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    3.24.2010 3.24.2010 Document Transcript

    • 3.24.2010 – Mitosis<br />
      • Identifying which stage of interphase is the cell in?
      • Label the cells with
      • Tritiated Thymidine
      • Bromodeoxyuridine (BRDU)
      • There is an antibody to detect BRDU in labeled DNA
      • Labels incorporated into the DNA
      • Tells how much DNA is in the cell during a stage
      • Stages of the Cell-Cycle
      • Precedes Mitosis
      • Has checkpoints to make sure cell is prepared in terms of size and chromosome copies.
      • Signaling at the Checkpoints
      • Can measure amount of DNA in the cell for measuring which step cell cycle is in
      • Labels used
      • Can be incorporated into DNA as it’s being copied.
      • To determine how much DNA is present
      • Radiography
      • Cells can be imaged and Trimidium label can be measured and labeled
      • Can determine which cells are at a certain stage
      • Majority of cells are in G1 phase > S phase > G2
      • Cell sorting can be done for separating the populations
      • FACS – fluorescent activated cell sorting
      • Can take labeled cells, run them through the FACS machine and separate the cells with the labels
      • BRDU label used primarily
      • What questions can be looked at?
      • What regulates the checkpoints?
      • What is happening at Mitosis?
      • Checkpoints
      • Between G1 and S: is environment favorable for entering DNA synthesis?
      • Can enter G0 stage until environment is favorable to continue to S phase
      • Cell can also undergo apoptosis if signals for ideal environment are not given at a certain time frame
      • G2 before Mitosis
      • Cell is looking for whether or not DNA has been replicated, and if so, correctly?
      • In Mitosis
      • Makes sure sister chromatids are attached and separate properly at mitotic spindle
      • Key Transition Points in the Cell Cycle
      • G1 S phase checkpoint – “restriction point” – requirements for S phase transition
      • Growth factors
      • Nutrients
      • Increase in cell size
      • Absence of DNA damage
      • Can enter G0
      • “G2 – M transition”
      • Cell size
      • DNA damage
      • DNA replication
      • Can enter G0 again and signal sequences can make cell go back to repair damage
      • Mitosis – “Metaphase-Anaphase transition”
      • Chromosome attachments to spindle so the chromatids can be pulled apart
      • What Did People do to determine that there were proteins that influenced cell splitting
      • Separate cells from different stages fused
      • The fused cell is called a heterokaryon (monoclonal antibodies in previous lectures) cells with two nuclei.
      • To fuse cells:
      • Can use viral infections
      • Chemical treatment
      • Electroporation (Electrical pulses)
      • Often used to put DNA into a cell as well
      • Then fuse separated cells
      • S and G1 fusion – G1 nucleus is activated to undergo DNA synthesis
      • M and G1 fusion – mitosis activated
      • Experimenting with oocytes to determine what was driving the cycle in fused cells
      • Stalled in Meiosis II
      • Cytoplasm injected and cell doesn’t divide
      • Side Note: Progesterone from female will move cell into mitosis and generate egg in resting phase. The signal begins to activate the completion of mitosis at the end of meiosis I
      • Fertilization would complete meiosis II, it would be the signal
      • Increased concentrations of certain proteins in mitosis
      • Saw two peaks in a graph
      • MPF = maturation promoting factor (green peak) (rose at onset of mitosis and fell very fast during mitosis)
      • Will mature the egg if being stalled in meiosis II aka. M phase promoting factor because it moved the cell into mitosis
      • Temperature Sensitive Yeast Experiments
      • Generated by random mutagenesis
      • Looked for mutations in mitosis at higher temps than what they normally grow at (ideal is 23 C for a normal cell)
      • At restrictive temp, some yeast cells will not move through mitosis, so the cell is stalled at some point in cell cycle.
      • Can see what isn’t happening at G1 and ask what protein is mutated or signaling molecule is absent that is not allowing the cell to move to S phase.
      • Can use for all stages of mitosis
      • Lee Hartwell
      • Used a particular yeast strain to identify mutations that didn’t allow mitosis to continue normally.
      • Wild Type yeast could divide into daughter cells normally wherease mutated ones did not divide into daughter cells
      • There was a mutation in cell cycle division (cDC2 cell division cycle)
      • Important for fission of mitotic event and without it, or mutated, the cells don’t divide
      • cDC2 is a protein that allows division of cells during mitotic events
      • without cDc2, the cells don’t divide.
      • We know it is involved in G2 to M phase transition point, but not sure of the specifics
      • CdC2 is involved in moving cell from G2 to M phase.
      • MPF is a cyclin-dependent kinase (CDK) because it doesn’t function until cyclin reaches a certain level.
