The cell cycle and its control

Question 1

Different cells divide at different rates. What does this depend on?

Answer 1

• Whether the cell is embryonic or adult
  
  • Rate of proliferation in adult cells much less than embryonic cells
  • e.g. Early frog embryo cells divide every 30 mins
• Complexity of system
  
  • The less complex the system, the more rapidly cell division can occur?
  • e.g. Yeast cell divides every 1.5-3 hours
• Necessity for renewal (intestinal epithelium - every 20 hours, hepatocytes - every 1 year)
• State of differentiation (some cells never divide i.e. neurons and cardiac myocytes)

Tumour cells have an inability to regulate the cell cycle

Question 2

COME BACK What is the relevance of the appropriate regulation of cell division?

Answer 2

• Premature, aberrant mitosis results in cell death
• In addition to mutations in oncogenes and tumour suppressor genes, most solid tumours are aneuploid (abnormal chromosome number and content).
• Various cancer cell lines show chromosome instability (loose and gain whole chromosomes during cell division)
• Perturbation of protein levels of cell cycle regulators is found in different tumours - abnormal mitosis
• Contact inhibition of growth
• Attacking the machinery that regulates chromosome segregation is one of the most successful anti-cancer strategies in clinical use

Question 3

What is the cell cycle?

Answer 3

Orderly sequence of events in which a cell duplicates its contents and divides in two

The cell cycle involves:

• Duplication
• Division
• Coordination

Question 4

What are the two main phases of the cell cycle?

Answer 4

Interphase

• The major event that takes place in this phase is duplication of:
  
  • DNA
  • Organelles
• You also have a lot of protein synthesis in this phase
  
  • You need to produce the proteins necessary for cell division

M-phase = Mitosis

• This is the process of division
  
  • Nuclear division
  • Cell division (i.e. division of the whole cell) → cytokinesis

Question 5

Why is mitosis the most vulnerable period of the cell cycle?

Answer 5

• Cells are more easily killed
  
  • By things like:
    
    • Irradiation (exposure to radiation)
    • Heat shock (cells are warmed past their optimal temperature)
    • Chemicals
• DNA damage can not be repaired
• Gene transcription silenced
• Metabolism
  
  • The cell is focusing all the energy from metabolic pathways into cell division

NOTE:

• Basically during this period, because the cell is so focused on cell division, it can’t really cope with anything else which makes it more vulnerable
  
  • i.e. The cell is not prepared to face any other problem
• Because mitosis is the most vulnerable period of the cell cycle, it is short (relatively - compared to interphase)

Question 6

What is interphase divided into?

Answer 6

• Gap phase 1 (G₁)
  
  • ​Decision point
    
    • The cell decides whether or not to divide (enter S or G0)
• Synthesis phase (S)
  
  • The main thing that occurs in this phase is DNA replication
• Gap phase 2 (G2)
  
  • ​Decision point
    
    • The cell checks the DNA to make sure it was completely replicated and the DNA is not damaged
• Gap phase 0 (G0)
  
  • ​The cell cycle machinery is dismantled
    
    • The cell is in a resting state

Question 7

Describe what happens in the S phase.

Answer 7

This phase is where the replication for division takes palce

• DNA replication
• Protein synthesis
  
  • Initiation of translation and (polypeptide) elongation increased
  • Capacity is also increased
    
    • i.e. Increase in ribosomes and protein synthesis machinery to allow the cell to produce proteins very quickly
• Replication of organelles (e.g. centrosomes, mitochondria, Golgi, etc)
  
  • In order for mitochondria to replicate, you need to replicate its mitochondrial DNA
  • mtDNA replication is coordinated with DNA replication of the cell

NOTE:

Protein synthesis

• In this phase you get synthesis of proteins such as histones
• This allows the newly formed DNA to be wrapped around histones and form chromatin, ready for cell division

Question 8

What is the centrosome?

Answer 8

• An organelle
• Consists of two centrioles
  
  • Centrioles = barrels of 9 triplet microtubules
  • The two centrioles are found at right angles to each other
• Is the main microtubule organising centre (MTOC)
  
  • Organises mitotic spindle
  • Not just this - also organises microtubules for things like cell motility, cellular transport

Question 9

Describe the process of centriole replication during the cell cycle.

Answer 9

NOTES:

• Step 3 - Centrosome engagement refers to each newly formed daughter centriole being orthogonally connected to its mother centriole
  
  • Orthogonally = at right angles
• Pericentriolar material (PCM):
  
  • It is essentially a matrix of proteins which acts as a scaffold for proteins which are necessary for the assembly of microtubules
    
    • Centrosome = centrioles + pericentriolar
    • material
  • The PCM contains proteins involved in microtubule formation

Question 10

Describe how microtubules grow from centrosomes.

