Cell Cycle (ch 17) Flashcards
What 3 processes most broadly outline the cell cycle?
- Cell growth/chromosome replication
- Chromosome segregation
- Cell division
Are nuclear division and cell division always linked? Please elaborate.
No, these processes can be independent as in drosophila melanogaster embryos.
How long does chromosome duplication take in humans? What about prokaryotes?
Humans: 10-12 hours
Prokaryotes: 20 minutes
How long does the mitotic phase of the cell cycle take in humans?
~1 hour.
What differentiates mitosis and cytokinesis?
Mitosis: duplication
Cytokinesis: splitting
What 4 main phases comprise the cell cycle? Which of these correspond to interphase?
Cell cycle: G₁, S, G₂, M
Interphase: G₁, S, G₂ only
What is the G₀ phase during cell division?
A quiescent phase which may be permanent in which a cell no longer divides or grows.
In yeast, the beginning of the cell division is called the “start point”. What is this called in mammals?
The “restriction point”.
After G₁, can cells go back to their pre-division state?
No. Once a cell has begun cell division there’s no turning back.
What 3 characteristics make yeast a good model organism for studying the cell cycle?
- Reproduce rapidly
- Very easy to do molecular analysis on
- Can proliferate in a haploid state
Why is it tricky to isolate mutants affecting the cell cycle? How do we get around that?
Because they continue to propagate at normal temperatures. Avoid this by growing at high temperature to arrest cell division but continue growth.
What 2 characteristics make Xenopus laevis embryos a good model system for studying cell division?
- Eggs are huge and easy to manipulate
2. Lots of cytoplasm means its easy to purify (to replicate cell cycle in a test tube)
What is the major disadvantage of using cultured mammalian cells to study the cell cycle? How do we get around this?
They undergo replicative senescence after ~30 replications. Avoid this by using “immortal” cell lines (cancer and viral origin).
What molecular methods are used to observe the cell cycle in cultured mammalian cells?
Specific stains (like BrdU) as well as flow cytommetry or FACS.
What are the 3 main characteristics of the cell cycle control system (checkpoints)?
- Binary on/off, irreversible
- Robust and reliable (many backups)
- Adaptable (cell-specificity)
What are the 3 cell cycle checkpoints?
- G₁/S (start) transition
- G₂/M transition
- Metaphase to anaphase transition
What condition must be met for the cell cycle to proceed past the G₁/S (start) transition checkpoint?
Environment must be favourable.
What 2 conditions must be met for the cell cycle to proceed past the G₂/M transition checkpoint?
- All DNA must be replicated
2. Environment must be favourable
What condition must be met for the cell cycle to proceed past the metaphase to anaphase transition checkpoint?
All chromosomes must be attached to the spindle.
What are the 2 major roles of cyclins during cell division?
- Activate cyclin-dependent kinase partner (Cdk)
2. Direct Cdk partner to specific target proteins
Outline which cyclin is active during each phase of the cell cycle.
G₁: G₁/S-cyclin, S-cyclin
S: S-cyclin
G₂: S-cyclin, M-cyclin
M: S-cyclin, M-cyclin
When does S-cyclin activity begin during the cell cycle?
At the “start” during the G₁ phase.
When does S-cyclin and M-cyclin activity end during the cell cycle?
At the metaphase-anaphase transition during M phase.
What is the function of the anaphase-promoting complex/cyclosome (APC/C)?
Targets cyclins for degradation.
How are cyclin-dependent kinases (Cdks) activated?
Partly active when cyclin binds to the active site. Fully active when Cdk-activating kinase (CAK) phosphorylates the active site.
How does Wee1 kinase affect the activity of cyclin-dependent kinases (Cdks)?
Adds second phosphate, which inhibits Cdk activity
How does Cdc25 phosphatase affect the activity of cyclin-dependent kinases (Cdks)?
