MolBiol #6-7
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  • XV. 1 XV) cell cycle ( Alberts Ch. 17) A) Phases of the cell cycle (M, G1, S, G2) 1) M = Mitosis 2) G1, S, and G2 make up interphase B) review of mitosis 1) M phase includes mitosis and cytokinesis 2) several events occur during mitosis 3) M phase can be divided into 6 stages a) prophase b) prometaphase c) metaphase d) anaphase e) telophase f) cytokinesis C) Cell cycle time varies D) Regulation of the Cell Cycle by cell growth and extracellular signals 1)G1 restriction point a) occurs late in G1 b) controls progression from G1 to S c) first defined in Saccharomyces cerevisiae - START d) passage through START in yeast is highly regulated e) most animal cells have a G1 restriction point analogous to START XV. 2 2) Some cell cycles are controlled via a G2 restriction point a) Schizosaccharomyces pombe b) oocytes E) Checkpoints 1) G2 checkpoint a) senses unreplicated DNA b) senses damaged DNA 2) G1 checkpoint a) also recognizes damaged DNA b) mediated by p53 3) M phase checkpoint F) Identification of MPF 1) experiments with xenopus oocytes 2) experiments in mammalian cells 3) experiments in yeast 4) studies in sea urchin embryos G) purification and characterization of MPF from xenopus 1) MPF contains two components a) a cyclin dependent kinase (CDK) called Cdc2 b) a mitotic cyclin - cyclin B 2) MPF activity cycles H) MPF (CyclinB/cdc2) controls G2-M transition XV. 3 1) Cyclin B synthesis begins in S phase 2) Cyclin B accumulates and forms complexes with Cdc2 throughout S and G2 3) Cdc2 is phosphorylated on three regulatory positions 4) Thr 15 and Tyr 15 get dephosphorylated by cdc25 5) active cdc2 phosphorylates a variety of target proteins a) nuclear lamins b) Histone H1 c) MAPs d) other targets 6) Cdc2 activity also triggers the degradation of cyclin B 7) cdc2 is then inactivated 8) cells exit mitosis, undergo cytokinesis, return to interphase I) Cyclin B and cdc2 are members of large families of proteins 1) yeast contain multiple cyclins 2) higher eukayrotes have multiple cyclins and cdc2 related kinases ( CDKs) J) The activities of the Cdks are regulated by multiple mechanisms 1) association of the Cdks with their cyclin partners 2) stimulatory phosphorylation sites 3) inhibitory phosphorylation sites 4) binding of inhibitory proteins ( CKIs - Cdk inhibitors) K) Growth factors and the D type cyclins 1) cells require growth factors to pass from G1 to S 2) Cyclin D synthesis is induced in response to growth factors 3) Cyclin D concentration falls if growth factors are removed XV. 4 4) Cyclin D/Cdk4 drives cells through the restriction point 5) Cyclin D overexpression is associated with cancer 6) CKIs such as p16 can inhibit Cdk4/Cyclin D complexes L) Rb is a key substrate of cyclin D1 1) Rb is a product of the gene responsible for retinoblastoma 2) Rb is also associated with other cancers 3) Rb is a tumor suppresser gene 4) Rb phosphorylation is regulated throughout the cell cycle 5) Rb becomes phosphorylated by Cdk4/Cyclin D complexes as cells pass through G1 restriction point 6) unphosphorylated Rb binds members of the E2F family 7) binding Rb inhibits E2F’s ability to activate transcription 8) phosphorylation of Rb results in its dissociation from E2F 9) E2F then activates target genes M) p53 acts to stimulate expression of p21 1) p21 inhibits the cell cycle progression in two ways a) it is a CKI that inhibits several Cdk/cyclin complexes b) It may also directly inhibit DNA replication in S phase 2) expression of the p21 gene is induced by p53 N) TGF  inhibits cell proliferation in many epithelial cells 1) TGFB arrests cells at G1 2) TGFB induces the CKI p15 and p27 3) p15 and p27 bind Cdk4/Cyclin D 4) In the absence of Cdk4 activity, Rb phosphorylation is blocked and the cell cycle is arrested at G1 Figures from Alberts: 17-14, 17-19, Panel 18-1

MolBiol #6-7 MolBiol #6-7 Presentation Transcript

  • Chromosomes and Mitosis Lecture 6
  • 1 Chromosomal Basis of Heredity
    • A gene is a unit of heredity
    • Genes are carried on DNA
    • DNA is contained within chromosomes as chromatin
  • Chromosomes replicate during cell division
  • The chromosome complement
  • Chromosome analysis Cri Du Chat results from loss of a small piece of chromosome 5
  • Gene Map
  • Chromosome pairs
  • Non-identical genes
  • Sex chromosomes
    • These determine the sex of an individual
      • Two X chromosomes make a female
      • One X and one Y a male
  • Two types of Cell Division
    • Cells divide for two reasons
      • To create genetically identical copies of themselves
        • This is
        • mitosis
      • To create gametes that contain half of the chromosomes of the original cell
        • This is meiosis
    46 46 46 46 23 23 23 23
