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Cell Biology > Lecture 1&2 - The cell cycle and cell cycle control > Flashcards

Lecture 1&2 - The cell cycle and cell cycle control Flashcards

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1
Q

What are the four phases of the cell cycle and what happens in each?

A

G1: Cell grows and carries out normal metabolism, cell receives external signals to enter the cell cycle and the organelles duplicate
S: Dna replication and synthesis
G2: Cell grows and prepares for mitosis, resolves problems
M: Mitosis and Cytokinesis

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2
Q

What is Quiescence?

A

When a cell is in an inactive state and not actively dividing, a cycling of G1 (G0)

  • can last for years
  • must receive signals to exit G0
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3
Q

How did Walther Flemming illustrate the major stages of the cell cycle?

A

Stained cells genetic material

-Observed that in each cell cycle the interphase chromatin is condensed into transmissible chromosomes then decondensed

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4
Q

What are the major componants of DNA condensation?

A
  • Nucleosomes (DNA wrapped around histone protein cores)
  • Solenoid (Winding of the nucleosomes on top of each other)
  • Mitotic chromosome scaffold
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5
Q

What occurs in the first stage of DNA condensation? (Nucleosome)

A
  • 146bp length of DNA is wrapped around an octamer of histone subunits, made up of pairs of: H2A, H2B, H3, H4, forming a Nucleosome
  • H1 tightens the connections between DNA wrapped around the histone octamer
  • Creates a ‘beads on a string’ structure
  • results in a 6-fold reduction in length
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6
Q

What is the function of the H1 molecule in the nucleosome?

A

Tightens the connections between DNA wrapped around the octamer by binding to linker DNA 20-80bp long
-stabilises chromatin structure

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7
Q

What occurs in the second stage of DNA condensation? (Solenoid)

A

Nucleosomes wind up on top of each others, forming chromatin fibres 30nm thick = Solenoid
-results in a 40 fold reduction in length

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8
Q

What occurs in the third stage of DNA condensation?

A

After coiling and supercoiling, chromosome fibres attach to the mitotic chromosome scaffold

  • results into 46 chromosomes with total length ~200µm
  • 10,000 fold reduction in length
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9
Q

For what percentage of time is a cell in M phase?

A

5%

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10
Q

What is the structure of microtubules in Interphase and the processes in the 5 stages of Mitosis?

A

Interphase: Microtubules are long and diffuse, not attached to centromere
Prophase: Chromosomes condense
Prometaphase: Nuclear envelope breaks down
Metaphase: Chromosomes align at the spindle equator
Anaphase: Sister chromatids separate along the spindle as the astral microtubules grow
Telophase: Cleavage furrow constricts around the central microtubules and central spindle as nuclei reassemble

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11
Q

What is the cleavage furrow?

A

An indentation of a cells surface which begins the progression of cleavage leading to cytokinesis

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12
Q

What are the three types of astrotubules formed when the spindle apparatus forms, and what are they?

A
  • Kinetochore microtubules: attaches to kinetochores of condensed chromosomes and moves towards centrosomes
  • Polar microtubules: emanates from 2 centrosomes and overlap with each other. Push against each other to force centrosomes towards cell plates
  • Astral microtubules: extend from centrosome to cell membrane and anchor to the periphery of the cell. Pull centrosome towards one of the poles
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13
Q

What is the kinetochore?

A

Sequences of DNA in the centromere where microtubules bind

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14
Q

What experimental benefits can be gained from the natural syncrony in fly embryos?

A

can study many cells at the same stage at the same time

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15
Q

Give two examples of chromosome segregation gone wrong

A
  • Anaphase bridges: when telomeres of sister chromatids fuse together and fail to completely segregate into their respective daughter cells
  • Aneuploidy: inaccurate segregation of chromosomes
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16
Q

What is the chromosome complement at each of the four stages of the cell cycle?

