Lecture 16 - Mitosis and Mitotic Exit Flashcards
What are the events in prophase?
- Chromosome condensation
- Centrosome migration and spindle formation
- Nuclear envelope breakdown marks the beginning of prometaphase
- All events stimulated by Cdk1 (increasing levels)
What do condensed chromosomes in interphase allow for?
Transcription and DNA replication at S-phase
What is required for proper chromosome segregation in mitosis?
Chromosomes must first be packaged into a highly condensed format
What promotes chromosome condensation in prophase?
Condensin
How does the condensin complex promote chromosome condensation in prophase?
It may act as a ring connecting two parts of a chromsome
What activates the condensin complex?
Cdk1 phosphorylation of condensin subunits
What are the events in prometaphase?
- Nuclear envelope breakdown (NEB)
- Spindle microtubules start attaching to kinetochores
What is the key to initiating mitosis?
Nuclear envelope breakdown
What does NEB allow?
- Cytoplasmic proteins access to the nucleus
- Nuclear proteins access to the mitotic spindle
- Microtubules from the mitotic spindle access to kinetochores
What is NEB triggered by?
Cdk phosphorylation of numerous targets, including the nuclear pore complex and nuclear lamins (intermediate filament proteins that support the nuclear envelope)
What kind of Cdk1 activity is required for NEB to occur?
High levels
What microtubules does the mitotic spindle have?
- Astral microtubules
- Interpolar microtubules
- Kinetochore microtubules
What do astral microtubules do?
- Grow and shrink in prometaphase
- Capture kinetochores on chromosomes to become kinetochore microtubules
What do interpolar microtubules do?
- Microtubules from either pole that interact in the spindle midzone
- Stabilize bipolar spindle
- Sliding of interpolar microtubules relative to each other allows pole separation in anaphase
- Establish site of cytokinesis
What do kinetochore microtubules do?
- Microtubule plus ends associate with kinetochores at centromeres of chromosomes
- Kinetochore microtubules can shrink and grow by polymerization/depolarization at plus end while still holding onto kinetochores
What are replicated sister chromatids held together by?
Cohesins
What is the kinetochore?
A multiprotein complex that assembles at the centromere of both sister chromatids for each chromosome
What does the separation of sister chromatids require?
The loss of Cohesins and pulling of kinetochore microtubules
What occurs during kinetochore attachment?
- Inner kinetochore binds to the centromeric region of chromosomes
- Outer kinetochore is thought to act as a collar
What does kinetochore attachment lead to?
Depolymerization of kinetochore microtubules from plus ends
- The outer kinetochore remains attached during polymerization
- This depolymerization pulls kinetochores and associated microtubules to poles
When are kinetochore attachments unstable?
Prometaphase
What captures kinetochores?
Astral microtubules with their plus ends
What do captured chromosomes do?
Move towards the pole depending on depolymerization of kinetochore microtubules
Why do chromosomes appear to move back and forth in prometaphase?
Chromosomes frequently lose kinetochore/microtubule contacts, remake them, or make a new contact with microtubules from the other pole
When are kinetochore attachments stabilized?
When a chromosome is successfully captured by microtubules from both poles
What does proper segregation require?
Amphitelic attachment
- Capture of both sister chromatids by microtubules from the opposite poles
What incorrect kinetochore attachments often occur?
- Monotelic: Single attachment
- Syntelic - Both kinetochores attached to microtubules from the same pole
- Merotelic - One kinetochore attached to microtubules from opposite poles
What does Aurora B kinase do?
Localizes to kinetochores and breaks microtubule/kinetochore contacts
What does the Aurora B kinase phosphorylate?
Kinetochore components leading them to bind microtubules less efficiently
How does Aurora B kinase get inactivated?
By tension that occurs when both kinetochores are captured as a result of pulling from both poles on the attached kinetochores
What happens if Aurora B is missing?
Monotelic, syntelic, and merotelic attachments are not destabilized
- These chromosomes are pulled to one pole
In metaphase, why do chromosomes remain at the midzone?
Because they are attached to each other via cohesins
When does anaphase occur?
When cohesin bonds are broken
What can occur when cohesin bonds are broken?
Pulling forces can now separate sister chromatids
What does the completion of anaphase require?
Inactivation of Cdk1
What does the separation of sister chromatids (breaking of cohesins) depend on?
The destruction of Securin
What must occur for the mitotic spindle to be able to pull chromatids to spindle poles?
Destruction of Cyclin B
What does the destruction of Secrurin and Cyclin B depend on?
A ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC/C)
How was cyclin experimentally destroyed?
Deletion of the first 90 amino acids of cyclin B
- Still functional (binds and activates Cdk1) but extracts or eggs arrest in mitosis
- If they add the full length cyclin B to this extract, it is destroyed, while the delta 90-cyclin B remains stable
What can be interpreted from the cyclin destruction experiment?
- MPF (Cdk1-cyclin B) is necessary for entry into mitosis
- MPF activates the cyclin destruction machinery
- Destruction of cyclin B is necessary for exit from mitosis and inactivation of the cyclin destruction machinery
How is cyclin B targeted for destruction?
