1. Introduction to the Cell Cycle
Promega Education Resources
Unit 7

2. Overview
In order for adult multicellular organisms to develop from a single fertilized egg, cell growth and division has to occur at the appropriate times and in the appropriate places.
When cell cycles proceed inappropriately (e.g., cells divide uncontrollably), pathological conditions like cancer can result.

3. Cells must accomplish two basic things during the cell cycle:
Copying cellular components
Dividing the cell so that components are distributed evenly to the daughter cells
The alternating “growth” and “division” activities of the cell is called the “cell cycle”.
The division activity corresponds to “M phase”.
The “growth” activity corresponds to “Interphase”.

4. Interphase Interphase can be subdivided into G1, S and G2 phases.
In yeast “Start” is at the end of G1; at this point the cell is committed to DNA synthesis.
In mammals, this is called the “restriction point”. This point late in G1 is a “checkpoint”; a cell will exit the cell cycle if certain requirements to proceed to synthesis are not met.
A second restriction point occurs in G2 before entry into mitosis.
Interphase M Interphase M Interphase
G S G2 M G S G2 M G1 S G2

5. Interphase: G1 Events during G1 Cell growth
Preparation of chromosomes for replication
Duplication of cellular components
G1 checkpoint (or restriction point); cell commits to division or exits from cell cycle

6. Interphase: S phase DNA replication Duplication of the centrosome
The centrosome is located near the nucleus of the cell and contains the microtubule organizing center MTOC in animal cells. It contains two centrioles that migrate to the poles before cell division and serve to organize the spindle.

7. Interphase: G2 Cell growth
Checkpoint (restriction point) for entry into M phase

8. M phase Cell division (mitosis or meiosis for germ cells)
Can be subdivided into four subphases:
Prophase
Metaphase
Anaphase
Telophase
Factors that influence M phase entry
Cellular Mass
Growth Rate
Time (During early embryogenesis, divisions may proceed rapidly, essentially alternating M and S phases, with little growth between them.)
Completion of DNA Replication

9. Studying the Cell Cycle
Much of what we know about the cell cycle comes from the study of model experimental systems:
Genetics
Yeast: Schizosaccharomyces pombe (fission yeast) and Saccharomyces cerevisae (budding yeast)
Short article from HHMI (
Biochemistry
Frog eggs
Mammalian cell culture
Some of our first clues about the genetic control of the cell cycle came from studies of yeast conducted in the 1970s by Lee Hartwell. He took advantage of the fact that baker’s yeast, Saccharomyces cerevisae, cells only stop growing and producing buds if they are abnormal.
Hartwell and other scientists were able to isolate temperature-sensitive mutations that specifically affected the cell cycle in yeast. Temperature sensitive mutations are particularly usefulfor understanding the genes involved in critical pathways like cell division. At one temperature (the permissive temperature), the mutation does not significantly affect function and cellular processes proceed normally or nearly normally, but at the restrictive temperature, the effects of the mutation are clearly seen.
With the cell division cycle (cdc) mutations in Saccharomyces cerevisae, budding and cell division proceeded normally at the lower temperature, but at the higher, restrictive temperature, yeast cells bearing a particular cdc mutation always arrest in the same point in the cell cycle.
Many of the genes that are responsible for the cell division disruption in yeast were later found in other multicellular organisms, including humans. Even more interesting, in multicellular organisms, disruption of many of these genes was linked to cancer.
Much of our understanding of the biochemistry of the cell cycle came from experiments using frog eggs. Specifically oocytes, eggs and embryos obtained from the African frog, Xenopus laevis, have been used to investigate cell cycle biochemistry. For instance, extracts from unfertilized Xenopus extracts contain all the materials required for multiple cell cycles. Such extracts have been used to demonstrate the role of MPF and cyclin B in cell cycle entry and exit from mitosis. (More in a later slide).

