Lecture 1: 22/09/2020 The cell Cycle

Mechanical view of mitosis

Temporal view of the cell lifecycle

• Mitosis occupies very little time
• The rest is called interphase
• There are 4 phases
• M: mitosis
• In most cells it takes 1 to 1 ½ hours to complete
• G1: Gap 1= growth
• S: DNA replication
• G2: Gap 2 prep for mitosis, spindle growth etc.
• G1 + S + G2 = interphase
•  

  Q. What drives cell cycle progression?

  A. two proteins called cyclin and cyclin dependent kinase (CDK)

  - Cyclin levels rise and fall during the various phases of the cell cycle

  - CDK levels are constant, sometimes it is active and sometimes it is not, whenever cycling is around the CDK is active because the cyclin binds to the CDK to make it active therefore must have cyclin to have active CDK (CDK works all the time but when cyclin is added to the equation CDK increases in action and specificity)

  - Cyclin regulates CDK by changing its substrate specifically at particular stages of the cell cycle – whenever the cyclin concentration is higher the CDK is affected

  - CDK activity is terminated by cyclin degradation (generally) ensuring uni-directionality as degradation is irreversible

  - What is a kinase – takes a phosphate off of ATP and phosphorylates a protein – adds a phosphate onto something else

  - Phosphatase removes a phosphate from a protein

  
  

  Proteasomes: protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases.

  
  

  The cell has safeguard mechanisms that control the order, integrity, and fidelity of the cell replication process… Called checkpoints

  
  

  The cell cannot go on to the next phase without the requirements for these checkpoints being completed.

  If you stop the cell in the middle of synthesising DNA the cell will die … so if you want to kill a cancer cell it is most susceptible during S phase … most cancer treatments target S phase… which is why cancer patients hair falls out because the hair cells have a high portion of cells in the S phase at any one time therefore they are very susceptible in the S phase

  To safely stop a cell the best place is at the end of G2

  Checkpoints

  the decision to precede from one part of the cell cycle to another depends on:

  • Growth
  • DNA replication
  • DNA integrity
  • Cellular integrity

• Therefore, the control system must be able to respond to the external and internal conditions that indicate the need for cell growth and division.

  Checkpoint ‘monitor progress’ through negative regulation – sending a signal to STOP the progression of the cell cycle – this is easier than encouraging a new pathway for positive msg..

  They ensure that eh events associated with each phase of the cell cycle are carried out at the appropriate time and in the appropriate sequence

  They make sure that each phase of the cycle has been properly completed before the next phase is initiated

  THERE ARE 3 CHECKPOINTS

  Q. Name the 3 checkpoints

  A.

  • G1/S restriction checkpoint
  • Check for:
  • Cell size
  • Nutrients
  • Growth factors
  • DNA damage
  • G2 Checkpoint
  • Check for: cell size
  • DNA replication
  • Metaphase or Spindle assembly checkpoint
  • Check for:
  • Chromosome attachment in spindle

• Cyclins

  • Four classes A, B, D, E (many subclasses) – this labelling depends on the subject/ sample
  • Class defined by phase of the cell cycle in which they bind their CDK
  • G1/ S phase cyclins
  • Bind CDKs at the end of G1, fall at the beginning of S phase and commit to cell DNA replication (cyclin E)
  • S phase cyclins
  • Bind CDKs during the S phase, requires to initiate replication (cyclin A) active through G2
  • G2/M phase cyclins
  • Bind CDKs immediately before M phase, initiate early mitotic (or meiotic) events (cyclin B)
  • Metaphase checkpoint  
  • Causes cyclin B to rapidly degrade
  • G1 cyclins
  • Does not behave like other cyclins. Coordinates cell growth with the entry to a new cell via external growth regulatory signals (cyclin D)

• How do People study the cell cycle?