  • The Cell Cycle
  • S phase Replication Condensation Schematic
  • DNA replication Duplex DNA begins Replicating Replication bubbles merge creating two duplexes
  • Mitosis
  • The stages of Mitosis
  • Prophase Detail
  • Prometaphase
  • Metaphase
  • Anaphase
  • Telophase
  • The sum total of the process
  • Karyotypes
  • Chromosome Length
  • Chromosome appearance
  • Meiosis and Gametogenesis
  • Somatic and Germline cells
    • Development of a fertilized egg into an adult results in two distinct types of cells
      • Somatic cells
        • These create all tissues and organs of the adult except for cells destined to become sperm or egg
        • They can only undergo mitosis
      • Germline cells
        • The final differentiated form of these cells are mature gametes: the sperm and egg
        • These cells undergo mitosis until gametogenesis
          • They then undergo meiosis
  • Meiosis
  • Meiosis is required for gametogenesis
  • Meiosis I Somatic cells Germline Cells
  • Interphase I and Prophase I Leptotene
  • Prophase I Zygotene
  • Prophase I Pachytene
  • Prophase I Diplotene
  • Recombination
  • And on the molecular level
  • Metaphase I and anaphase I
  • Meiosis I is the reduction division
  • Non-disjunction
  • Telophase I
  • Cytokinesis sperm formation oocyte formation
  • Meiosis II
  • A comparison of meiosis and mitosis Germline Somatic Cells involved 4 2 Cells resulting 2 1 Nuclear Divisions Reduces Maintains Chromosome number Meiosis Mitosis
  • Relationship to Gametogenesis
  • Sperm and Egg formation
  • Gametogenesis
  • Fertilization
    • Entry of a single sperm into an egg prevents entry of other sperm
    • The DNA of sperm and egg are initially kept separate in “pronuclei” of the zygote
    • Timing of a pregnancy extends from the “last menstrual period” (LMP) rather than the time of fertilization
  • Mitotic Non-disjunction
  • Cell cycle and apoptosis
    • Cells undergo 3 controlled processes
      • The first two are part of the cell cycle, the last an exit from the cell cycle
      • Division (the cell cycle)
      • Quiescence
        • This is where most of the work of being a cell lies
          • During division the energy of the cell is devoted to making a new cell
      • Death
        • This can be a normal process creating a final functional form or an induced suicide
          • Epithelium and reticuloendothelial cells undergo active transitions towards terminally differentiated states in which the final forms are unable to divide
            • The stratum corneum consists of cells that have become bags of crosslinked keratin protein with no internal metabolism
          • Suicide can be induced because the organism senses a threat to the entire organism
            • Infection, cancer, avoidance of autoimmunity
  • Control of entry into cell cycle and apoptosis
    • Cell cycle is initiated by phosphorylation of transcription factors
    • These activate transcription of a set of proteins known as cyclins
    • The appearance of cyclins is progressive and determines the types of proteins that will be phosphorylated at a particular point during the cell cycle
  • Cyclins and CDK’s
    • CDK levels don’t change while cyclins are destroyed at the end of each phase
    • There are 3 general groups of each
      • G1 cyclins
        • Cyclin D
      • S-phase cyclins
        • Cyclin A
      • G2 cyclins
        • Cyclin B (maturation promoting factor MPF)
      • Cyclin E is shared between G1 and M phase
      • Cyclin A is shared between M phase and G2
  • Cyclins bind CDK’s
    • CDK’s are C yclin D ependent K inases
    • Association with cyclins activates their kinase function
      • A cyclin tethers a target protein to the CDK
    • The targets of CDK’s are transcription factors among other proteins
      • CDK’s are serine/threonine kinases
  • The exit from Go
    • Go is a quiescent state
    • Activation of G1 CDK occurs due to a rising level of G1 cyclins
    • G1 cyclins are transcriptionally activated by growth factors
  • Events during G1
    • A rising level of G1 cyclins increases the activity of G1 CDK’s
    • CDK’s in turn activate proteins and in turn genes that prepare the cell to begin DNA replication
    • At the G1 S boundary, the cell encounters a checkpoint
  • G1/S checkpoint
    • This is controlled by the activity of the transcription factor E2F
      • E2F is a family of related proteins (E2F 1 to E2F5)
    • E2F is found complexed throughout the cell cycle to another family of proteins: Rb
      • At the G1/S checkpoint, Rb is phosphorylated by CDK2/cyclinA
      • E2F is freed from sequestration and activates transcription at genes containing an E2F consensus sequence
  • And those genes are
    • Three groups