Answer 10

They grow outwards

Question 11

What are the 6 different phases of mitosis?

Answer 11

1. Prophase
2. Prometaphase
3. Metaphase
4. Anaphase
5. Telophase

NOTE: Cytokinesis takes place after telophase - part of the M phase but not part of mitosis

Question 12

What happens in prophase?

Answer 12

• Chromatin condenses to form chromosomes
• Duplicated centrosomes migrate to opposite sides of the nucleus and organize the assembly of spindle microtubules
• Mitotic spindle forms outside nucleus between the two centrosomes

Question 13

Describe the process of DNA condensation?

Answer 13

NOTE: In the diagram, the direction of arrows is showing deconsdensation, but in mitosis you have condensation (i.e. the opposite)

Question 14

Describe the structure of the condensed chromosomes during prophase?

Answer 14

• Each chromosome consists of 2 sister (identical) chromatids
• Each sister chromatid has as a kinetochore

NOTE:

Centromere = specialized DNA sequence of a chromosome

• Site of assembly of kinetochore - main function
• Links sister chromatids

Question 15

Describe the process of spindle formation.

Answer 15

• Radial microtubule arrays (asters) form around each centrosome
  
  • Asters - i.e. shaped like a star
• The radial arrays meet
• Polar microtubules form
  
  • Polar microtubules are the microtubules which overlap with each other in the center of the cell
  • The polar microtubules are essentially made so they are long enough to overlap
  • This allows them to slide over each other to push the centrosomes apart, towards opposite poles of the cell

REMEMBER:

Microtubules are in a dynamic state

• In a population of microtubules, at any point in time, a subset of microtubules are rapidly growing while others are quickly shrinking
• Individual microtubules switch randomly between growing and shrinking states
  
  • Growing = polymerisation
  • Shrinking = depolymerisation

NOTE:

There are two other microtubule types:

• Astral microtubules
  
  • Attach to plasma membrane to pull the centrosomes toward one of the poles
• Kinetochore microtubules
  
  • Attach to chromosomes via kinetochore

Question 16

What happens in prometaphase?

Answer 16

Early prometaphase:

• Breakdown of nuclear membrane
  
  • So chromosomes are now in the cytoplasm
• Spindle formation largely complete
• Attachment of chromosomes to spindle via kinetochores
  
  • Kinetochores assemble at the centromere region of the chromosome

Late prometaphase:

• Microtubule from opposite pole is captured by sister kinetochore
  
  • So in early prometaphase, the one sister chromatid is attached to the microtubule via its kinetochore
    
    • ​e.g. Already attached on left side by microtubule from left centrosome
  • So the other sister chromatid also becomes attached to the microtubule emanating from the opposite centrosome
    
    • ​e.g. Gets attached on right side by microtubule from right chromosome
• Chromosomes attached to each pole congress to the middle
• Chromosome slides rapidly towards center along microtubules

EXPLANATION (extra) - chromosome congression:

• Even if the chromosomes were not captured in the middle of the cell, they have to migrate to the equator so all the chromosomes can line up there
• This chromosomes are pulled towards the centre of the cell until microtubule tension is balanced on both sides of the centromere
  
  • This is how the cell senses that the chromosome is at the equator
  • Sensed by CENP-E
• The chromosomes move laterally towards the equator so it looks like the chromosomes are sliding

NOTE: Prometaphase = late prophase

Question 17

What is the role of CENP-E in prometaphase?

Answer 17

CENP-E = centromere-protein E

MAIN:

• Motor protein - essential for the movement of the chromosomes towards the equator
• Senses the tension (stretch) between microtubules and chromosome which allows

Also…

• Attaches to both the kinetochore and the spindle microtubules
• Play an important role in the formation of stable attachments between kinetochores and spindle microtubules
• Important checkpoint protein

Question 18

What happens in metaphase?

Answer 18

Chromosomes are aligned at equator of the spindle

Question 19

What happens in anaphase?

Answer 19

Overall:

• Paired chromatids separate to form two daughter chromosomes
• Cohesin holds sister chromatids together
  
  • Cohesin is a protein complex
  • Cohesin complex present at centromere

Anaphase split into two parts - A and B

Question 20

What happens in anaphase A?

Answer 20

• Breakdown of cohesin
• Microtubules get shorter
• Daughter chromosomes pulled toward opposite spindle poles
  
  • Essentially the kinetochore microtubules get shorter to pull the sister chromatids apart

Question 21

What happens in anaphase B?

