Tag: Cell Cycle
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Cell Cycle: Description, Diagram, Stages, and Checkpoints
Continue ReadingCell Cycle: Description, Diagram, Stages, and Checkpoints
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Cell Cycle: Definition, Stages, and Examples
Continue ReadingCell Cycle: Definition, Stages, and Examples
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Cell Cycle: Definition, Description, Stages, and Checkpoints
Continue ReadingCell Cycle Introduction
Beings of earth are programmed to be born, grow, reproduce, and die. Multicellular eukaryotes develop from a unit cell to an array of trillion celled creature by a simple mechanism of cell division.
The capacity to divide is the measure of growth of an organism and the capacity to grow differs for every species are limited by internal and external factors.
Proliferation of cell can be infinite which might have resulted in an overwhelming cornucopia of cells.
The cell proliferation and growth are finite and regulated by an organism by spatial and temporal means according to its functional ability.
The increase in cell corresponds to the growth an organism exhibits; regulated or stopped after a particular period.
Plants tends to show a regulated unlimited growth; developing over lifetime a distinctive character of plants to support the sedentary life; but the growth is governed by internal and external cues of development.
Specialized regions such as meristem plays a primary role in plant cell division and growth.
Growth is regulated at cellular level by the cell cycle and growth regulators plays a significant role in inducing growth and supporting the structural composition of growth.
Periodic stages of cell cycle involve the major function of a single cell over a lifetime following a repetitive phase of development and function.
What is Cell Cycle
Cell cycle is the sequential process taking place to regulate the growth of organism; cell divides to produce a genetic replica and enters the stage of cell growth.
Cell growth involves the synthesis of organic material and integrates information across its counter parts for synchronous development of the whole body.
The cell synthesis phase lasts till a cell reaches its maturity; on initiation the cell again divides to produce new cell and the process continues.
Cell cycle is a sequential development of cell between two cell divisions.
The cycle is genetically controlled and are programmed in every cell and are specific for each region.
Varied species has variable time length of cell cycle decided by physiological and influences pertaining to their niche.
Cell Cycle History
Discovery of microscope was a breakthrough in understanding the physiology at cellular level.
Robert Hooke was first to observe cork cells; from there microscopic observation revealed the world of microbiota.
The cell was discovered and components, functions and the significance were found. In late 19th century; it was deducted that new cell develops from the pre – existing cell by the process of cell division.
Cell division led to elucidation of cell cycle processes which was determined to be the principal process regulating growth and development of the cell and the organism.
But the DNA replication restricted to S phase of the cell cycle was not discovered until 1950 by Alma Howard and Stephane Pele.
The conservative nature of the cell cycle was identified in 1980’s; were scientists found out that the molecular processes of cell cycle is similar in all eukaryotic organisms; from experimenting various research on organisms.
Cell cycle regulators and genes “START” was discovered by Leland Hartwell.
Paul Nurse discovered CDK which runs the cell cycle based on the genetic expression.
Timothy Hunt discovered cyclin activating subunit of CDK’s. Leland Hartwell, Paul Nurse and Timothy Hunt shared Nobel Prize for Medicine in 2001.
Characteristic of Cell Cycle
o The sequential progress of cell growth and cell division to form new daughter cells are termed as cell cycle.
o The sequences are divided into 4 divergent phases falls in 2 majors they are: Interphase and Mitotic Phase.
o The stages of cell cycle is common for eukaryotic organisms.
o The cell cycle through evolution is conservative because of the similarities of the phases in all organisms.
o The cycle is well regulated by genes and protein kinases.
o Protein kinase action is same in all eukaryotic species signifies the conserved evolution and a proof that all organisms arise from a common ancestor who diverse from single celled unicellular organism to complex human beings.
o The stages of cycle are governed by time at which cells enter the cell cycle.
o Cell cycle is asynchronous in a living organism.
o The cell division is regulated and controlled by the protein kinases to maintain the structural integrity and functionality of the organism.
o As the organism grows few regions remains constant with no cell growth or maintained to a threshold level where cell death is balanced by cell division.
o In few parts the cells the growth might be rapid or enter the cycle first and other cells may enter later result in asynchronous cell division.
o Length of cell cycle varies from species to species. Few organisms have rapid replication system compared to other organism.
