BIOL 286 Chapter 18: Cell Division Cycle C.

Mouzakitis

The human body has ~3 x 1013 or 30 trillion cells.

There are about 10 billion mitoses occurring every second. Most division is in bone marrow (red blood cells
live 4 months) and the epithelial lining of the gut (cells in harsh environments are replaced every 2-3 days).
Human feces is 20-30% dead epithelial cells and 50% bacteria.

The cell division cycle is also called the cell cycle.

Cells grow, chromosomes replicate, chromosomes are
segregated, and the cytoplasm divides. Cells must grow
in size because a newly formed daughter cell is half the
size of its parent. Some cells, immediately after
fertilization of an egg, do not increase in size as they
divide. Each rapid division makes the cells smaller and
smaller.

The Cell Cycle

After dividing, a cell enters the G1 (Gap 1) phase.
Most cells are usually in this phase. After all organelles
have been divided, the cell enters the S phase, during
which the DNA replicates. The cell enters the G2
phase, in which it continues growing and preparing for
mitosis. The cell then enters the M phase, which
includes mitosis and cytokinesis.
• M phase lasts 30-60 minutes
• Interphase (G1, S, G2) lasts hours, days, or weeks
• Three categories of cells
o No division (stay in G1): red blood cells, muscle, neurons
o Divide only when signaled (waiting in G1 for signal): liver (hepatocytes), lymphocytes
o Active dividers: blood stem cells (precursor of RBC and WBC), intestinal epithelial cells,
spermatogonia
• When the cells are waiting in G1 for a signal to divide, they are said to be in the G0 phase, a phase
where nondividing cells arrest just before S phase. The G0 phase can last many weeks.

Different eukaryotic cells have different cell cycle

times. The early frog embryo cells divide every 30
minutes. Yeast cells divide every 1.5 hours.
Mammalian intestinal epithelial cells take 12 hours to
go through a cell cycle. Mammalian fibroblasts in
culture take about 20-24 hours to go through the cell
cycle. Bacteria divide every 10-15 minutes.

About 25 Xenopus (frog) eggs were fertilized simultaneously in a petri dish. Coordinated cell division is
observed, even though the zygotes are not touching each other. This is evidence for some kind of cell cycle
control, which regulates the times for every cell division. These early stages of cell cycle have repeated S and
M phases with very short or no G1 or G2 (no growth). This means that the early cells get smaller with each
division.

21:25 in Lecture 21
Checkpoints in the cell cycle control system and ensure
that a phase has been satisfied before the next one begins.
• In G1: ensures that the environment is favorable
for the start of division
• Just before M Phase: ensure that DNA and
organelles have been replicated in an orderly
manner
• During M phase: ensure that chromosomes are at
metaphase plate and have attached to mitotic
spindles
• Feedback mechanism for the process being
performed. If corrections must be made, the cycle
will stop.
• Checkpoints can pause the cycle at a transition
point to prevent the next step from starting. If
corrections cannot be made, the cell initiates
apoptosis to die.

An experiment was conducted to see if the cytoplasm

contained regulatory factors affecting the cell cycle.
A cell in S phase was fused with a cell in G1 phase.
Is there something in the S phase cytoplasm that will
tell the G1 cell to begin S phase? Is there something
in the G1 phase cytoplasm that blocks replication in
the S phase? When the S phase and G1 phase cells
are fused, the G1 cell immediately goes into S phase.
There must only be a regulatory factor driving the G1
cell into S phase.

When an M phase cell is fused with a G1 phase cell,

the cell in G1 immediately enters mitosis, even though
it has not gone through S phase and G2. The G1
condenses its chromosomes, breaks it nuclear envelope, and forms spindles, even though its DNA has not been
replicated. The M phase cytoplasm can drive any phase cell toward mitosis. When M phase cytoplasm is
added to S phase, the chromosomes in S phase are so fragile that they fall apart.

Mature Xenopus egg is a good system for studying cell division.

• Large (~1 mm)
• Completed DNA replication; arrested at G2
• All of the mRNAs and most of the proteins required for early division already made
• Fertilized cells divide in synchrony so isolating cytoplasm from a whole batch of eggs from the same
mother frog will be at the same point of the cell cycle

A micropipette is used to extract cytoplasm from mature eggs arrested in metaphase II. When this cytoplasm is
injected into a cell that’s in interphase (an immature oocyte), meiosis is triggered. This work in frog oocytes
identified maturation promoting factor (MPF) to trigger transition from immature oocyte to mature egg.
Oocytes are a good source of cytoplasm.

