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BIOM 3500 - Midterm 3: The Cell Cycle > Lecture 16 - Mitosis and Mitotic Exit > Flashcards

Lecture 16 - Mitosis and Mitotic Exit Flashcards

1
Q

What are the events in prophase?

A
  • Chromosome condensation
  • Centrosome migration and spindle formation
  • Nuclear envelope breakdown marks the beginning of prometaphase
  • All events stimulated by Cdk1 (increasing levels)
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2
Q

What do condensed chromosomes in interphase allow for?

A

Transcription and DNA replication at S-phase

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3
Q

What is required for proper chromosome segregation in mitosis?

A

Chromosomes must first be packaged into a highly condensed format

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4
Q

What promotes chromosome condensation in prophase?

A

Condensin

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5
Q

How does the condensin complex promote chromosome condensation in prophase?

A

It may act as a ring connecting two parts of a chromsome

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6
Q

What activates the condensin complex?

A

Cdk1 phosphorylation of condensin subunits

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7
Q

What are the events in prometaphase?

A
  • Nuclear envelope breakdown (NEB)
  • Spindle microtubules start attaching to kinetochores
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8
Q

What is the key to initiating mitosis?

A

Nuclear envelope breakdown

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9
Q

What does NEB allow?

A
  • Cytoplasmic proteins access to the nucleus
  • Nuclear proteins access to the mitotic spindle
  • Microtubules from the mitotic spindle access to kinetochores
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10
Q

What is NEB triggered by?

A

Cdk phosphorylation of numerous targets, including the nuclear pore complex and nuclear lamins (intermediate filament proteins that support the nuclear envelope)

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11
Q

What kind of Cdk1 activity is required for NEB to occur?

A

High levels

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12
Q

What microtubules does the mitotic spindle have?

A
  • Astral microtubules
  • Interpolar microtubules
  • Kinetochore microtubules
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13
Q

What do astral microtubules do?

A
  • Grow and shrink in prometaphase
  • Capture kinetochores on chromosomes to become kinetochore microtubules
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14
Q

What do interpolar microtubules do?

A
  • 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
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15
Q

What do kinetochore microtubules do?

A
  • 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
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16
Q

What are replicated sister chromatids held together by?

A

Cohesins

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17
Q

What is the kinetochore?

A

A multiprotein complex that assembles at the centromere of both sister chromatids for each chromosome

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18
Q

What does the separation of sister chromatids require?

A

The loss of Cohesins and pulling of kinetochore microtubules

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19
Q

What occurs during kinetochore attachment?

A
  • Inner kinetochore binds to the centromeric region of chromosomes
  • Outer kinetochore is thought to act as a collar
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20
Q

What does kinetochore attachment lead to?

A

Depolymerization of kinetochore microtubules from plus ends
- The outer kinetochore remains attached during polymerization
- This depolymerization pulls kinetochores and associated microtubules to poles

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21
Q

When are kinetochore attachments unstable?

A

Prometaphase

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22
Q

What captures kinetochores?

A

Astral microtubules with their plus ends

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23
Q

What do captured chromosomes do?

A

Move towards the pole depending on depolymerization of kinetochore microtubules

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24
Q

Why do chromosomes appear to move back and forth in prometaphase?

A

Chromosomes frequently lose kinetochore/microtubule contacts, remake them, or make a new contact with microtubules from the other pole

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25
Q

When are kinetochore attachments stabilized?

A

When a chromosome is successfully captured by microtubules from both poles

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26
Q

What does proper segregation require?

A

Amphitelic attachment
- Capture of both sister chromatids by microtubules from the opposite poles

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27
Q

What incorrect kinetochore attachments often occur?

A
  • Monotelic: Single attachment
  • Syntelic - Both kinetochores attached to microtubules from the same pole
  • Merotelic - One kinetochore attached to microtubules from opposite poles
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28
Q

What does Aurora B kinase do?

A

Localizes to kinetochores and breaks microtubule/kinetochore contacts

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29
Q

What does the Aurora B kinase phosphorylate?

A

Kinetochore components leading them to bind microtubules less efficiently

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30
Q

How does Aurora B kinase get inactivated?

A

By tension that occurs when both kinetochores are captured as a result of pulling from both poles on the attached kinetochores

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31
Q

What happens if Aurora B is missing?

A

Monotelic, syntelic, and merotelic attachments are not destabilized
- These chromosomes are pulled to one pole

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32
Q

In metaphase, why do chromosomes remain at the midzone?

A

Because they are attached to each other via cohesins

33
Q

When does anaphase occur?

A

When cohesin bonds are broken

34
Q

What can occur when cohesin bonds are broken?

A

Pulling forces can now separate sister chromatids

35
Q

What does the completion of anaphase require?