o For Example: Yeast completes its cell cycle in 90 minutes (about 1 and a half hours) whereas in human cell cycle lasts for 24 hours and in Drosophila it is about 8 minutes.
o Phase of cell cycle is sequential in continuously dividing cells were one phase follows the other in a constant order becomes inevitable for a proper functioning of the cell cycle are regulated by the controls of the cycle.
o In multicellular organisms, higher level of cell differentiation reduces the capacity of a cell to proceed the cycle.
o A post – mitotic differentiated cell restricts it at G1 phase or enter G0 phase a quiescent phase.Â
o Stem cells are an ideal example for cell cycle where it keeps on proliferating to maintain the integrity of the organism replacing the dead cells by new ones.
o Most of the cells completes the cycle whereas few other remains in the synthetic phase their lifetime without entering the dividing phase.
o Proceeding the phases of cell cycle is determined by the synthesis phase where a cell cycle sets up an upper threshold level which must be crossed to enter the division phase.
Cell Cycle Phases
The cell cycle is common for all eukaryotic organisms; travelling through 2 major phases based on the cell division: Interphase and Mitotic Phase.
Interphase consists of 3 phases Gap 1 phase, Synthesis Phase and Gap 2 Phase.
Similarly, mitosis has four phases Prophase, metaphase, anaphase, and telophase.
The development of cells through these phases are influenced and facilitated by heterodimeric protein kinases – Cyclin and Cyclin Dependent Kinases.
I. Interphase
Interphase of the cell cycle is non dividing phase where the cells undergo major synthesis and other integrating functions transducing signals and coordinate with other cells to produce an overall reaction to the stimulus.
Biosynthetic activity is more prevalent in this phase; absence of cell division marks a resting period; varies for every differentiated cells.
Certain cells might have a short resting period and certain cells such as neurons loses the power of differentiation and remains constant.
II. Gap1 Phase
The first gap phase starts just after the 2 daughter cells formed by mitosis is long and are species specific.
Intermediate to Mitosis and the synthesis phase the cell increases in size by synthesizing proteins and RNA for Synthesis Phase.
In a cell cycle, G1 phase is regulated by the external and internal factors.
External limiting factor is the availability of the nutrients in general eukaryotic organism and additional hormonal induction in plants.
Most of the organism restrict their cycle at G1 or enter G0, few cells on proper hormonal induction will reenter the cell cycle from G0 phase; specifically, plants have a specialized region called the meristem which continuously divide to produce cell growth.
The G1 phase determines the fate of the cell whether to enter cell cycle or to retain in G1 phase.
The cell must grow to an appropriate size and must synthesize proteins and RNA to enter S phase, and this is regulated by Cyclins and CDK’s.
III. Synthesis Phase
The threshold point – restriction point in mammals and START in other organisms; when crossed the cells enter the synthesis phase.
The G1 phase prepared the cell and its components to synthesis phase to get committed to cell division.
The cell division is arrested when external aids are less or unavailable.
The DNA replication doubles the genetic material, but the chromosome is not condensed and replicated material remains as chromatin.
Synthesis of genetic material is significant in this phase where cells are committed to divide and produce 2 daughter cells in most of the metazoans.
The S phase does not induce the increase in chromosomal numbers but doubles the genetic material.
Endoreduplication a landmark in plant endosperm is the different process of the metazoans permits DNA replication without mitosis results in ploidy.
In animals, Drosophila’s salivary glands and mammal’s hepatocytes exhibit endoreplication.
IV. Gap2 Phase
Synthesis phase and Mitosis phase is separated by G2 Phase; marked by absence of synthetic function.
This phase is a preparatory phase where the cell again enters the mitotic cycle for division.
Decondensed chromatin starts a preliminary condensation marks the start of mitotic phase.
Condensation inhibits the RNA synthesis gradually as the synthesis sites are being compactly wound for replication.
G2 Phase has minimal RNA synthesis which reduces or absent in Mitotic phase.
V. Mitotic Phase
The changes in above phases are minimal or not clearly visible in microscopes whereas the changes in M Phase are easily detectable.
The phase has 4 parts in which the division takes place systematically and continuously.