A

G1: 2N
S: 2N/4N
G2: 4N
gametes: N

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17
Q

How can multiple cell cycle synchrony be achieved?

A

Chemical inhibition
-blocks cell cycle before mitosis or before S phase leading to aggregation of cells at a particular stage in the cell cycle
-check by flow cytometry - measures the DNA content of a cell (SS, labelling, fluorescent peak) Normally smallest amount of light scattered in G1, smear for S, largest in G2
Natural synchrony - use process of quiescence to synchronise

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18
Q

What are the chemicals that can be used to attain multiple cell cycle synchrony?

A 
Nacodazole (G2)
Cytochalasin B
Thymidine (G1) 
Aphidicolin 
Flow cytometry analysis - Nacodazole shows biggest fluorescent peak for larger cells (G2); Thymidine shows biggest fluorescent peak for smaller cells (G1)
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19
Q

What are the benefits of Natural synchrony over chemical inhibition when achieving experimental cell cycle synchrony?

A
  • adding chemical may prevent validity

- more natural and can stimulate cells with a chemical signal to enter cell cycle at the same time

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20
Q

What are five classic experiments that identified the cell cycle?

A 
Cell fusion experiments - Roa and Johnson
Discovery of cyclins - Hunt
Discovery of MPF - Lokha and Masui
Discovery of cell cycle genes - Hartwell
Discovery of Cdc2 - Nurse
21
Q

Describe the Cell Fusion Experiments done by Roa and Johnson

A

Aim - (1)discover what happens when take a cell in one stage of cell cycle and fuse with one in another; (2)what happens when cells in cell cycle phases are exposed to different diffusible factors

(1) combined:
Cells in S + G1 => all cells were in S phase. Hypothesised that it was by an S phase specific factor (SPF)

Cells in S + G2 => No change. Therefore, SPF factor not recognised by G2 nucleus

found there was a difference in G1 cells being able to respond and G2 not.

(2)MPF (mitosis promoting factor)

Combined G1 and G2 cells => neither produce any dominant chemical signal

M phase cells fusing with any other cell in a different phase the M phase is dominant, acts on all cells in any phase

Consequently M phase cells produce MPF

22
Q

What is MPF?

A

Mitosis promoting factor or Maturation promoting factor: diffusible factor that triggers mitosis

23
Q

Why use Xenopus eggs as an experimental organism?

A
  • rapid cleavage divisions
  • lay hundreds of eggs at the same time
  • fertilisation and maturation is external and visable
  • only get one gamete, all the nutrients go to one cell resulting in a robust cell with high volume of enzymes and nutrients
24
Q

What is the process of Oocyte maturation in frogs that Masui observed, and what did he conclude?

A

Oocyte is arrested at G2 phase, where an influx of progesterone stimulate progression into Meiosis I followed by meiotic interphase. The egg is released and the cell cycle is arrested again at metaphase meiosis II. Fertilisation then occurs and the cell cycle continues and undergoes the first cleavage. (post fertilisation colours segregate to show first division)
Concluded - there are 2 key points of regulation in meiotic cell cycle

25
Q

How did Lokha and Masui identify MPF?

A

1- Took cytoplasm from an egg in Metaphase of MEiosis II, injected it into a G2 arrested oocyte. => Initiated the maturation process and G2 arrested oocyte matured into a metaphase meiosis II arrested egg

Concluded - must be a chemical in the cytoplasm of metaphase meiosis II arrested egg that promotes meiosis = MPF

2- Used serial cytoplasmic transfers to show that this was always the case, and that MPF was always present (Developed a robust assay)
Used this to test for MPF activity over time

26
Q

What test did Lokha and Masui do to test MPF activity of a frog oocyte over time and what did they find?