      • Timothy Hunt and sea urchins
      • Found that there were cycling proteins through the period of the cell cycle and it peaked at the onset of mitosis. (G2 Mitosis)
      • Named it cyclin (also by Lee Hartwell at the same time) because it cycled to increase levels and dropped during the cell cycle
      • MPF and Cyclin
      • Both peak at onset of Mitosis
      • MPF is a Cdk – a cyclin dependent kinase because it doesn’t function until the cyclin concentrations reach a certain level
      • Cyclin increases during interphase
      • At G2, will activate Cdk as a complex
      • Complex is a functional protein that moves cell past G2 – M transition and into mitosis
      • Complex stays functional until cyclin levels drop
      • Relationhsip between CDk and Cyclin
      • Cdk is by itself in cell at all times, stable
      • As cyclin increases, more Cdk binds to it.
      • There are a series of inactivating phosphorylating events to activate complex
      • Kinases put Pi on Tyrosine and Threonine, which keeps complex inactive
      • Activation phosphorylation event: kinase comes in and Pi an activating site on cyclin.
      • Activating phosphatase after Cdk component is Pi’ed, is dephosphorylated and strips away inactivating Pi
      • Results is a cyclin Cdk complex as a functional protein
      • At G2/M transition, MPF is actually the cyclin Cdk complex which regulates G2 M.
      • Active MPF allows cell to move past G2 transition point and go into M phase
      • Table in text –
      • M-Cdk (cyclin Cdk complex)
      • Cyclin B (cyclin identified by Timothy Hunt) and Cdk is Cdk1
      • Cdk1 is CdC2 in yeast experiments
      • Between G2 and M
      • (((Future terminology)))
      • G2 M
      • M-cdk
      • Cyclin b and Cdk1
      • G1-Cdk – cyclin D and Cdk4 or Cdk6
      • G1/S-Cdk cyclin E
      • Ready to synthesize DNA
      • Makes sure ready to commit to S phase
      • S-Cdk
      • Commits cell to DNA synthesis
      • M-cdk is active between G2 and M
      • M-Cdk plays a part in the events between G2 and M
      • Nueclar envelope breaks down
      • G2 M
      • Lamins are phosphorylated by M-Cdk
      • Chromosomes must condense for mitosis
      • Condensin condenses them only when Pi’ed by M-Cdk
      • Recognition of amino acids on M-Cdk
      • Inactivating Kinase
      • Wee
      • Comes in first
      • Activating Kinase
      • MO15
      • Comes in next
      • Activating phosphatase (from wee and MO15)
      • Stimulated by recognizing Pi events on Cdk complex and remove inactivating Pi to alieve an active M-Cdk
      • As you make active M-Cdk, it will Pi the inactive phosphatase to make it active so there is a + feedback loop
      • More M-Cdk made, the more active phosphatase made to continue cycle.
      • Recognize amino acids to Pi on Cdk
      • Activating Phosphatase is sitmulated by recognizing Pi’ing events
      • As active M-Cdk is made, it will Pi the inactive phosphatase to make it active. Positive feedback.
      • Concentration of cyclin drops after transition to M phase, which inactives functional M-Cdk
      • Cyclin is ubiquiniated
      • Ubiquitin ligases which recognize a sequence on cyclin when it is in complex form
      • Cyclin binds to Cdk
      • Exposes site where ubiquitin can be placed onto cyclin
      • Ligases place ub. On cyclin and then ub acts as signal to degrade protein in proteosomes (organelles in cell that will target ub-linked proteins for degredation)
      • Cyclin is ubiquitinized by ub-ligases so that they are destroyed.
      • Once cyclin is lost, the M-Cdk is inactive and Cdk is left, which will stay constant
      • Ligases place ubiquitin on cyclin and that acts as a signal to degrade proteosomes (organelles in cell that target ubiquitin-linked proteins for degredation)
      • CDK IS FAIRLY CONSTANT THROUGHOUT CELL (concentration – but it may change as cell size increases)
      • CDK regulation
      • Important amino acids required to be Pi for Cdk to be functional
      • Tyrosine 15 and threonine 14 Pi’ed by Wee
      • Threonine at 161 Pi by MO15
      • Cdk Inhibitor (Cki)
      • Bind complex at the ATP binding site for the proteins
      • Wipes out abilities to be kinases
      • Laminine or condensin, so without binding to ATP, cannot function
      • Active MCdk cannot bind ATP and can’t use Pi from ATP to Pi target
      • Synthesis of Cyclin B
      • Increases during interphase
      • Interacts with Cdk 1
      • Activatin/inactivating Pi events
      • Inactivating Pi’s need to be removed
      • Activated phosphatase removes Pi events at Threonine 14/Tyrosinse 15 leaving only threonine 161 Pi’ed
      • Active M-Cdk complex!