Chapter 14- Cellular reproduction

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Chapter 14- Cellular reproduction

• Where were going
• Review of cell cycle
• Mostly focusing on regulation of processes
• Kinases, CDKs will be critical, along with
  checkpoints
• Some review from genetics
• Mitosis in a bit more depth

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What weve learned from bacteria (Ill skip for
now ?

• Bacteria grow at a variety of rates, depending
  upon nutrients available. The cells have to
  increase to a certain size, and they also have to
  replicate and partition their DNA.
• Initiation of DNA replication is triggered by
  increased cell size as the mass increases, DNA
  replication is initiated. Some cells have
  multiple initiation events, and the amount of DNA
  in a cell may vary
• however, the origincytoplasm ratio tends to
  remain constant over a two-fold range.

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• Once replication has begun, the cell won't divide
  until it is completed. If you block replication
  you block cell division you produce snakes.
• The models are consistent with the production of
  a protein that builds up to a certain level in
  the cell. Initiation then destroys that
  protein. The cell may also produce a cell
  division inhibitor, that prevents division until
  replication and partitioning is complete.

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Go resting cell, unable to undergo division
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How do we determine these stages?

• 1. Generation time how long does it take to
  make a new cell? Easily answered by doing a
  growth curve seeing how long it takes to double
  the cell number.
• 2. Mitotic index once you know the generation
  time, the mitotic index will tell you how long
  mitosis is e.g., if 5 of the cells are in
  mitosis at any one time, and there is a 20 hr
  generation time, then mitosis lasts 1 hr, approx.
  (there is a correction factor, because there is
  always more younger than older cells in a
  culture

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• 3. S phase can be calculated similarly add 3H
  thymidine briefly, then autoradiograph. The of
  cells in S-phase the length of S phase.
• 4. G2-phase can be calculated by labeling
  briefly, and then determining the earliest time
  that labeled cells enter mitosis. (14.2)

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Important! Fairly short G2 of 3-5 hrs
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Models for how cell cycle is controlled- washing
machine! Fig. 17-14, MBOC
How is the cell cycle like a washing machine? A
washer doesnt start agitating until its full of
water! It does NOT simply start a few minutes
after you hit start.
Am I big enough?
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Cool experiments tell us about the checkpoints

• Fuse G1 and S phase cells- G1 goes into S phase.
  Something is in the S phase cell that will drive
  the G1 cell into S phase.
• Fuse G2 and S phase- nothing! The G2 cell is now
  resistant to the S phase factor
• Fuse M phase and other cells- promote condensation

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S phase chromosomes are PULVERIZED!!!
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Key players CYCLINS and CDKs- Cyclin-dependent
protein kinases
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Active cdc2 kinase actually sets up a positive
feedback loop- activating more cdc25, and
inactivating wee1. Thus, the amt of active cdc2
kinase EXPLODES!
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Some activity, but amplified by active M-CDK
M-CDK phosphorylates Wee1, inhibiting the
inhibitor!
From MBOC, Fig. 17-23
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• These switches are progressive After mitosis,
  there's cell growth G1 cyclin builds G1 START
  S phase mitotic cyclin builds---M-phase
  promoting factor is activated mitosis.

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These move things into Mitosis. Destruction of
MPF allows reentry into G1!
These move things through S
These move the cell through G1-S
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Controls to the system

• There are several controls, such that, even if
  other conditions are met, the cell won't go on
  with the cell cycle.

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To leave S phase, DNA must be replicated fully,
but only once

• Stop replication with a poison, and the cell
  won't enter mitosis.
• After S-phase the re-replication block cell
  fusion studies mentioned previously. DNA, once
  replicated, won't replicate again until after
  mitosis and the next G1-S transition.
• http//www.ncbi.nlm.nih.gov/books/bv.fcgi?ridmboc
  4.figgrp.3217
• Fig 17-22 MBOC. Theres a protein that sits on
  the origin that, in one form, activates the
  origin, but, after that, it inhibits the origin.