The cell stages are easily visible in plant parts as the specialized dividing region – MERISTEM is prevalent in roots and shoots are continuously dividing providing a mechanical support and functional integrity to the plants.
The 4 phases are: Prophase, Metaphase, Anaphase and Telophase.
Each phase has a distinctive change to be identified and Eukaryotic cells replicates in the same order in most of the organisms.
Prophase: The prophase is marked by chromosomal condensation and disintegration of cellular components and assembly of cytoskeletons for cell division. RNA synthesis is inhibited.
Metaphase: Nuclear membrane is eliminated completely chromosomes are completely condensed. The cytoskeleton – spindle fibers attach to the kinetochores. The chromosomes are aligned in the equatorial plate.
Anaphase: Chromosomal split forms daughter chromatids; travels to the opposite poles. The chromosomes are V – Shaped as they are dragged to the opposite sites.
Telophase: Microtubules disappear and chromosomes decondense to chromatin mass. Nuclear envelope starts to form. The disintegrated organelles form again.
These phases mark the Karyokinesis were the nucleus and other cell parts are newly formed.
Cytokinesis is the formation of daughter cells after mitosis; indicated by furrow which starts to differentiate two daughter cells grows gradually forming a cell plate while the organelles formed gets segregated. Cell plate represents the lamella between 2 cell walls.
Cell Cycle Regulation
The cell cycle is regulated by heterodimeric protein which was first discovered in sea urchin; the cyclic appearance named the compound CYCLIN; is the regulatory subunit activating Cyclin dependent Kinases – Catalytic subunit.
The involvement of CDK’s promotes an intricate network of mechanism triggering positive and negative response creates a loop controlling the cell cycle.
The protein kinases phosphorylate other repressors of the cell cycle traversing cell over distinct phases of development.
The cell cycle and the basic protein kinases were conserved throughout the eukaryotic cells, but the CDK types of serine – threonine special class are not conserved; varying from quantity to Plant types
The Animals has a distinctive CDK types; CDK1 – CDK7 are present in animals and CDK A – CDK E in plants.
Cyclins also falls under 5 types A, B, C, D, and H. The cyclin is transcript from most conserved part of the human genome preserving its integrity over their period.
CDK and Cyclin combines to regulate the cell cycle at each phase; characterized by specific combination of Cyclin and CDK promoting the cell to reproduce for the growth of an organism.
Distinct phases have different cyclin – CDK complex varying in animals and plants.
Monomeric CDK does not participate in regulation of cell cycle; CDK require cyclin subunit to be activated by CDK activating kinases.
These heteromeric protein can be inhibited by inhibiting proteins – Kip-related proteins by inhibitory phosphorylation.
The CDK’s and cyclins are dependent on the rate of turnover, transcription, and translational control.
Cell cycle Regulation in Plants
The CDK – CYC (cyclin) complex regulates the cell cycle. Each phase expresses specific complex for the cells to be accustomed to proceeding through cell cycle. CDK – A;1 is expressed in all phases but are activated by D type cyclins in G1 Phase to traverse the restriction point.
Transcription of CDK A – CYC D accumulates and facilitates the preparation of cell to transverse G1/S phase to commit to cell cycle.
The synthesis of CDKA – CYCD complex when increase in quantity reaches a restriction point and when it exceeds the point the cell commits to cell division.
When the point is not crossed the cell remains in G1 phase and eventually enters G0 phase indicating maturation and takes part in regular cellular activities.
In S phase CDKA – CYCA is expressed during the synthesis of DNA duplicate.
Traversing from S phase the CYCD starts expressing with the entry of G2 phase along with CYCA2 – CDKA later this followed by the expression of CDKB – CYCB which on reaching a threshold limit indicates the cells are ready for the cell division.
Again, at the end of G2 phase the positive feedback of CDKB-CYCB induces the CDKA-CYC3 to be produced all along the mitosis and prevails in G1 PHASE to increase the growth of the cell.
Cell Cycle Plant Hormones
Plant growth hormones Auxins, cytokinin and brassinosteroids has a significant role in control of the cell cycle.
Auxin and Cytokinin are essential for cell proliferation and are widely expressed in the meristematic tissues of plants promotes plant growth by cell division induces the cell cycle.
