Cell Division and Cell Cycle

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Cell Division and Cell Cycle

• 22.228
• Dr. Bill Diehl-Jones

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Reading list

• Today The cell cycle.
• Topic Karp third edition Karp fourth edition
• Cell cycle mitosis and meiosis pp 590 611 588 -
  609
• cell cycle pp 580-582 578 - 580
• control of cell cycle pp 582 - 590 ,601 580
  588, 601
• Tomorrow the cell nucleus and how DNA is
  organized.
• Topic Karp third edition Karp fourth edition
• the Nucleus, structure pp 494 492
• membranes and pores pp 494 501 492 - 498
• DNA packaging pp 501 507 498 -507
• nuclear matrix pp 515 516 514 -516
• gene structure pp 521 539 520 -537

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Agenda

• Overview of Cell Cycle
• Mitosis and Cell Division
• Control of the Cell Cycle

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The Life of a CellThe Cell Cycle

• Divided into the following phases
• M Phase
• Mitosis occurs here
• G1 Phase
• Cell grows in size and differentiates
• S Phase
• Synthesis of DNA yields double-stranded
  chromosomes
• G2 Phase
• Cell synthesizes material needed for mitosis

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The Cell Cycle
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Cell Cycle Continued

• G0
• cell rests metabolic and synthetic activity
  slows
• Restriction point
• A decision is made on whether to complete cell
  cycle
• Mitosis
• We will not review mitosis herein, although you
  should familiarize yourself with the events of
  cytokinesis and karyokinesis
• An understanding of mitosis provides a basis for
  understanding cancer chemotherapy

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Cell Division
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Agenda

• Overview of Cell Division
• Mitosis cytokinesis
• Cytokinesis
• The mitotic apparatus
• Kinetocores, microtubules and motors
• Mechanics of cytokinesis
• Roles of Actin and Myosin II

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• Finally, therefore, we state the conclusion that
  all the cells or their equivalents in the fully
  developed organism have arisen by a progressive
  segmentation of the egg-cell into morphologically
  similar elements .
• Remak,
  1855

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Cytoskeletal Dynamics During Mitosis

• Mitosis
• essentially the process of partitioning
  newly-replicated chromosomes into separate parts
  of the cell
• Occurs as the last step of the cell cycle
• Last approx. 1 hour
• During that time
• Cell must build, then disassemble mitotic
  apparatus

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A Brief Summary of Mitosis
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Association of Tubulin with Chromatin
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Mitotic Apparatus at Metaphase

• Two Parts
• (1) Central mitotic spindle
• Bilateral bundle of microtubules separated by
  metaphase chromosomes
• (2) Asters (x2)
• Tuft of microtubules at each pole

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The mitotic spindle has 3 sets of microtubules

• (1) Astral microtubules
• Radiate from centrosome, form aster
• (centrosome is essentially an MTOC)
• Help position mitotic apparatus, determine
  cleavage plane
• (2) Kinetochore microtubules
• Attach to chromosomes at kinteochore
• (3) Polar microtubules
• Interdigitate with opposing pair

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Microtubules have a polarity

• Minus (-) ends
• all point toward kinetochore
• Plus () ends
• All point away from kinetochore
• Recall () and (-) ends of microtubules differ
  in their rates of assembly

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Mitotic Apparatus at Metaphase

Centrosome
Aster
Aster
Spindle
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Attachment of Microtubules to Chromosomes
Kinetochore

• Occurs at Kinetochore
• In animal cells
• A 3-layer, plate-like structure
• Yeast are different (next slide)
• () ends of MT attach to outer layer
• Centromeric chromatin have binding factors
  mediating MT attachment

Fibrous Corona
MT
Inner Plate
Outer Plate
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The trouble with yeast
Kinetochore MT
Nucleus

• Celll wall remains intact
• Kinetochore MTs attach attach to spindle pole
  bodies in nuclear membrane
• Yeast centromere
• 3 contiguous units (DCE I-III)
• Centromeric binding factors (CBF 2 and CBF 3)

Spindle Pole Body
Polar MT
DCE I
CBF 2
DCE II
DCE III
CBF 3
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Two Key Events in Spindle Assembly

• (1) Formation of Poles
• Spindle microtubules must attach to poles
• Cytoplasmic Dynein likely involved
• Evidence from micro-injected anti-dynein
• (2) Capture of Chromosomes

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Events of Early Spindle Formation

