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BIOM 3500 - Midterm 3: The Cell Cycle > Lecture 15 - Into to Cell Cycle, Discovery of Cdks and Cyclins, and Events of Mitosis > Flashcards

Lecture 15 - Into to Cell Cycle, Discovery of Cdks and Cyclins, and Events of Mitosis Flashcards

1
Q

What occurs during entry into mitosis?

A

Prophase, prometaphase, and metaphase

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2
Q

What happens in prophase?

A
  • Chromosome condensation
  • Centrosomes move to opposite poles
  • Spindle starts to form
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3
Q

What happens in prometaphase?

A
  • Nuclear envelope breakdown (NEB)
  • Spindle microtubules start attaching to kinetochores
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4
Q

What happens during metaphase?

A

Chromosomes are attached to kinetochore microtubules from each pole

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5
Q

What occurs during exit from mitosis?

A

Anaphase, telophase, and cytokinesis

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6
Q

What happens during anaphase?

A
  • Sister chromatid separation
  • Movement of sister chromatids towards spindle poles
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7
Q

What happens during telophase?

A
  • Contractile ring formation at spindle midzone
  • Nuclear envelope starting to reform
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8
Q

What happens during cytokinesis?

A
  • Cleavage furrow formation and separation of two daughter cells
  • Spindle disassembly
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9
Q

What drives the cell cycle?

A

Cyclin-dependent kinases (Cdks)

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10
Q

What does NEB depend on?

A

Cdk1-cyclin B activity, which causes the phosphorylation of nuclear lamins and disassembly of nuclear lamina

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11
Q

What does assembly of mitotic spindle and capture of chromosomes by spindle microtubules depend on?

A

Cdk1-cyclin B activity, which causes the phosphorylation of microtubule-associated proteins, kinetochore proteins, and others

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12
Q

What requires the inactivation of Cdk1?

A

Spindle elongation and separation of sister chromatids in anaphase

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13
Q

Why doesn’t Cdk1 activation allow for spindle elongation and separation of sister chromatids?

A

Cdk1 phosphorylates proteins that maintain the metaphase state

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14
Q

What are genetic and biochemical approaches to studying the cell cycle?

A
  • Genetic screens in S. cerevisiae and S. pombe
  • In vitro assays in Xenopus and sea urchin egg extracts
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15
Q

What did the genetic screens in S. pombe identify?

A

The cdc2 gene

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16
Q

What kind of regulator is S. pombe cdc2?

A

G2/M regulator

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17
Q

What do cdc2-ts mutant colonies and cells do?

A

Colony fails to grow at restrictive temperature and all cells appear to arrest in G2

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18
Q

How is S. pombe cdc2 cloned?

A

By complementation of cdc-ts
1) Transform individual mutant cells with individual plasmids from S. pombe cDNA library
2) Plate out at a restrictive temperature
3) Identify colonies
4) Isolate plasmid from these cells
5) Determine sequence of cDNA
*These cells carried a cDNA that could complement the cdc2-ts mutation

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19
Q

What did the genetic screens in S. cerevisiae identify?

A

The cdc28 gene

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20
Q

What kind of regulator is S. cerevisiae cdc28?

A

G1/S regulator

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21
Q

What did S. cerevisiae cdc28 cells do at restrictive temperature?

A

Arrested in G1

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22
Q

How was S. cerevisiae cdc28 cloned?

A

By complementation of cdc28-ts

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23
Q

How identical is S. cerevisiae cdc28 to S. pombe cdc2?

A

60%

24
Q

What does cDNA for cdc28 complement?

A

S. pombe cdc2 mutant

25
Q

What does S. pombe cdc2 and S. cerevisiae cdc28 encode?

A

A 34 kDa protein with sequence similarity to known kinases

26
Q

What are cdc2 and cdc28?

A

Functional homologues

27
Q

What was later found about both cdc2 and cdc28 genes?

A

They are required for both G1/S and G2/M

28
Q

Identification of Cdk1: 1974-76

A

Genetic screens in S. cerevisiae and S. pombe
- Identified temperature-sensitive mutants that caused arrest at a specific stage in the cell

29
Q

Identification of Cdk1: 1981

A

cdc2 in S. pombe initially described as being necessary for G2/M (later found to be also necessary for G1/S)

30
Q

Identification of Cdk1: 1982

A

1) cdc28 from S. cerevisiae required for G1/S (later found to be also necessary for G2/M)
2) cdc2 and cdc28 clones, sequences, and found to be functional homologues

31
Q

Identification of Cdk1: 1988

A

IP’d cdc28 acts as a protein kinase in vitro - later named Cdk1

32
Q

Identification of Cdk1: 2001

A

Nobel prize to Hartwell, Nurse (with Hunt - Xenopus/sea urchin)

33
Q

How was it discovered that cdc28 was a kinase?