These were discovered from the mutational studies inhibiting the hormones and observing cell cycle.
Cytokinin helps in G1/S transition and G2/M transition by dephosphorylating CDK’s thereby activating the cell cycle or inhibits WEE 1 kinase by down regulation.
Abscisic acid inhibits cell cycles by promoting inhibitors such as KRP1 in certain plants.
Similarly, Jasmonic Acid inhibits DNA replication when administered during G1 phase and has minimal inhibition effects in later stages.
Ethylene promotes cell death in the developing cell cycle.
Cell Cycle Citations
- Cell cycle checkpoints. Curr Opin Cell Biol . 1994 Dec;6(6):872-6.
- Cell Cycle Control: A System of Interlinking Oscillators. Methods Mol Biol . 2016;1342:3-19.
- Cell cycle development. Dev Cell . 2004 Mar;6(3):321-7.
- Linking the Cell Cycle to Cell Fate Decisions. Trends Cell Biol . 2015 Oct;25(10):592-600.
- The cell cycle: a review. Vet Pathol . 1998 Nov;35(6):461-78.
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Cell Cycle: Phases, Diagram, Stage, and Checkpoints
Continue ReadingCell Cycle Phases
o Every cell has a life cycle that begins with the birth of the cell and ends with the death or division of the cell.
Cell Cycle Diagram
o M ⇒ Mitosis
o G1 ⇒ Gap 1
o G2 ⇒ Gap 2
o S ⇒ Synthesis
o G0 ⇒ Gap 0/Resting.
Quiescent/ Senescent
Gap 0 / G0
o The cell has left the cycle and has stopped dividing.
o In humans, liver cells spend a great deal of time in G0.
o Mature neurons and muscle cells are in G0 permanently.
o Liver (hepatic) and pancreatic cells are normally in G0 but can reenter normal division
Interphase
Gap / G1 Phase
o G1 is normally the longest phase.
o Cells increase in size in Gap 1, regions of heterochromatin has been unwound and decondensed.
o RNA synthesis and protein synthesis is very active.
o The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis.
o The main factor in triggering the beginning of S is cell size based upon the ratio of cytoplasm to DNA.
Cell Cycle Checkpoints
Synthesis / S Phase
o DNA replication occurs during this phase.
o Organelles and proteins are produced more slowly.
o Each chromosome is exactly duplicated, but the cell is still considered to have the same number of chromosomes, only now, each chromosome is made of two identical sister chromatids.
Gap 2 / G2 Phase
o During the gap between DNA synthesis and mitosis, the cell will continue to grow.
o Cellular organelles continue to duplicate.
o RNA and protein (especially tubulin for microtubules) are actively synthesized.
o The G2 checkpoint control mechanism ensures that everything is ready to enter the M (mitosis/meiosis) phase and divide.
o It checks for mitosis promoting factor (MPF), when the level is high enough, mitosis is triggered.
Cell Division / Mitosis / Meiosis / M Phase
o Cell growth stops at this stage and cellular energy is focused on the orderly division into two daughter cells.
o A checkpoint in the middle of mitosis (Metaphase Checkpoint) ensures that the cell is ready to complete cell division.
Cell Cycle Citations
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Cell Cycle Assay With Flow Cytometry: Detailed...
Continue ReadingAbout PhD In Psychology
Cell cycle analysis by quantitation of DNA content was one of the fantastic applications of flow cytometry.
The mammalian DNA, yeast, DNA, plant DNA or bacterial DNA can be easily stained by a variety of DNA binding dyes such as propidium iodide.
The premise of these DNA binding dyes is that they are stoichiometric, i.e. they bind in proportion to the DNA amount present in the cell.
In this way if cells are in S phase will have more DNA than cells in G1 phase.Â
They will take up proportionally more dye and will fluoresce more brightly until they have doubled their DNA content.
The cells in G2 phase will be approximately twice as bright as cells in G1. DNA-binding dyes include propidium iodide (PI), Hoechst 33342, 33258 and S769121, 7-aminoactinomycin-D (7-AAD), DRAQ5™ and DRAQ7™, TO-PRO-3, 4’6’-diamidino-2-phenylindole (DAPI).
In general, cells must be fixed and permeabilized to allow entry of the DNA binding dye which is otherwise actively pumped out by living cells.