• Centrosomes must separate and migrate to poles
  for orientation of mitotic apparatus
• Two cytosolic proteins are likely involved
• Kinesin-related proteins (KRPs)
• Dyneins
• Other MT motors
• Studies in yeast and flies
• Abs to KRPs (() or (-) ended) manipulate
  centrosome positioning

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Model for centrosome movements during late
prophase
1. Centrosome Alignment
Polar MTs


Aster MTs
(-) end directed MT motor
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Model for centrosome movements during late
prophase
1. Centrosome Separation
(-) end Dynein
() end directed MT motor
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Question

• What would you predict is the effect of the
  following
• an antibody to ()-ended KRP?
• An antibody to (-)-ended KRP?

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Chromosome Capture

• Rapid fluctuations in spindle MTs
• Occurs during prohase as nuclear envelope break
  down
• Dynamic MT acts as a poker

Gotcha!
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What generates chromosomal movement?

• A combination of microtublar dynamics and motors
• Rapid polymerization/depolymerization occurs at
  the () end of microtubules
• Microtubule motors assist in
• i) maintaining flow of tubulin subunits
• Ii) tethering the kinetocore

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Model for Chromosome Congression
A. Treadmilling
-

Tubulin cycling via motors
In this scenario, there is no net elongation /
shrinking of the MT
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Model for Chromosome Congression
B. Depolymerization
-

In this scenario, there is a net LOSS at the ()
end, and the chromosome moves to the right
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Model for Chromosome Congression
C. Polymerization
-

In this scenario, there is a net gain at the ()
end, and the chromosome moves away
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Model for Chromosome Congression
D. MT motors
-

The yellow lollipop depicts Dynein, a (-)
directed MT motor
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Putting the Sequence Together
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Mitosis without Cell Division

• Occurs in functional syncytia
• Eg the developing Drosophila embryo
• During early fly development, mitotic complexes
  termed synergids share a common cytoplasmic
  compartment in the outer blastoderm of the embryo
• These can be studied by GFP-labelling of
  chromosomes

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Cell division in Drosophila Embryos
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A Fundamental Question in Cell Biology

• The Drosophila model presents many opportunities
  for studying developmental and cellular processes
• For example why do all of the synergids undergo
  mitosis synchronously?

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Cytokinesis
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Association of Actin with the Cleavage Furrow

• In addition to forming stress fibres, filamentous
  actin is a polymer that contributes to
  cytokinetic movements
• These confocal images show F-actin throughout the
  cell, but especially at the cleavage

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Plant and Animal CellsA Fundamental Difference
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New Tools for Studying an Old Process
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Actin and Myosin II Form the Contractile Ring
Contractile Ring
Pole
Pole
Myosin I
Aster Microtubules
Actin
Myosin II
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Actin and Myosin II Form the Contractile Ring
Cleavage Furrow
Pole
Pole
Aster Microtubules
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What is the Evidence for Myosin II in Cytokinesis?

• Treatments
• Myosin II Knock-outs
• Gene for myosin II is deleted
• Antisense inhibition of myosin II mRNA
• Antimyosin II Antibodies
• Treatments results
• cells that can replicate their chromosomes, but
  are able to divide, resulting in large,
  multinucleate cells

Control
Treated
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Incomplete Cytokinesis

• Best example
• Germ cell syncytium
• Eg Drosophila ovariole
• Other syncytia do not result from incomplete
  cytokinesis
• Eg skeletal muscle cells
• Result from fusion of myoblasts
• Eg Osteoclasts

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Food for Thought

• What are some of the control points for the
  mitosis and cytokinesis?
• What might occur when these are disrupted?

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Cell Cycle
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Checkpoints in the Cell Cycle

• Checkpoints in the cell cycle
• (1) Progression from G2 to M
• by maturation promoting factor (MPF)
• (2) A checkpoint for DNA damage
• cell cycle halts, allows time for repair
• (3) The spindle checkpoint

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First, we need to define a term

• Kinase a protein that adds a phosphate group to
  a protein. (usually at serine , tyrosine or
  threonine amino acids). This is a way the cell
  uses to activate (or sometimes - inactivate)
  proteins

kinase
ser

phosphatase
Active
inactive
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Summary of the Cell Cycle
interphase
G1
G2
M
S
prophase metaphase anaphase Telophase cytokinesis
I. experiments show that cytoplasmic factors
control progress of the cell cycle
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How is the Cell Cycle Controlled?