Removes inhibitory phosphate added by Wee1 kinase, re-activating the Cdk.
How does a cyclin-dependent kinase inhibitor protein (CKI) function?
Inhibits Cdk activity by binding to both the Cdk and the cyclin.
What 3 things must occur for a cyclin-dependent kinase (Cdk) to activate?
- Inhibitory phosphate must be removed
- Activating phosphate must be present
- Cyclin must be bound
During the cell cycle, when does the Anaphase-Promoting Complex (APC/C) become active? What helps this activation?
Midway through mitosis at the metaphase-anaphase transition. Phosphorylation by M-Cdk can help Cdc20 bind.
During the cell cycle, what triggers activation of the Anaphase-Promoting Complex (APC/C)? How?
Binding with Cdc20 activates the APC/C.
Once activated during the cell cycle, what does the Anaphase-Promoting complex (APC/C) do?
Carries out the ubiquitylation and degradation of securins, S-cyclins, and M-cyclins.
What 2 main proteins is the Anaphase-Promoting Complex (APC/C) responsible for degrading?
- Securin
2. S- and M-cyclins
What mitotic event is triggered by the activation of the Anaphase-Promoting Complex (APC/C)? How does this happen?
Separation of the 2 sister chromatids. Happens when activated separase cleaves the cohesins holding the chromatids together.
During the cell cycle, how is separase activated? How does it cause separation of the sister chromatids?
The APC/C degrades securin (bound to separase), activating separase. Separase cleaves cohesins which hold the chromatids together.
Regarding cell cycle control, what is the purpose of the Skp/Cullin/F-Box (SCF) complex?
To ubiquitilate CKIs in G₁ and to degrade G₁/S-cyclins once S-phase begins.
Regarding cell cycle control, how does phosphorylation of a CKI affect Skp/Cullin/F-Box (SCF) activity?
Phosphorylation enables targeting by specific F-Box proteins.
How does Cdh1 activity affect the Anaphase-Promoting Complex (APC/C)?
It maintains APC/C activity after anaphase and throughout G₁.
What 2 requirements are critical for DNA replication during S phase of the cell cycle?
- DNA replication must be highly accurate
2. Every nucleotide must be replicated only once
Give a broad description for the S phase of the cell cycle.
DNA is replicated for inheritance by the two daughter cells.
During S phase of the cell cycle (initiation phase), what is the 1st step for DNA replication? How is this initiated?
Forming the prereplicative complex (pre-RC). Initiated by the APC/C.
During S phase of the cell cycle (Initiation phase), what is the 2nd step for DNA replication? How is this initiated?
Forming the preinitiation complex. Initiated by S-Cdk.
What 2 (sub?)phases make up the S phase of the cell cycle?
- Initiation phase
2. Replication phase
How is formation of the prereplicative complex (pre-RC) during S phase inhibited?
By Cdk activity (all about timings).
During the cell cycle, what holds sister chromatids together? How do these interact?
Cohesins. Form a ring structure around the sister chromatids.
What are the 4 proteins which make up a cohesin (involved in the cell cycle)?
- Smc1
- Smc3
- Scc1
- Scc3
What stimulates the production of cohesins which then bind the sister chromatids together during the cell cycle?
S-Cdk complexes, present during S phase of the cell cycle.
What is DNA catenation? How must this be resolved?
The interweaving of DNA strands as a result of replication. Must be unwoven prior to strand separation.
How is DNA catenation resolved to allow strand separation?
Topoisomerase II recognizes entanglement and reversibly cuts one strand to allow the other strand to pass through.
During what phase of the cell cycle does DNA catenation occur?
S phase.
What are the 5 traditional stages of mitosis?
- Prophase
- Prometaphase
- Metaphase
- Anaphase
- Telophase
What are the 2 major parts of mitosis as divided by regulatory steps?
- Increase in M-Cdk activity
2. Metaphase-to-anaphase transition
What happens during the first part of mitosis?