  • Had to find a way to synchronize cells to ensure there was enough cell extract to do meaningful work
  • Used organisms with unusual/ useful properties
  • Used chemicals that synchronizes cells
  • Very few cells will allow themselves to be synchronized
  • Focus on eukaryotes
  • Yeast: a fungi – critical for studies:
  • S. cerevisiae
  • Divided by budding termed the ‘budding yeast’ meaning they are in the early stages of the cell cycle
  • Isolating the buds allows you to synchronize the cells
  • S. pombe
  • They have haploid stages in mitosis therefore mutants that arrest in different stages of the cell are easy to spot – many of them are temperature mutants allowing the genes to be ‘switched’ on and off with the change in temperature
  • Ease of manipulation
  • Block and release through mutants
  • Centrifugation elutriation – through size – can get them to come off as they are being centrifuged through orifices in the machine – separating the smaller buds from the larger buds
  • Ease of genetic engineering (tools available to modify gene expression)
  • Stag of the cell cycle can be inferred from the size of bud in budding yeast
  • Cell cycle proteins are so key they have not evolved much over time therefore they are in many ways similar to the human cells in this cycle
  • xenopus laevis
  • Through the use of the African clawed toad (xenopus laevis) oocytes and eggs can be used to mimic events of the cell cycle with a slight difference
  • To synchronize oocyte development, the frogs can be injected with PMSG/ human chorionic gonadotrophic (HCG) which induces ovulation and egg laying
  • The eggs are 1.3mm diameter
  • Progesterone triggers oocyte maturation
  • Oocytes progress through 2 cycles of meiosis and arrest until fertilization
  • The egg will rapidly divide every 10 minutes when fertilized (and the egg extract will also ‘cycle’)
  • Stockpiled material which gathers during the maturation period can be used for multiple cell divisions (up to 11)
  • Xenopus are very useful for biochemical studies due to the large amount of synchronous material available for examining precipitation of interacting proteins due to the availability of cell free extracts
  • A cell extract is obtained by lysing the cell of interest and centrifuging out the cell walls, DNA genome, and other debris.. effectively creating a cell environment in the test tube
  • Invertebrates
  • Such as Drosophila melanogaster have shown us the interplay between the cell cycle and development
  • Drosophila has 13 rapid synchronous divisions straight after fertilization due to stockpiling of the necessary regulators in the egg
  • For all intents and purposes there are NO gap phases and no increase in cell size at this point in embryogenesis (as in xenopus)
  • Growth only follows when the G phases appear after this point
  • Mammalian Cells
  • Populations of mammalian cells can be followed during the cell cycle by fluorescence
  • By microinjection (small highly-manipulated populations) and staining and by confocal microscopy in living cells
  • Microinjection is most commonly very disruptive to cells and time consuming … imagine microinjecting a population of 100 cells
  • By flow- cytometry (large real- time populations) and where equipment allows … FACS
  • Most often, DNA is fluorescently labelled as it is easy to see the % of cells in G1 have ½ as much fluorescent DNA than cells in G2, while S-phase has intermediate levels.
  • These cells must be synchronized
  • Factors can be added or removed which arrest the mammalian cells in certain stages of their cell cycle
  • Hydroxyurea
  • Arrest in S-phase
  • Rapamycin
  • Arrest in G1
  • Taxanes/ Nocodazole/ Vincristine – flower/ colcichine
  • Arrest in M phase

• Cells DO NOT cycle with the same frequency

  • Epidermis is about 12 cells thick
  • Cells in the basal layer divide just fast enough to replenish cells that are shed

• The cell cycle and cancer

  • Cell cycle is really important in cancer research
  • Proteins that are important in inhibiting the cell cycle were identified as ‘Tumour suppressors’ 
  • Names such as p53, p105 – they prevent the cell progression – preventing passing from G1
  • In the case of p53
  • When the cell’s DNA is damaged, a sensor activates p53
  • This halts the cell cycle at G1 by triggering production of an inhibitor allowing time for DNA repair (the repair enzymes are also activated by p53)
  • When the damage is fixed, p53 will release the cell, allowing it to continue through the cell lifecycle
  • If the damage is not fixed, p53 will trigger apoptosis (programmed cell death) so that the damaged DNA is not passed on
  • P53 is not present or fully functional
  • When p53 is defective, a cell with damaged DNA and can proceed with cell division
  • Thus the daughter inherits the mutation
  • Over generations, cells with non-functional or absent p53 tend to accumulate mutations allowing the cancer to progress in gravity over time
  • P53 is regulated by phosphorylation by cyclin/ CDK

• How do cyclins get activated in succession? – important question

  • We known cyclins are in charge of different parts of the cell and decide what gets phosphorylated (activated or inactivated) and when
  • How do cyclins get switched on in succession[EM1] ?
  • One example of how this occurs is shown by the tumour suppressor (anti-oncogene) p105, also known as the retinoblastoma protein (RB)
  • In order to move from early G1 to late G1 the cell must male cyclin E
  • The cyclin E gene requires the transcription factor E2f to produce RNA transcripts and ultimately the protein product
  • The G1 cyclin/ CDK phosphorylates and inactivated RB allowing cyclin E to be produced
  • If RB is mutated…
  • Can occur randomly (sporadically), or it can be congenital (by germline mutation or inherited)
  • Children develop retinoblastomas – that gave the gene its name
  • Used to always mean removal of the affected eye but not anymore
  • Can affect sight
  • If it is a germline mutation the consequences can be more severe

• At G1 the environment has a massive effect, telling the cycle is to go ahead or it needs to be inhibited  

   

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  [EM1]Look for a video for more info and a simplified explanation