• Cytoplasmic factors control cell division
• Experiment
• Rao and Johnston (1970) fused mitotic and
  interphase cells

Mitotic Cell
Found if non mitotic cells were in G1, you get
premature chromosomes
- the non-mitotic cell tried to go into mitosis
Conclude. a factor present in the mitotic
cell sends cells into mitosis
Cell in G1
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How is Cell Cycle Controlled?

• Another experiment (Rao and Johnson, 1970)
• Fused mitotic and interphase cells

Cell in M
Found if non mitotic cells were in G2, you get
premature, double chromosomes
The non-mitotic cell tried to go into mitosis
Conclude a factor in the cytoplasm sends cells
into mitosis, - after S the chromosomes are double
Cell in G2
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Still More Experiments

• Rao and Johnson (1970) (yes, again!)
• Fused mitotic and interphase cells

Cell in M
Found if non mitotic cells were in S, you get
pulverized chromosomes (see Karp, figure 14.3)
The non-mitotic cell tried to go into mitosis
Conclude A factor in the cytoplasm sends cells
into mitosis, but the DNA is being replicated so
all heck breaks loose (a mess!)
Cell in S
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Control of the cell cycle at G2/M

• Progress requires a protein complex called
  Maturation Promoting Factor or MPF
• (Rao and Johnstons factor)

MPF is a complex of two proteins
1 Cyclin b - regulates Cdk1 activity
2 Cdk1 (cyclin dependent kinase)
a Kinase is a protein which adds a phosphate to
other proteins. the activity of the target
protein is then modified.
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MPF, phosphorylates, activates proteins of
mitosis
Cdk1
MPF increased conc.
activity
Cyclin increased conc.
Cyclin B
G1
S
G2
M
time
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• 1) Active MPF initiates entry to mitosis
• 2) The Cyclin dependent kinase Cdk portion turns
  on other proteins involved in mitosis
• - by phosphorylating them
• 3) MPF controls entry to G2/M

Details on how Cdk is activated a multistep
process.
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Cdk activation is a multi-step process involving
a number of factors

• 1) Cyclin b levels increase
• - due to increased gene transcription.
• 2) As concentration increases it binds to Cdk,
  producing the MPF complex
• MPF is a kinase, which activates other proteins
• But it is inactive when it is first made

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• 3) Three other proteins are required to activate
  MPF (these are also kinases).
• i) Cdk activating kinase (CAK) which adds a
  phosphate group (thr-161) needed to activate MPF
• ii) wee1 which adds a phosphate (tyr-15) which
  inhibits the MPF activity
• iii) a phosphatase cdc25 recognizes and removes
  the inhibiting phosphate (tyr-15), at the end of
  G2

(Still inactive!)
(MPF is now active!, mitosis starts)
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• 4) The MPF is now active, coordinating protein
  actions involved in mitosis. The active MPF
  phosphorylates the following proteins.
• a) lamin proteins cause nucleus to disappear
• b) histone proteins altered DNA packing as
  chromosomes condense
• c) proteins of the mitotic spindle are
  phosphorylated, activated
• d) Ubiquitin ligase, a protein which causes
  other proteins to break down when activated
• Adds a tag called ubiquitin to cyclin b
• This marks it for removal
• Proteolysis, proteasome
• ii. A self-terminating effect of MPF

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• 5) Inactivation of MPF ends M phase
• - the remaining CdK is dephosphorylated.

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MPF activation and the cell cycle
G2
M
inactive MPF
inactive MPF
active MPF
Cdk1
Bind together
cyclin b
CAK (activates) wee1 (inactivates)
cdc25
cyclin levels increase
(activates)
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Cyclin action is turned back off by
ubiquination, which terminates its activity by
proteolysis
M
G1