      • Cell cycle regulators
        • Cyclin A
        • E2F, Rb, myc, myb
          • Note that these are not all positive regulators of cell cycle
      • Enzymatic machinery for DNA synthesis
        • DNA polymerase
        • PCNA
        • Enzymes involved in nucleotide metabolism
      • DNA synthesis regulators
        • Enzymes that recognize the origins of replication for example
  • Other Checkpoints
    • These monitor the completion of DNA synthesis
      • The presence of Okazaki fragments prevents entry into G2
    • DNA damage
      • This occurs before, during and after completion of S phase
    • Spindle attachment
      • Failure to attach spindle to centromere results in blockage of mitosis at metaphase
      • Failure to align the spindle during cytokinesis results in blockage at anaphase
  • Downregulation of cyclin influenced CDK activity
    • This is accomplished through proteolysis of the cyclins
      • G1 phase cyclins disappear during S and G2 phase
      • M-phase promoting factor (CDK2 + cyclin B) concentrations rise just prior to onset of mitosis
        • Cyclins associated with MPF are degraded by anaphase promoting complex
          • Cyclin B levels peak at G1/M
            • Degradation during anaphase
          • APC promotes polyubiquitination of cyclin B
          • Ubiquitinated cyclin B is degraded by a proteosome
    • Cyclin transcription is also turned off and the mRNA is unstable
      • So no new cyclin is made until transcription is restored
  • MPF activates APC which ubiquitinates cyclin B
  • In the overall
    • Stimulated entry into G1 results in appearance of an initial level of cyclins that promote the progressive activation of genes enabling the cell to synthesize DNA
    • A series of progressive steps result in
      • Activation of genes further into the cycle
      • Degradation of cyclins that promoted earlier steps
      • Passage through checkpoints that insure mechanistic fidelity of each step
  • Apoptosis (apo – toe – sis)
    • This is programmed cell death
      • Distinguish it from necrosis
      • Necrosis results from traumatic forces outside the cell
      • Necrotic tissue provokes inflammation as the immune system moves in to clear out damaged and dead cells
    • Apoptosis is an ordered stepwise self-destruction that permits surrounding cells to utilize the breakdown products of the dead cell
      • There is no inflammation involved
  • The apoptotic cell
    • Mitochondria break open
    • DNA fragments in a regular way
    • The cell loses a regular shape
      • Undergoes blebbing
      • This is an irregular bubbling appearance of the plasma membrane
  • The mechanisms of apotosis
    • Can be classified as externally or internally signaled
    • One internal route involves p53
    • p53 is a transcription factor that is involved in cell cycle control and sensing the presence of DNA damage
    • The central role p53 plays is at the G1/S checkpoint
  • P53 controls entry into S-phase
    • P53 can sense DNA damage by binding mismatches
    • In the presence of damage, p53 activates transcription of p21
      • P21 binds and inactivates CDK2-cyclin E complexes
      • The complex is unable to phosphorylate Rb and free E2F
      • Thus entry into S phase is inhibited
      • If the damage is repaired, p53 levels and p21 levels drop and S phase ensues
  • But if the DNA damage is extensive
    • P53 induces apotosis by activating transcription of Bax
      • BAX protein competes with BCL-2 to form pores in mitochondrial membranes
        • BCL-2 prevents the release of cytochrome c from mitochondria into the cytoplasm
        • BAX permits release of cytochrome c
      • When released, cytochrome c stimulates caspase activation
  • The caspases
    • These are proteolytic enzymes that are held in check by external or internal inhibitors
    • Activation results in an explosive proteolytic cascade
      • Caspase 9 cleaves and activates other caspases
      • The caspases also activate quiescent nucleases
  • External apoptotic mechanisms
    • Involve external “death signals”
    • Cells may be recognized as a threat to the whole organism
      • The immune system moves in to kill them
      • One mechanism of killing involves a command to the cell to initiate apoptosis
  • Fas/Fas ligand signaling
    • Fas ligand (FasL) is a membrane bound cell surface protein
    • It binds to Fas receptor
    • Binding results in trimerization and activation of APAF
    • APAF in turn activates caspase 8 by proteolysis of a caspase 8 zymogen
      • Caspase 8 cleaves a BCL-2 family member BID
      • BID translocates to the mitochondria and binds BAX
      • Bax permits leakage of cytochrome c and activation of the caspase 9 cascade via APAF-1 again