In vitro mitotic cell cycle using CSF extracts
- Extracts taken from CSF arrested oocytes
- These can be made to cycle by adding Ca2+ (along with sperm DNA and ATP) - cycling extracts
- Add 35-S-cyclin B —> it gets degraded
- Make deletions from N-terminus —> assay for degradation when added to cycling extract
- delta-13-CycB causes CycB to be degraded and the extract goes through mitosis and into interphase
- delta-90-CycB is not degraded and extract arrests in mitosis (specifically, in anaphase)
How do we determine if the N-terminal 90 amino acid deletion is sufficient for cyclin B degradation?
- Fusion of the N-terminal 90 amino acid of cyclin B to protein A which makes the protein unstable in mitotic extracts
- N-terminal 90 amino acid of cyclin B is sufficient to mediate cyclin B destruction
How do we determine what sequence within the 90 aa is important for cyclin destruction?
By looking for conserved motifs within the region amongst cyclins from different species
What amino acid sequence is conserved within the 90 aa important for cyclin destruction?
A 9 aa sequence: RxxLxxxxN
How do we determine if the 9 aa sequence is necessary and sufficient to mediate cyclin B destruction?
- Fusion of the N-terminal 90 aa of cyclin B to Protein A makes this protein unstable in mitotic extracts
- Changing a single amino acid within the RxxLxxxxN motif (R42C) makes protein A stable again
- Protein A fused to destruction box is destroyed over time
What happens as Protein A is being destroyed?
A ladder of higher molecular weight forms and is detected
What does western blotting using antibody against ubiquities reveal?
That the protein A isoforms contain ubiquitin
- Ubiquitin chains are being assembled on the protein prior to destruction
Discovery of the APC/C: 1995
Biochemical purification from Xenopus extracts of a protein complex that is necessary for cyclin ubiqutination
- Complex contains homologues of yeast genes cdc16, 23, and 27
What are budding yeast genes cdc16, 23, and 27 required for?
Anaphase progression and destruction of B-type cyclins
- These proteins physically interact throughout the cell cycle
What activates the machine that eventually destroys cyclin B?
Cyclin B
What does Cdk1-cycB do?
- Promotes NEB and other events of mitosis
- Phosphorylates and activates APC/C
What targets cycB for destruction?
APC/C
What does the destruction of cycB result in?
Loss of Cdk1 activity
What is the destruction of cycB necessary for?
The movement of sister chromatids to poles in late anaphase
What is the destruction of Securin by APC/C necessary for?
Cohesin removal and separation of sister chromatids at the onset of anaphase
When are cohesin complexes assembled?
In S-phase
What do cohesins do?
Keep sister chromatids together until anaphase of mitosis
What are cohesins necessary for?
To ensure proper chromosome segregation in mitosis
What cleavage does the segregation of sister chromatids at anaphase by breaking cohesin complexes depend on?
Scc1
What experiment was carried out with regard to securin in 1996?
- cenV-GFP labelling of single chromosome
- Wild type cells complete mitosis and each daughter (sister chromatid) gets a single dot
- In APC mutants that arrest in mitosis with a single GFP dot, metaphase arrest is seen
- Delta 90-cycB causes a mitotic arrest with 2 GFP dots —> anaphase arrest
*APC mutants arrest in metaphase with sister chromatids still associated
*Stabilized cycB results in anaphase arrest with sister chromatids separated
In 1996, where was securin identified?
In S. pombe (cut2) and S. cerevisiae (pds1)
- Mutants segregate chromosomes incorrectly and prematurely, before anaphase
What part of securin is targeted for destruction by the APC?
Destruction box (D-box)
What do both D-box mutants and APC mutants have in common?
They result in failed sister chromatid segregation
What is the main function of APC?
To target securin
What is the main purpose of securin?
To prevent anaphase
When do APC mutants arrest?
Metaphase (1 GFP dot)
When do APC, securin double mutants arrest?
Late anaphase (2 GFP dots)
How does securin prevent anaphase?
By protecting Scc1
How does APC promote anaphase?
By promoting securin destruction
When is Scc1 (Cohesin) stably associated with DNA?
In APC mutants
When is Scc1 (cohesin) unstable?
In securin mutants and in securin APC double mutants
When do mutants with stabilized Scc1 arrest?
Metaphase
What is separase necessary for?
- Sister chromatid separation
- Scc1 destruction
What identified separase?
Affinity purification of securin interacting proteins
Prior to anaphase, what does securin do?
Binds to and inhibits separase
At anaphase, why does APC ubiquitinate securin?
To target it for proteasomal destruction causing separase to activate and cleave Scc1
What does the loss of Scc1 break up?
The cohesin complex and sister chromatids can separate
What needs to occur for APC to ubiquitinate securin (or cyclin B)?
All chromosomes are properly attached to kinetochores microtubules via bipolar attachments
- A single unattached kinetochore is enough to delay anaphase