• - Cyclins are low again
• Cdk1 is inactive again

active MPF
Cdk1
destroys the cyclin
Ubiquitin lig.
P
phoshporylates - lamins - histone
- microtubules - ubiquitin ligase
target for proteolysis by proteasome
Ubiquitin ligase
P
Ubiquitin ligase
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Why does it work this way? Because a number of
things all have to be properly coordinated before
mitosis starts.
- is DNA replicated - is the cell big enough ? -
does it have enough stuff ?
MPF
M
G2
G1
G0
e.g. if wee1 isnt working the cells go on too
soon and end up small.
S
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MPF was the first of several cell cycle
checkpoints,
- is DNA replicated - is the cell big enough ? -
does it have enough stuff ?
MPF
metaphase are chromosomes aligned ?
M
- is DNA intact?
G2
G1
G0
S
START or RESTRICTION is cell big enough ? is
environment ok ?
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cyclins and cyclin dependent kinases appear at
different stages of the cell cycle
Karp figure 14.8
M
cyclin b/A Cdk1
G2
G1
cyclin Ds Cdk4 Cdk6
S
START or RESTRICTION
cyclin E Cdk2
cyclin A Cdk2
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checkpoint. cell cycle stops if there is DNA
damage
damage!
(if in G2)
ATR
Chk1
cytoplasm
interior of nucleus
P
Chk1
cdc25
cdc25
cdc25
ser216
adaptor protein
if not present it cannot activate Cdk
see Karp, fig. 14.9 both eds.
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DNA damage checkpoint

• this checkpoint is inoperative in patients with
  Ataxia-telangiectasia (AT),
• - makes them susceptible to cancer
• and extremely sensitive to injury
• Ionizing radiation (ATM protein)
• Ultraviolet radiation (ATR protein)

Note to students in the third edition of the
textbook, no distinction is made between ATR and
ATM (in figure 14.9 the G2 pathway is shown as
coming from ATM, when in fact it comes from ATR).
These are two separate proteins as correctly
shown in the fourth edition (fig.14.9) and in the
diagram above.
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• How does it work? (see fig. 14.9, first half).
• 1. ATR protein detects a break in DNA strand
• 2. ATR is a kinase. It phosphorylates another
  kinase Chk1, thus activating it.
• 3. Chk1 is a kinase. It phosphorylates the
  protein cdc25 (serine 216)
• 4. When phosphorylated cdc25 binds to adaptor
  protein in the cytoplasm
• 5. The adaptor protein binds the cdc25, keeping
  it in the cytoplasm
• 6. the nuclear supply of cdc25 is depleted

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• 7. cdc25 is necessary for progress from G2 to M.
• - it is the posphatase which removes the
  inhibiting phosphate from the cyclin activated
  kinase Cdk (see slide 12 this lecture).
• - if not available in the nucleus the cell cant
  activate
• 8. The cell therefore cell gets stuck and
  cannot enter mitosis until the DNA is repaired
• a) this means the cell waits for repair
• b) if repair is impossible it is prevented from
  dividing into a potential cancer cell

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• Components of a checkpoint
• 1. Sensor. is DNA damaged ?
• - ATM protein. One break stops the whole cell.
• 2. Transducer (s) create a signal
• -Cdc25 phosphorylated at ser 216
• - the MPF proteins
• this is one reason that the cell works in a
  multi-step fashion different control systems
  interact.
• 2. Effector change key enzyme action
• - the phosphorylated histones, lamins, ubiquitin
  and other proteins whose activity drives the
  process of mitosis

(not covered in class, but please know)
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Another example of a checkpoint The Spindle
Checkpoint

• Cell monitors the status of events in metaphase
  before going on to anaphase
• It is important that the chromosomes are all
  evenly lined up as the cell goes on to anaphase,
  so that the separation process goes smoothly.
• If one of the chromosomes is not at the metaphase
  plate it waits, to give it a chance to get there.

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Mechanism of the spindle checkpoint

• MAD2 protein is continuously released from the
  kinetochore of un-aligned chromosomes
• Mad2 normally inhibits a protein called cdc20
• Until it is connected to TWO microtubules and is
  placed under tension
• MAD2 protein stops being made. No longer
  inactivates cdc20
• Cdc20 is active now, it, in turn, activates
  ubiquitin ligase

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Spindle checkpoint continued.

• The ubiquitin ligase tags securin for removal
• it is broken down by proteolysis
• Securin breaks down, so sister chromatids
  separate and cell enters anaphase
• Note that there are many chromosomes, they all
  have to be lined up because they supply MAD2 for
  each other, even a single non-aligned chromosome
  prevents the others from separating

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4. Allows activation of ubiquitin ligase
Ubiq.lig.

• - centromere
• with kinetochore
• And with securin glue

3. This frees up the cdc20
cdc20
Mad2
Spindle fiber
1. tension
Mad2
2. Mad2 stops being produced
5. Securin is ubiquitin-tagged and then removed,
sister chromatids separate.