A

Inactivation of Cdk1

36
Q

What does the separation of sister chromatids (breaking of cohesins) depend on?

A

The destruction of Securin

37
Q

What must occur for the mitotic spindle to be able to pull chromatids to spindle poles?

A

Destruction of Cyclin B

38
Q

What does the destruction of Secrurin and Cyclin B depend on?

A

A ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC/C)

39
Q

How was cyclin experimentally destroyed?

A

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

40
Q

What can be interpreted from the cyclin destruction experiment?

A
  • 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
41
Q

How is cyclin B targeted for destruction?

A

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)

42
Q

How do we determine if the N-terminal 90 amino acid deletion is sufficient for cyclin B degradation?

A
  • 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
43
Q

How do we determine what sequence within the 90 aa is important for cyclin destruction?

A

By looking for conserved motifs within the region amongst cyclins from different species

44
Q

What amino acid sequence is conserved within the 90 aa important for cyclin destruction?

A

A 9 aa sequence: RxxLxxxxN

45
Q

How do we determine if the 9 aa sequence is necessary and sufficient to mediate cyclin B destruction?

A
  • 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
46
Q

What happens as Protein A is being destroyed?

A

A ladder of higher molecular weight forms and is detected

47
Q

What does western blotting using antibody against ubiquities reveal?

A

That the protein A isoforms contain ubiquitin
- Ubiquitin chains are being assembled on the protein prior to destruction

48
Q

Discovery of the APC/C: 1995

A

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

49
Q

What are budding yeast genes cdc16, 23, and 27 required for?

A

Anaphase progression and destruction of B-type cyclins
- These proteins physically interact throughout the cell cycle

50
Q

What activates the machine that eventually destroys cyclin B?

A

Cyclin B

51
Q

What does Cdk1-cycB do?

A
  • Promotes NEB and other events of mitosis
  • Phosphorylates and activates APC/C
52
Q

What targets cycB for destruction?

A

APC/C

53
Q

What does the destruction of cycB result in?

A

Loss of Cdk1 activity

54
Q

What is the destruction of cycB necessary for?

A

The movement of sister chromatids to poles in late anaphase

55
Q

What is the destruction of Securin by APC/C necessary for?

A

Cohesin removal and separation of sister chromatids at the onset of anaphase

56
Q

When are cohesin complexes assembled?

A

In S-phase

57
Q

What do cohesins do?

A

Keep sister chromatids together until anaphase of mitosis

58
Q

What are cohesins necessary for?

A

To ensure proper chromosome segregation in mitosis

59
Q

What cleavage does the segregation of sister chromatids at anaphase by breaking cohesin complexes depend on?

A

Scc1

60
Q

What experiment was carried out with regard to securin in 1996?

A
  • 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
61
Q

In 1996, where was securin identified?

A

In S. pombe (cut2) and S. cerevisiae (pds1)
- Mutants segregate chromosomes incorrectly and prematurely, before anaphase

62
Q

What part of securin is targeted for destruction by the APC?

A

Destruction box (D-box)

63
Q

What do both D-box mutants and APC mutants have in common?

A

They result in failed sister chromatid segregation

64
Q

What is the main function of APC?

A

To target securin

65
Q

What is the main purpose of securin?

A

To prevent anaphase

66
Q

When do APC mutants arrest?

A

Metaphase (1 GFP dot)

67
Q

When do APC, securin double mutants arrest?

A

Late anaphase (2 GFP dots)

68
Q

How does securin prevent anaphase?

A

By protecting Scc1

69
Q

How does APC promote anaphase?

A

By promoting securin destruction

70
Q

When is Scc1 (Cohesin) stably associated with DNA?

A

In APC mutants

71
Q

When is Scc1 (cohesin) unstable?

A

In securin mutants and in securin APC double mutants

72
Q

When do mutants with stabilized Scc1 arrest?

A

Metaphase

73
Q

What is separase necessary for?

A
  • Sister chromatid separation
  • Scc1 destruction
74
Q

What identified separase?

A

Affinity purification of securin interacting proteins

75
Q

Prior to anaphase, what does securin do?

A

Binds to and inhibits separase

76
Q

At anaphase, why does APC ubiquitinate securin?

A

To target it for proteasomal destruction causing separase to activate and cleave Scc1

77
Q

What does the loss of Scc1 break up?

A

The cohesin complex and sister chromatids can separate

78
Q

What needs to occur for APC to ubiquitinate securin (or cyclin B)?

A

All chromosomes are properly attached to kinetochores microtubules via bipolar attachments
- A single unattached kinetochore is enough to delay anaphase

BIOM 3500 - Midterm 3: The Cell Cycle (7 decks)

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