Alcohol or aldehyde are commonly used to fix the cells.
Alcohol is a dehydrating fixative which also permeabilizes cells of interest. This will allow easy access of the dye to the DNA and gives good profiles (low coefficient of variation, CV).
The disadvantage of alcohol fixation is that it is often incompatible with fluorescent proteins and some surface markers.
For simultaneously proteins or surface markers examination, use of an aldehyde (cross-linking) fixative, such as paraformaldehyde is more efficient and appropriate.
Aldehyde fixation in cell cycle analysis may lead to poorer quality profiles (higher CVs) but will allow simultaneous detection of other fluorochromes and membrane-bound surface proteins.
Another disadvantage of paraformaldehyde fixation is, generally paraformaldehyde does not permeabilize the cell membrane, and so further sample processing is required.
With fixed cells, samples may be processed, stained and analyzed at the conclusion of an experiment.
Alcohol-fixed cells are stable for several weeks at 4°C and several weeks in -20 °C.
Aldehyde fixed cells are stable for 2 to 3 days. An alternative method to allow the DNA dye into the cells is to permeabilize them with a detergent such as Triton X-100 (0.1%) or NP40 (0.1%).
Usually, it is also necessary to combine a fixation (paraformaldehyde) and permeabilization (Triton X-100) for the intracellular staining.
On the other hand, methods are available, e.g. use of citrate buffers (in combination with detergent), although these are not so widely used.
There are also some dyes that will enter live cells and quantitatively bind to DNA, these include Hoechst 33342, DRAQ5â„¢ (ab108410) and the DyeCycle dyes
Adopted from BioRender
Cell Cycle Assay Reagents
70% Ethanol
Propidium iodide (stock solution 50 µg/ml)
Ribonuclease I (stock 100 µg/ml)
Cell Cycle Assay Procedure
1. Seed appropriate number of cells (e.g. 1.5×105 MDA-MB-231, 1X105 MCF-7, and 1X105 SK-BR-3) in 6 well plates in 2 ml complete media.
Seed less number of cells in control wells if your cells are fast growing like MDA-MB-231 so that they will not die due to over confluence.
Incubate in 5% CO2 incubator for 18-24 hours.
2. When the cells are at 40-50% confluence, discard the media and add 1 ml of serum deprived media (containing 0.5% FBS) to synchronize the cells.
Incubate in 5% CO2 incubator for 6 hours.
3. After this period discard the media and add 1 ml complete media.
Then, treat the cells in same media at different doses of your test compound in triplicates. Make a negative control with no treatment.
Incubate in 5% CO2 incubator for 18-24 hours.
4. Harvest the cells (both dead and live cells) after the incubation period by trypsinizing them (add 500 μL Trypsin per well).
Pool triplicate wells into single 15 ml tube.
Centrifuge at 1000 rpm for 5 minutes to pellet down.
5. Wash the pelleted cells with 2-3 ml of PBS.
Re-centrifuge the cells at 1000 rpm for 5 minutes.
6. Give second washing with 2-3 ml PBS if necessary.
Again centrifuge cells at 1000 rpm for 5 minutes.
7. Discard the PBS. Gently mix the pelleted cells in the remaining PBS and fix each cell suspension in 1-3 ml of 70% ethanol (depending on the number of cells in the suspension).
8. Keep them at -200C for a minimum of 1 day (although they can stay for up to 2-3 days).
9. Spin down the cells at 1800 rpm for 5 minutes.
Discard the supernatant.
Wash the pellet in 2-3 ml PBS.
Again spin down at 1800 rpm for 5 minutes.
10. Give a second wash with PBS if necessary.
Spin down the cells at 1800 rpm for 5 minutes to remove the traces of ethanol.
Mix the cell pellet in remaining PBS.
11. Make PI-RNase master mix by taking 5μl of PI (2mg/ml stock) and 2μl of RNase (10mg/ml stock) in 300 μl PBS per sample.
12. Add 300 μl of master mix per sample. Mix well.
13. Incubate for 30 minutes in dark at RT.
Transfer to FACS flow tubes. Keep the tubes on ice.
14. Analyse the samples via FACS as soon as possible and interpret using software
Cell Cycle Assay Citations:
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