Assembly of mitotic spindle and attachment to sister chromatids. Nuclear pore complex is phosphorylated and disassembled.
What happens during the second part of mitosis?
APC/C activation leads to chromatid separation. APC/C triggers cyclin degradation.
What occurs in the cell during prophase?
Chromatin condensation.
What occurs in the cell during prometaphase?
Nuclear envelope breaks down.
What occurs in the cell during metaphase?
Chromosomes are aligned along the metaphase plate.
What occurs during anaphase?
Sister chromatids are pulled to opposite ends of the cell.
How is M-Cdk activated during the cell cycle? When is it activated?
By dephosphorylation in early mitosis.
During prophase of mitosis, what protein complex assists in chromosome condensation? What stimulates this?
Condensin, which is activated through phosphorylation by M-Cdk.
What changes occur to the number/size of microtubules as the cell is entering mitosis?
There is an increase in the number of shorter microtubules at the entry of mitosis.
How are Microtubule(MT)-dependent motor proteins and MT-associated proteins (MAPs) activated during mitosis. What does this cause?
They are phosphorylated by M-Cdks. Triggers assembly of the mitotic spindle.
What is the mitotic spindle during cell division?
A bipolar array of microtubules to separate the sister chromatids.
What are the 3 classes of microtubules which make up the mitotic spindle?
- Astral microtubules
- Kinetochore microtubules
- Interpolar microtubules
During mitosis, where are the astral microtubules?
Branching out from the centrosomes in all directions but not attaching to the kinetochore.
During mitosis, where are the kinetochore microtubules?
Extending from the centrosomes and binding to the sister chromatids at the kinetochore.
During mitosis, where are the interpolar microtubules?
Extending from each centrosome to bind interpolar microtubules from the other centrosome.
Describe the function of a kinesin during mitosis.
Moves towards the +ve end of the microtubule, binds sister chromatids or other microtubules.
Describe the function of a dynein during mitosis.
Moves towards the -ve end of the microtubule. Binds to the membrane.
What does kinesin-14 bind to during mitosis?
Interpolar microtubules.
What does kinesin-5 bind to during mitosis?
Interpolar microtubules.
What do kinesin-4 and kinesin-10 bind to during mitosis?
Sister chromatids and kinetochore microtubules
In what orientation are chromosomes attached to the kinetochore microtubules? Is this constant throughout mitosis?
Before metaphase: attached parallel
At/after metaphase: attached perpendicularly
During mitosis, are the chromosomes always attached to the microtubule via the kinetochore?
No, can be connected to other parts of the chromatid.
During mitosis, what direction will a chromatid be pulled if it is only attached to the kinetochore microtubule?
Towards the centrosome (-ve direction).
During mitosis, what direction will a chromatid be pulled if it is only attached to an astral or interpolar microtubule?
Away from the centrosome (+ve direction).
What is a kinetochore?
A complex of proteins that attaches the microtubules to the chromatid at the centromere.
Describe the general structure of a kinetochore and how it attaches to the microtubule.
Like a hollow cylinder which fits on the end of the microtubule. The end comes to a point which binds to the chromatid.
During mitosis, can a kinetochore bind to any nucleosome on the chromatid?
No, it must bind to a nucleosome with a centromere-specific H3.
Why is the unique attachment method of a kinetochore to a microtubule necessary?
It leaves the end of the microtubule free to depolymerize as necessary.
Give an example of a protein which is a component of a kinetochore.
Ndc80.
What is meant by “incorrect attachment” of the mitotic spindle to the kinetochores on the chromatids?
Any deviation from each spindle pole binding to one centromere (on opposite sides of the chromatid).
What enzyme is responsible for sensing the tension produced by correct attachment of the mitotic spindle to the kinetochores on the chromatids?
Kinase aurora-B.
Under what circumstance is kinase aurora-B activated during mitosis?