A

In vitro kinase assay
1) IP using alpha-cdc28 antibody (or control antibody)
2) Add radioactive ATP (y-P32-ATP)
3) Add purified Histone H1 (a protein that can be phosphorylated in vitro by many different kinases)
4) Incubate
5) SDS-PAGE
*Radioactive band at about 29 kDa (Histone H1)

34
Q

Histone H1 kinase activity: 1988

A

Cdc2/Cdc28 (Cdk1) immunopurified from yeast cells phosphorylates Histone H1 in vitro

35
Q

Histone H1 kinase activity: 1989

A
  • Synchronize yeast cells (Hydroxyurea block then release)
  • Cdc2 from yeast cells in G2 showed low Histone H1 kinase activity
    Cdc2 from yeast cells in M showed high Histone H1 kinase activity
  • Cdc2/Cdc28 kinase activity varies through the cell cycle, peaking at M phase
36
Q

What oscillations drive the cell cycle?

A

The kinase activity of Cdc2/Cdc28

37
Q

What about meiosis is similar in many respects to entry into mitosis?

A

Nuclear envelop breakdown (NEB) [a.k.a. maturation]

38
Q

How does the 1st meiotic division differ from a mitotic division?

A

Homologues segregate

39
Q

What phase is not between meiosis I and meiosis II?

A

S-phase

40
Q

What is meiosis II very similar to?

A

A mitotic division because sister chromatids segregate

41
Q

How is meiosis in many species regulated?

A
  • Prophase –> NEB (progesterone)
  • Metaphase II –> anaphase II (fertilization)
42
Q

What is meiosis followed by?

A

Zygote formation and specialized rapid mitotic cell division (early embryo)

43
Q

What is discovered about meiosis in Xenopus?

A
  • Oocytes arrest in prophase of meiosis I until progesterone triggers maturation
  • Following maturation, the egg proceeds and arrests in metaphase of meiosis II until fertilization, which triggers Ca2+ release leading to egg activation
  • Activation results in meiosis continuing into anaphase of meiosis II
44
Q

How was the maturation-promoting factor (MPF) identified?

A

Assay for MPF by Masui in 1971
1) Remove cytoplasm from mitotically cyclin embryos (eggs that have been released from meiosis II arrest by fertilization or in vivo activation)
2) Inject the cytoplasm into prophase I arrested oocytes
*Eggs can now complete meiosis
*The extracts contain MPF, a factor required for oocyte maturation

45
Q

Characterization of MPF: 1977

A

Cytoplasm taken from embryos at given times before or after activation - has different abilities to induce maturation in arrested oocytes
- After activation: Time 1 has low MPF activity (interphase) and time 2 has high MPF activity (mitosis)

46
Q

How do we know protein synthesis is required in each cell cycle for MPF?

A

Cycloheximide, which blocks protein synthesis, was added to dividing embryo lead to arrest before NEB (prophase arrest)
*Extracts from these embryos lack MPF activity

47
Q

When is MPF activity present and inactive?

A

Present upon entry into mitosis and inactive at completion of meiosis

48
Q

How was cyclin B identified?

A

1) 35S-Met added to eggs to label all proteins
2) Identified a protein that cycles

49
Q

How was it concluded that cyclin B was the maturation-providing factor?

A

Cycloheximide added to dividing embryo which lead to prophase arrest and the embryos lacked MPF activity, but when cyclin B protein was added, cycling continued

50
Q

In 1989, what happened in Cdk1 affinity purification from starfish oocytes?

A

Cyclin B copurifies
- Cdk1 is only active when cyclin is bound to it
- Cdk1-cyclin B = MPF

51
Q

What Cdks do higher eukaryotes have?

A
  • Mitotic Cdk - Cdk1 (=MPF)
  • S-phase Cdk - Cdk2
  • Other Cdks as well
52
Q

How many Cdk’s does yeast have?

A

One that functions throughout the cell cylce

53
Q

What are Cdks and what do they depend on?

A

Ser/Thr kinases that depend on a cyclin partner for activity and specificity

54
Q

What are the cyclin-cdk complexes?

A

1) G1-Cdk (cyclin D/Cdk4)
2) G1/S-Cdk (cyclin E/Cdk2)
3) S-Cdk (cyclin A/cdk2)
4) M-Cdk (cyclin B/cdk1)

55
Q

How are Cdks activated?

A

1) Cyclin binding
2) Phosphorylation of T-loop by a Cdk-activating kinase (CAK) –> Cdk7
3) Dephosphorylation of two specific residues by Cdc25 phosphatase
4) Release from Cdk inhibitors - p27, p21, or p16 in mammals and Sic1 in S. cerevisiae

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

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