Transcript Introduction to the Cell Cycle

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:
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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.
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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
S
M
G2 M
Interphase
G1
S
G2
M
M
Interphase
G1
S
G2
Interphase: G1
• Events during G1
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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
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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:
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Prophase
Metaphase
Anaphase
Telophase
• Factors that influence M phase entry
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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:
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Genetics
• Yeast: Schizosaccharomyces pombe (fission yeast) and
Saccharomyces cerevisae (budding yeast)
• Short article from HHMI (http://www.hhmi.org/genesweshare/a300.html)
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Biochemistry
• Frog eggs
• Mammalian cell culture
Control of the Cell Cycle
• Intrinsic Control: Cyclins
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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
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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
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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
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Extracts resume cycling activity
• Add cyclin B mRNA that encodes a protein with a
mutated protein-degradation signal to parallel extracts
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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
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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
Cell Cycle Checkpoints
• G1 (Restriction) Checkpoint
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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
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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)
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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
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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
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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
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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.