A

1- Took cytoplasm from different stages of frog egg maturation and tested for MPF activity by the ability to induce egg maturation when injected

Found - High MPF activity:

  • After G2 arrest when cell stimulated with progesterone at Meiosis I
  • During Meiosis II Metaphase arrest, declines at fertilisation
  • Then continued ossilations of high MPF activity during embyronic mitosis cycles
27
Q

Why use sea urchins as an experimental organism for developmental studies?

A
  • large eggs that develop externally

- visible early divisions

28
Q

How did Tom Hunt discover cyclins?

A

-SDS-PAGE analysis of proteins present at each stage of the cell cycle after the fertilisation of Sea urchin eggs.
Identified that:
-cyclin levels ossilate after fertilisation whilst other proteins remained constant
-cyclin peaks precede cell division, correlating with the cell cycle

(intensity of bands were tested by densitometry)

29
Q

How did Hunt show that oscillating levels of cyclin (falling during cell division) were achieved by periodic destruction?

A

1- labelled proteins either:

a) continuously to visualise the total amount of protein at any one time (SDS-PAGE)
b) in short pulses to visualise newly synthesised proteins (SDS-PAGE)

Found - a) different level of cyclins compared to other proteins in the total amount of proteins
b) but the same level of cyclins were being newly synthesised

Conclusion - that cyclins were synthesised constantly but degraded at specific points during the cell cycle

30
Q

How did Hunt check that the oscillating levels of cyclin were directly related to the cell cycle?

A

blocked the cell cycle and analysed the total and contiually synthesised products by SDS-PAGE

found - no destruction occurs if the cell cycle is blocked

31
Q

How did Hunt’s cyclin observations match up to previous observations about MPF?

A

Cyclin disappears as anaphase begins, correlating with MPF activity at cyclin peaks

32
Q

Why use Sacromyces cerevisae as an experimental organism in developmental biology?

A
  • Unicellular eukaryote, life cycle = cell cycle
  • can grow and visually mark the stages of the cell cycle
  • can analyse gametes
  • start of the cell cycle = restriction point in mammalian cells
33
Q

How did Lee Hartwell identify the cell cycle genes in Sacromyces cerevisae?

A

1-Created temperature sensitive mutants, identified their mutant phenotype and revealed a complex set of interconnecting pathways
e.g.
action of Cdc8, Cdc10 and Cdc 24 mutants

2- once identified, placed the point of action of mutants in order and identified the secondary mutations (supressors) that counteract them

34
Q

How do temperature sensitive mutants work?

A
  • at permissive temperature the growth of all mutants is the same as the WT
  • restrictive temp cell arrests at a certain point in the cell cycle
35
Q

What temperature sensitive mutants did Hartwell use?

A

cdc (cell division cycle) mutants = arrest before phase in which its gene product is needed
non-cdc mutants = arrest immediately irrespective of what cell cycle stage it is at

36
Q

What did Hartwell observe about Cdc8, 10, 24 mutants and what could he conclude about the involvement of their mutated genes in the cell cycle

A

Cdc8 mutants arrested before DNA replicated => gene product is involved after initation of DNA synthesis but before full synthesis could occur

Cdc10 stops during cyctokinesis => gene product involved in cytokinesis

Cdc24 arrests before fusion during yeast mating => involved in bud formation

37
Q

What were the supressor genes that Hartwell identified for some of the cell cycle genes?

A

Cdc8 - Cdc21

Cdc10 - Cdc3, Cdc11

38
Q

How did Nurse discover Cdc2 (key cell cycle regulator) in Schizosaccharomyces pombe (fission yeast)?

A
  • found temperature sensitive mutants that did not preogress through the cell cycle at the restrictive temperature
  • identified mutant gene through plasmid rescue
  • identified cyclin dependent protein kinase 2
39
Q

What is the process of plasmid rescue?