Answer 21

Spindle poles (i.e. the centrosomes) migrate apart due to:

1. Sliding of polar microtubules over each other
2. Pulling force generated on centrosomes
   
   • By astral microtubules attached to the plasma membrane

Question 22

What happens in telophase?

Answer 22

• Daughter chromosomes arrive at spindle poles (centrosomes)
• Nuclear envelope reassembles at each pole
• Assembly of contractile ring
  
  • Actomyosin ring (actin and myosin filametns) → inward contraction → forms cleavage furrow

Question 23

When does the spindle assembly checkpoint happen?

Answer 23

In the transition period out of metaphase (just before anaphase)

Question 24

What happens in cytokinesis?

Answer 24

• Contraction of the actin-myosin ring results in division of the cytoplasm to form two new daughter cells
• Near the end of cytokinesis you get midbody formation
  
  • Midbody = thin intercellular cytoplasmic bridge formed by microtubules (of cytoskeleton), surrounded by the contracted acto-myosin ring
  • At the final stage of cytokinesis, the midbody is cleaved, and two daughter cells are formed
    
    • This process is known as abcission

Question 25

What does the spindle assembly checkpoint check?

Answer 25

Senses completion of chromosome alignment and spindle assembly

• Does this by monitoring kinetochore activity

Question 26

How does the spindle assembly checkpoint work?

Answer 26

• There is a kinetochore associated with each sister chromatid of a chromosome
• So any unattached kinetochores generate checkpoint signals which shows that the cell is not ready to move onto anaphase

This requires two proteins:

• CENP-E
• BUB protein kinases

How they work:

• BUBs dissociate from kinetochore when chromosomes are properly attached to the spindle
• When all dissociated, anaphase proceeds
• CENP-E role:
  
  • Regulates (mediates) BUB activity
  • Also, CENP-E senses tension in the microtubules to make sure that the chromsomes are properly aligned at the equator

Question 27

What is aneuploidy?

Answer 27

Abnormal chromosome number in a cell

Question 28

What two things lead to aneuploidy?

Answer 28

• Mis-attachment of microtubules to kinetochores
• Abberant (abnormal) centrosome/DNA duplication
  
  • i.e. Abnormal centrosome duplication but both of these duplicate at the same time

Question 29

What are the different types of attachements between microtubules and kinetochores? Which ones are the mis-attachments?

Answer 29

Amphelic

• Normal attachment
  
  • Each kinetochore hooked onto microtubule from the centrosome on its corresponding side
• Both kinetochores do not produce a checkpoint signal

Monotelic

• Normal attachment before amphelic attachment
  
  • Only one of the kinetochores of one chromatid is attached to a microtubule
  • The other kinetochore is unattached
• The attached kinetochore does not produce a checkpoint signal
• The unattached kinetochore does produce a checkpoint signal

MIS-ATTACHMENTS

Merotelic

• Attachment:
  
  • One of the kinetochores has two microtubules, one from each centrosome attached to it
    
    • So this chromatid is being pulled in two different directions
• Both kinetochores do not produce a checkpoint signal since they still both have microtubules attached
  
  • And you have enough tension from both sides

Syntelic

• Attachment:
  
  • Both the kinetochores on each chromosome are hooked by a microtubule from the same centrosome
• The kinetochore may OR may not produce a checkpoint signal
  
  • Whether or not a checkpoint signal is generated is due to the tension in the microtubules sensed by CENP-E
  • If the attachment is just from one side, the tension could be weaker, which means the checkpoint signal is still generated
  • More tension = more stable kinetochore attachment

Question 30

How do the mis-attachments lead to aneuploidy?

Answer 30

Question 31

How does aberrant centrosome/DNA duplication lead to aneuploidy?

Answer 31

• Abnormal duplication leads to too many centrosomes - instead of just two
• So you get abnormal attachment of kinetochores to microtubules
  
  • Due to multipolar spindle assmbly rather than bipolar
• This could lead to abnormal cytokinesis

Question 32

Describe how anti-cancer therapy can target cell division?

Answer 32

REMEMBER: You want to induce cell death of the cancer cells

Inducing gross chromosome mis-segregations:

• Normally, when there is a mis-attachment, these may be detected by checkpoint kinase proteins
• The checkpoint kinase would stimulate the attachment-error-correction mechanism
• So by inhibiting the checkpoint kinase you are making the attachment error more likely, thereby making aneupoidy more likely
  
  • Due to unequal chromosome segregation
• Aneuploidy could lead to inviable daughter cells being formed, so they undergo apoptosis

Inhibiting the G₂ checkpoint

• G₂ checkpoint involves checkpoint kinase 1 (CHKE1) and checkpoint kinase 2 (CHKE2)
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