When the mitotic spindle is improperly attached to the kinetochore on the chromatids.
How does kinase aurora-B prevent improper attachment of the mitotic spindle with the kinetochore on the chromatids.
By phosphorylating the Ndc80 ends of the kinetochore to prevent attachment.
What differentiates anaphase A from anaphase B?
A: driven by spindle depolymerization
B: driven by kinesin and dynein motor proteins
What effect does phosphorylation have on replication machinery during telophase.
Promotes the breakdown of phosphorylated replication machinery.
What occurs in the cell during telophase?
The nuclear envelope reforms and the mitotic spindle is disassembled.
What 4 steps outline cytokinesis?
- Initiation
- Contraction
- Membrane insertion
- Completion
Broadly, describe the regulation of the contractile ring formation during cytokinesis.
Actin-myosin ring formation regulated through activation of RhoA by RhoGEF and inactivation of RhoA by RhoGAP.
How do higher plant cells produce two daughter cells?
Same as in animal cells, just split the cell with a new cell wall as well.
In the traditional model of mitosis, when does cytokinesis begin? When does it end?
Begins during anaphase and ends shortly after telophase.
Must mitosis always be paired with cytokinesis?
No. An example of this would be in Drosophila embryos, where mitosis occurs many times prior to cellularization.
Must cytokinesis always involve the symetrical separation of cellular components?
No, sometimes the stuff inside isn’t split evenly if the daughter cells aren’t fated to be the same tissue type.
How is cell cycle activity reset in embryonic cells which have no G₁ phase?
Cdc20-APC/C activity alone suppresses M-cyclin activity.
How is cell cycle activity reset in cells which have a G₁ phase?
CdC20-APC/C activity initially supresses M-cyclin, and Cdh1-APC/C keeps M-cyclin activity low during G₁.
During the cell cycle, when does Cdh1-APC/C activity increase? Why?
During late mitosis to suppress M-cyclin activity during the upcoming G₁ phase.
During the cell cycle, what triggers cytokinesis?
Dephosphorylation of Cdk targets.
How does a cell escape the stable G₁ phase to initiate a new cell cycle?
A rise in G₁/S-Cdk activity ends the suppression of other Cdk activity.
What factor determines the size of organs and bodies? What 3 processes is this dependent on?
Total cell mass, which is dependent on:
- Cell growth
- Cell division
- Cell death
What is a mitogen? What is their function?
An extracellular signalling molecule which stimulates cell division by activating G₁/S-Cdk.
What is a growth factor? How do they function?
An extracellular signalling molecule which stimulates an increase in cell mass via synthesis of proteins/macromolecules.
What is a survival factor? How do they function?
An extracellular signalling molecule which promotes cell survival by suppressing apoptosis.
How many different mitogens do we know of?
More than 50.
How can mitogens affect the levels of Myc in a cell? Also, what’s a Myc?
They can increase the levels of Myc by activating the Ras and MAPk pathways. Myc is a family of proto-oncogenes and transcription factors.
In broad terms, outline the pathway where DNA damage can cause the inactivation of Cdks.
DNA damage leads to p53 activation which triggers transcription of CKI p21. This Cdk inhibitor then inactivates Cdks.
What does a unicellular organism do if it can’t repair DNA damage?
It initially arrests the cell cycle, but then resumes it because it figures being mutated is worth continuing to live.
What does a multicellular organism do if it can’t repair DNA damage?
Decides between having that cell undergo permanent cell cycle arrest (G₀) or apoptosis.
What are 2 potential reasons for a cell to undergo cell cycle arrest (G₀) or apoptosis?
- Abnormal proliferation signals
2. DNA damage
How is APC/C activated? What about SCF?
APC/C: Cdc20 binding
SCF: always active, targets phosphorylated proteins
In what case is it most important to work with conditional mutations in a yeast cell?
When observing a loss-of-function mutation in a gene which is essential for survival.
