycle Synchronization at the G2/M Phase Border by Reversible Inhibition of CDK1|url=|journal=Cell Cycle|volume=5(22)|pages=2555-2556|via=Taylor & Francis Online}}</ref>

==== [[Roscovitine]] ====

Roscovitine can be used to inhibit the activity of [[Cyclin-dependent kinase|cyclin-dependent kinases]] (CDKs) by competing with [[ATP]] in the ATP-binding region of the kinase. It'sIts effects are highly potent, and arrestarresting 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.<ref>Azevedo, W. F., Leclerc, S., Meijer, L., Havlicek, I., Strnad, M., and 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, 518–526.</ref>

==== [[Colchicine]] ====

Colchine 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 checkpoint|spindle assembly checkpoint]].

=== Arrest in S (G1/S arrest) ===

Line 58:

==== Double [[thymidine]] block ====

High concentrations of thymidine interrupt the [[Nucleotide metabolism|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.<ref>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</ref> The process isbegins aswith followsa - treattreatment with thymidine, washwashing of the culture, treatfollowed withby another thymidine againtreatment.

==== [[Hydroxycarbamide|Hydroxyurea]] ====

Hydroxyurea decreases the production of [[Dideoxynucleotide|dNTPs]] by inhibiting the enzyme [[ribonucleotide reductase]]. This serves to halt DNA synthesis by depriving [[DNA polymerase]] of dNTPs at [[Replication fork|replication forks]].<ref>{{Cite journal|last=Koc|first=Ahmet|last2=Wheeler|first2=Linda J.|last3=Mathews|first3=Christopher K.|last4=Merrill|first4=Gary F.|date=21 October 2003|title=Hydroxyurea Arrests DNA Replication by a Mechanism That Preserves Basal dNTP Pools|url=|journal=Journal of Biological Chemistry|volume=279|pages=223-230|via=}}</ref> Hydroxyurea is also used to treat certain types of cancer orand blood disorders.

==== [[Aphidicolin]] ====

Line 67:

=== Arrest in G1 ===

A single commonly -used chemical method exists for synchronization of cells in G1. It involves [[Lovastatin]], a reversible competitive inhibitor of [[HMG-CoA reductase|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 rescueundo 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.<ref>{{Cite journal|last=Keyomarsi|first=Khandan|last2=Sandoval|first2=Larue|last3=Band|first3=Vilma|last4=Pardee|first4=Arthur B.|date=1 July 1991|title=Synchronization of Tumor and Normal Cells from G1 to Multiple Cell Cycles by Lovastatin|url=|journal=American Association for Cancer Research|volume=51(13)|pages=3602-3609|pmid=1711413|via=PubMed}}</ref>

== Other Methods of Synchronization ==

=== Mitotic Selection ===

Mitotic selection is a drug-free procedure for the selection of mitotic cells from ana exponentiallymonolayer growingundergoing monolayerexponential growth.<ref>{{Cite web|url=https://www.biology-online.org/dictionary/Mitotic_cell_selection|title=Mitotic cell selection|last=|first=|date=3 October 2005|website=Biology Online Dictionary|archive-url=|archive-date=|dead-url=|access-date=}}</ref> During mitosis, cells undergo changes in morphology, and mitotic selection takes advantage of this in adherant cells grown in a [[monolayer]]. The cells become more spherical, decreasing the surface area of cell membrane still attached to the culture plate. Mitotic cells can therefore be completely detached by gently shaking and collected from the [[Precipitation (chemistry)|supernatent]].<ref name=":0" />

===Nutrient/Serum Deprivation===

Elimination of [[Blood plasma|serum]] from the [[culture medium]] for about 24 hours results in the accumulation of cells at the transition between [[G0 phase|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 orwhile others proceed through the cell cycle at variable rates.<ref name=":1">{{Cite journal|last=Davis|first=Penny K|last2=Ho|first2=Alan|last3=Dowdy|first3=Steven F|date=June 2001|title=Biological Methods for Cell-Cycle Synchronization of Mammalian Cells|url=|journal=BioTechniques|volume=30(3)|pages=1322-1331|via=}}</ref>

=== 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.<ref name=":1" /> Because of the proliferation-promoting mutations thatintrinsic theyto gaincancer, tumor cell lines are not usually able to undergo contact inhibition, though there are exceptions.<ref>{{Cite journal|last=Zeng|first=Qi|last2=Hong|first2=Wanjin|date=11 March 2008|title=The Emerging Role of the Hippo Pathway in Cell Contact Inhibition, Organ Size Control, and Cancer Development in Mammals|url=|journal=Cancer Cell|volume=13(3)|pages=188-192|doi=10.1016/j.ccr.2008.02.011|via=Elsevier Science Direct}}</ref>

==External links==