A
  1. Make cDNA library of the whole genome of yeast against human
  2. Transform mutant Cdc2^ts cells with plasmid cDNA library and grow at the restrictive temperature
  3. Only cells that have been transformed with the WT copy of the mutated gene will grow
  4. Re-isolate cDNA and clone from rescued cells
  5. Sequence gene and predict protein
  6. Identify orthologs in other organisms
40
Q

How was it concluded that MPF is a complex of cyclin B and Cdc2?

A
  • Cdc13 and Cdc2 mutants have similar phenotype and therefore functions
  • the predicted amino acid sequence reveals that cdc2 is a protein kinase and cdc13 is a cyclin
  • cdc13 and cdc2 form a heterodimeric complex

-the activity of Cdc2 kinase correlated with rising level of cdc13 (like MPF, shown in previous experiments: Hunt - in sea urchin eggs cyclin levels fluctuate with the cell cycle; Masui - Xenopus MPF induces cell division and activity fluctuates with cell cycle)
Therefore MPF is made up of two subunits, a kinase and a cyclin

41
Q

The expression of what genes (and how) act as checkpoints in the cell cycle?

A

G1: CDK4/6 forms a complex with cyclin D
-expression falls as S phase cyclins expression increases
S: CDK2 complexes with either cyclin E or A
-expression falls as M phase cyclins take over, CDK1 (Cdc2) complexes with either A or B (MPF)

These molecules are expression sequentially and are dependent on what was previously expressed, regulating the cell cycle

42
Q

When is MPF involved in M-phase checkpoints (mainly in yeast and embryonic systems) and how is it regulated?

A

MPF is involved in G2 progressing to the M phase
Regulation relies upon:
Induction of MPF
-blocked until the S phase is complete and damage is repaired (phosphorylation of CDK1/Cdc2)
Destruction of MPG
-blocked until chromosomes are aligned on metaphase plate (spindle assembly checkpoint)

43
Q

What is the destruction of MPF controlled by?

A

APC/C (anaphase promoting complex), followed by destruction by the proteosome

44
Q

During the cell cycle what is MPF destruction required for?

A
  • for cells to separate

- phosphorylation only occurs when all chromosome kinetochores are occupied and chromosomes are aligned

45
Q

What is the main checkpoint in mammalian cells?

A

S phase checkpoint

-Roe and Johnson showed S phase cell contains a factor which acts upon G1 to progress to S phase

46
Q

Why can G1 cells respond to SPF and not G2 cells?

A

In mammalian cells, S phase cyclins (CDK2, cyclins E and A) confer specificity of the kinase
-Cyclin E synthesised first but levels drop as cyclin A levels rise (dependent upon the stability of the proteins, E less stable)

47
Q

How does the cell ensure that it’s genome is replicated only once in the cell cycle?

A

Tightly control the assembly and activation of proteins that initiate DNA replication

Early G1: origin of replication bound by the Origin recognition complex
Late G1: MCM complex forms (MCM with CDt1 and CDC6) [6 proteins in a ring structure] and binds to sites specified by ORC. MCM complex is a DNA helicase and opens up DNA to allow access. (Pre replication complex)
S phase: Replication complex is assembled and activated, DNA synthesis occurs (assisted by polymerases and ligases)

Movement from late G1 into S phase is under control of cyclins E and A - Cdk2

  1. Cyclin E/Cdk2 complex controls the assembly step by promoting phosphorylation events to support assembly
  2. As Cyclin A/Cdk2 levels rise they add different phosphorylation events to assembly proteins to prevent it assembling again
  3. As cyclin A/cdk2 levels rise futher they promote phosphorylation event that activate replication complexes
48
Q

How does the ORC select its binding site in lower eukaryotes and higher eukaryotes?

A
  • lower eukaryotes: ORC selects binding site based on well defined sequence of DNA
  • higher eukaryotes: ORC recognises binding site by DNA sequence, which nearby genes are active, whether packaged into chromatin
49
Q

What is Geminin?

A

Protein that regulates cell cycle, builds up during S phase to act as an inhibitor for assembly

Cell Biology (15 decks)

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