10. Control of the Cell Cycle
Intrinsic Control: Cyclins
Proteins whose levels rise and fall during the various phases of the cell cycle (primary regulators of the cyclin-dependent kinases)
Interact with the cyclin-dependent kinases (cdk)
Cdk levels are constant
Cdks must bind to cyclins to be activated
The complexes of cyclin+cdk act in concert. The cdks phosphorylate proteins that initiate or regulate cell cycle activities. Cyclins also may be involved in cdk target recognition.
Cdk activity is terminated by cyclin degradation (generally).
Cdk activity can be “fine tuned” by other mechanisms (i.e., inhibitory phosphorylation by Wee1 kinase, activation by cdc25 kinase. Cdk inhibitor proteins also can regulate the cyclin-cdk complexes.

11. Cyclins
Four classes
Defined by phase of the cell cycle in which they bind their cdk
G1/S phase cyclins-bind cdks at the end of G1, commit cell to DNA replication (cyclin E)
S phase cyclins- bind cdks during S phase, required to initiate replication (cyclin A)
M phase cyclins- bind cdks immediately before M phase, initiate early mitotic (or meiotic) events (cyclin B)
G1 cyclins- involved in progression through the checkpoint in late G1 (cyclin D)

12. M phase Promoting Factor (MPF)
Cytoplasmic protein factor responsible for initiation of meiotic and mitotic phases of cell cycles in eukaryotes
First detected in unfertilized amphibian eggs
Progesterone triggers oocyte maturation; MPF is important for progression from meiosis I)
MPF activity rises at beginning of meiosis II and before mitotic divisions after fertilization
MPF activation, oocyte activation and mitosis will not occur without protein synthesis*
Originally MPF was an abbreviation for Maturation Promoting Factor because it was first identified as critical for Xenopus egg maturation. Later it was found to be necessary for progression to mitosis as well as meiosis, so became known as M phase promoting factor.
References:
J. Gerhart et al Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs. J. Cell Biol. 98:1247.
A. W. Murray et al Cyclin synthesis drives the early embryonic cell cycle. Nature 339:275
*See Gerhart Use of cycloheximide (protein synthesis inhibitor) to prevent initial MPF activation. Cycloheximide does not affect auto-activation of MPF after the initiation step however.

13. What is MPF? MPF is a complex of proteins
In 1988, one of the proteins associated with MPF was shown to be homologous to the cdc2 gene of S. pombe
cdc2 gene encodes a protein kinase
Could one of the other components be cyclins?
Dunphy, W. G. et al. (1988) Cell. 54, 423.
Gautier, J. et al. (1988) Cell. 54, 433.

14. Xenopus oocyte maturation
Progesterone triggers oocyte maturation
Oocytes progress through two cycles of mieoisis, but the second meiotic division is arrested at metaphase II until fertilization
Oocytes are released from metaphase II arrest when exposed to sperm chromatin
Scientists often prepare extracts from oocytes that have been released from metaphase II arrest for their studies.
Cytoplasm extracted from oocytes will “cycle” when exposed to sperm chromatin

15. Key Experiment Murray and Kirschner (1989)
Determine that cyclin is involved (isolated “cycling” proteins from extract)
Show cyclin synthesis is required to drive cell cycle
Prepare extracts from unfertilized Xenopus eggs
Contain all materials required for multiple cell cycles
Can synthesize proteins from maternal mRNA in the cytoplasm
Can be mixed with chromatin from interphase sperm generates a haploid nucleus with sperm DNA, which will replicate, and the extract will go through “mitosis” (cycling extracts).

16. Experimental Details
Treat cycling extracts with limited amount of RNase A
Degrades all of the maternal mRNAs
Does not degrade the tRNAs or rRNAs necessary for protein synthesis (these RNAs are protected by proteins within the protein/RNA complexes)
Add RNasin Ribonuclease Inhibitor to inactivate RNase A
Add back cyclin B mRNA
Extracts resume cycling activity
Add cyclin B mRNA that encodes a protein with a mutated protein-degradation signal to parallel extracts
Extracts start to cycle, but get “stuck” in early mitotic events
Conclusion: Cyclin B synthesis and degradation are necessary for cycling.