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Figure 17-22. The initiation of DNA replication
once per cell cycle. The ORC remains associated
with a replication origin throughout the cell
cycle. In early G1, Cdc6 associates with ORC.
Aided by Cdc6, Mcm ring complexes then assemble
on the adjacent DNA, resulting in the formation
of the pre-replicative complex. The S-Cdk (with
assistance from another protein kinase, not
shown) then triggers origin firing, assembling
DNA polymerase and other replication proteins and
activating the Mcm protein rings to migrate along
DNA strands as DNA helicases. The S-Cdk also
blocks rereplication by causing the dissociation
of Cdc6 from origins, its degradation, and the
export of all excess Mcm out of the nucleus. Cdc6
and Mcm cannot return to reset an ORC-containing
origin for another round of DNA replication until
M-Cdk has been inactivated at the end of mitosis
. Similar to Fig. 13-20 in your text
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• d. Damaged DNA p53 is involved in damage
  repair cells with damaged DNA repair it before
  going into mitosis. 14.9. This is a story well
  want to learn.
• The key here is the ATM/ATR proteins- bind to DNA
  breaks, and becomes ACTIVE.
• Once active, it then can phosphorylate Chk2 (if
  the cell is in G1) or Chk1 (G2- go figure the
  names!) Chk2-P then phosphorylates p53- the
  FAMOUS p53- stabilizing it. P53 is a
  transcription factor- p21 is produced, which
  inactivates CDK- no movement into S phase.
• If the cell is in G2, ATR activates Chk1-P, which
  phosphorylates Cdc25- the activator of MPF.
  Once phosphorylated, its removed from the
  nucleus by RAD24- CDK remains inactive, no
  movement into M phase.

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P
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The metaphase-anaphase transition

• All the chromosomes have to be attached before
  anaphase can begin- not just most!
• A bit about chromosomes- theyhave securin on
  them, preventing anaphase
• The APC acts to ubiquitinate securin, marking it
  for destruction in the tunnel of death (aka
  proteasome)
• MAD2 inhibits APC until all the chromosomes are
  attached to securin!

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APC adds ubiquitin to proteins, destroying key
proteins that finish Mitosis. APCCdc20 is what
cuts securin, and APCCdh1 is what cuts M-CDK
SCF Is the ubiquinator during nomal cell growth
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Unattached chromosomes have MAD2, which stops
APC! When ALL are attached, MAD2 goes away!
MAD2
They add ubiquitin, which allows cutting!
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Other things

• Cell size- probably operates in G1, and may
  simply be tied to production of cyclin
• SEX IN YEAST! When yeast are induced to mate,
  they can send out a soluble signal that inhibits
  cell division in nearby yeast until they have
  mated with the signal producer! Amazing!

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Mitosis

• Where were going- well put a cell biology
  spin on this subject- adding some of the areas
  that weve already covered, mainly from what
  weve learned about the cytoskeleton, to this
  subject.

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The centrosome duplicates the centrioles, MTs
disassemble, then reassemble as spindle fibers
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The nucleus disappears- how this happens is not
clear. The centromeres assemble kinetochores.
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Kinesin-like!
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Cool picture of NEWT lung cells in early
prometaphase
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Once attached, the chromosomes head for the
middle theres much tubulin turnover at the plus
end until it reached the equator. VERY strange at
the molecular level!
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AKA kinetochore spindle fibers
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Lots of tubulin flux in the polar and
chromosomeal spindle fibers. Treadmilling!
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Anaphase A- Chr. Move to opposite poles Anaphase
B- poles move farther apart
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Anaphase A
Anaphase B
So the movement is due to new tubulin being added
(red)!
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The little dot on the unattached chromosome is
MAD2!
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Cool experiment that shows that tensions needed
for anaphase!
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The furrow is placed mid-way between the nuclei,
and is actin-requiring- two experiments
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Sea urchin egg treated with myosin antibodies!
Mitosis continues, but no cytokinesis!
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The split is between the nuclei, not halfway
through the cell!
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How plants do it- the phragmoplast

• The cell wall produces special requirements-
  cant just split the cytoplasm!

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Preprophase band!
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Preprophase band!
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Phragmoplast
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The vesicles bring in both membrane and content
to make the cell wall- cellulose, hemicellulose,
lignin,etc. The bundle of MTs starts from the
middle and spreads outward.
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So the vesicles produce both membrane and cell
wall-possibly at the same time
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Things to know

• Parts of the cell cycle
• How we determine the times of each
• CDK-cyclin story
• Controls- re-replication block, anaphase, DNA
  repair- p53, ATM, ATR, CHK12, p53,21,cdc25,
  etc. APC role in promoting anaphase and entry
  into G1 (fig. 14.26)
• Mitosis and the new things weve learned-
  anaphase AB, Kinetochore structure, etc.
• Preprophase band, phragmoplast, cell plate