Introduction to the Cell Cycle

Promega Education Resources

Unit 7

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.

 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.

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

G1

G2 M

Interphase

G1

G2

Interphase

G1

G2

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

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.

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

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

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 (http://www.hhmi.org/genesweshare/a300.html)

Biochemistry
Frog eggs
Mammalian cell culture

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.

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)

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*

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?

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

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).

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.

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
regulationsending a signal to stop the progression of the cell
cycle rather than dialing back a positive signal.

Schematic of Cell Cycle Checkpoints

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.)

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.

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.

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.

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 cdc20anaphase promoting complex (APC), and anaphase is delayed

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.

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.

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

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.