17. Cell Cycle Checkpoints
The decision to proceed from one part of the cell cycle to another depends on a variety of factors
Growth
DNA replication
DNA integrity
Cellular integrity
The mechanisms that the cell has to monitor these factors act at “checkpoints”
Generally, the feedback from checkpoints is through negative regulation—sending a signal to stop the progression of the cell cycle rather than dialing back a positive signal.
Schematic of Cell Cycle Checkpoints

18. Cell Cycle Checkpoints
G1 (Restriction) Checkpoint
End of G1, just before onset of the S phase (DNA replication)
Yeast “start”; other eukaryotes “restriction point”
The options for the cell at this point:
divide, delay division, or exit the cell cycle
Cells can exit the cell cycle at this point into an arrested stage (G0)
When this checkpoint is passed, cdk4 and cyclin D interact. This interaction results in phosphorylation of the retinoblastoma protein, which in turn allows activation of the transcription factor E2F. Active E2F promotes expression of the cyclin E gene. Cyclin E (protein) and cdk 2 interact to promote the G1 to S phase transition. (Figure available here.)

19. p53 Mutations at the G1 Checkpoint
The gene encoding the p53 tumor suppressor protein is essential for stopping cells with damaged DNA from entering S phase.
Mutations in the p53 gene are found in more than 50% of human cancers.
Many mutations in p53 gene eliminate its ability to activate expression of target genes like the p21 gene. The p21 protein is a cyclin kinase inhibitor (CKI) that inhibits activity of the G1 cyclin-cdk complexes.
Notes for discussion:
Why would mutations in p53 gene be associated with cancers?
p53 targets also include genes involved in apoptosis. Why do you think these genes are targets of p53?

20. Cell Cycle Checkpoints
DNA Replication Checkpoint (end of G2)
Cell will not proceed with mitosis if DNA replication is not complete
The way the cell senses this is not understood completely
This checkpoint involves signals that block the activation of M phase cyclin-cdk complex (MPF) by inhibiting the activity of cdc25 protein phosphatase.
Cells with mutations in this checkpoint pathway or cultured mammalian cells treated with caffeine will proceed through mitosis with unreplicated DNA.

21. Cell Cycle Checkpoints
Growth checkpoints
In budding yeasts, division produces a small daughter cell and a large mother cell. The daughter cell spends a longer time growing in G1 before it can divide again. There is a minimum size that must be reached before S phase can begin.
In animal cells, external growth factors play a major role in providing signals about cell growth and differentiation and regulating the cell cycle.

22. Cell Cycle Checkpoints
Spindle-attachment checkpoint
Before anaphase (separation of chromosomes) there is a checkpoint to ensure the chromatids are correctly attached to the mitotic spindle
The kinetochore (where the chromatids attach to the spindle) is the structure that is monitored
Unattached kinetochores negatively regulate the activity of cdc20-anaphase promoting complex (APC), and anaphase is delayed

23. Cell Cycle Checkpoints
Exiting Mitosis
Degradation of the M phase cyclin/cdk complex (aka MPF) is required to proceed with the final activities of mitosis (spindle disassembly and formation of the nuclear envelopes).
This degradation is accomplished by ubiquitinylation of the complex.
The cdc20-APC complex is responsible for signaling the degradation and exit from mitosis.

24. Some Cell Cycle Summary Figures
A table listing some of the players in cell cycle regulation and where they act.
A schematic summary of the cell cycle regulators.

25. Developmental Links
Because cell divisions have to start and stop at the right times during development, many developmental events are marked by changes in the cell cycles
Cell cycle regulation and regulation of developmental transitional events are often intertwined

26. Early Embryonic Divisions in Xenopus
After fertilization, the Xenopus embryo goes through 12 rapid and synchronous cell cycles which are essentially alternating M and S phases (G1 and G2 are dramatically reduced).
These cell cycles are driven by maternally supplied proteins and mRNAs
Mid-blastula transition (MBT): At cycle 13 maternal transcripts are destroyed; zygotic transcription begins; cell cycles change to include extended G1 and G2 phases
How is this developmental transition accomplished?
The answer lies in miRNAs, which bind to sequences in the 3´ untranslated regions of maternal RNAs and direct their deadenylation, leading to their degradation.
References:
Lund, E. et al Deadenylation of maternal mRNAs mediated by miR-427 in Xenopus laevis embryos. RNA 15, 2357.
Giraldez, A.J Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75.