33:45 in Lecture 21
Fluctuation of maturation promoting factor activity
during the cell cycle:
The cytoplasm in a mitotic cell has the ability to induce
mitosis in an interphase cell. MPF activity was
measured by its ability to induce mitosis in a recipient
cell. The cytoplasm of a cell in interphase cannot
induce mitosis in another cell. MPF only becomes
concentrated in the cytoplasm when the cell is in
mitosis.
Maturation Promoting Factor (MPF)
• Factor that controls the entry into mitosis/meiosis
• MPF has two subunits:
o Kinase – phosphorylates Serine and Threonine on other proteins
o Cyclin – regulatory subunit that activates the kinase so kinase is not always on in cell cycle
• When cyclin levels rise as the cell approaches mitosis, MPF levels rise. MPF and cyclin levels are
highest during mitosis and lowest in interphase. Cyclin is named because its levels fluctuate in a cycle.
• Kinase levels remain stable; only their activity increases and decreases as a result of cyclin levels
Cyclin was first identified for mitosis, but
cyclins and kinases control several steps in the
cell cycle.
S-Cdk is a cyclin-dependent kinase that
regulates S phase
M-Cdk is a cyclin-dependent kinase that
regulates M phase

Cdks (cyclin-dependent kinases)

As shown right, S-Cdk becomes
active at the start of the S phase. S-
Cdk will phosphorylate something
that drives S phase to completion.
When G1 cells were injected with S
phase cytoplasm, they were being
injected with active S-Cdk.

M-Cdk becomes active at M phase,

which phosphorylates nuclear
lamins, causing the nuclear envelope
to break, and well condenses chromosomes.
Distinct cdks associate with different cyclins to trigger different events of the cell cycle
The identity of the cyclin or cdk determines substrate specificity
Proteins that inhibit cdks can arrest the cycle at specific checkpoints to make corrections
Cdk activity regulated by controlled cyclin degradation:
Cyclin levels rise during M phase because synthesis of
cyclin is upregulated. When active cyclin-Cdk complex
has completed its job, the cyclin is degraded by
ubiquitin, a small protein covalently linked to a protein
(cyclin) to be degraded. Ubiquitination occurs as the
cell exists metaphase and enters anaphase. Ligases are
activated by the Cdks themselves and are told to add ubiquitin to the cyclin, which targets the cyclin to the
proteasomes for degradation.
Phosphorylation is also needed in addition to cyclin levels in order to control cdk activity

An inactive mitotic Cdk binds to an M-cyclin for partial activation as the level of cylin rises in the cell. Cyclin
binding is not sufficient to activate cdks. The Cdk has two phosphorylation sites that regulate its activity. For
M-Cdk to be active, it must be phosphorylated at one site and dephosphorylated at other sites. An activating
kinase, CAK (cdk activating kinase) activates Cdk by phosphorylating Thr161. There is also an inactivating
kinase, wee1, which phosphorylates Tyr15. Now the M-Cdk has cyclin bound and a phosphorylated Thr161
and Tyr15. In order to be fully active, the deactivating phosphorylated Tyr15 must be removed. Cdc25
phosphatase removes Tyr15-P at the end of G2. Cdc25 is only activated to remove Tyr15-P when the cell is
large enough. The cell then enters mitosis with a fully active M-Cdk.

Wee1 got its name because cells that lack the Wee1 kinase will not
grow in size before entering mitosis. They produce smaller and
smaller daughter cells (wee is Scottish for small).
As shown left, when active Cdc25 phosphatase removes the inhibitory
phosphate from M-Cdk, the M-Cdk becomes very active. In addition
to initiating M phase, M-Cdk will provide a positive feedback loop by
further activating (phosphorylating) Cdc25 phosphatase so the
phosphatase can more rapidly remove Tyr15-P. The activated M-Cdk
can also phosphorylate and inhibit wee1 inhibitory kinase, so the
kinase does not rephosphorylate Tyr15 once Cdc25 has
dephosphorylated the Try15-P
These inhibitions/activations drive an explosive M-Cdk activation.

Video on cyclin dependent kinases shown in lecture:

https://www.youtube.com/watch?v=tsc1LZKRWAY

59:18 in Lecture 21

What happens if the cyclin is not proteolyzed, and the kinase is allowed to remain on even when mitosis is
complete? An experiment was performed using a kind of cyclin that could not be degraded. Once lefts of
cyclin increased, they permanently remained high. The experiment used GFP-histone to label the DNA and
noticed that the cells without cyclin degradation got stuck in anaphase. Once the cells condense the DNA and
enter anaphase, they are never able to reenter interphase.

Dynamic behavior of cyclin B1-GFP in HeLa cells

• Interphase – cyclin localizes in cytoplasm and centrosomes
• Prophase – cyclin accumulates in the nucleus. Upon nuclear envelope breakdown, relocalizes from the
nucleus to the centrosomes and to the mitotic spindle
• End of metaphase – cyclin B1-GFP signal disappears and chromosomes segregates, consistent with the
view that degradation of cyclin B1 correlates with the transition from metaphase to anaphase
 Checkpoints (molecular brakes) in the cell cycle are
surveillance mechanisms that halt the cell cycle by
regulating kinase activity.

If cells in G1 have growth and do not have DNA damage, then the kinases that drive the S phase will be
activated. DNA damage will inhibit S-Cdk until corrections are made.
There is a spindle assembly checkpoint to ensure that the chromosomes are on the mitotic spindle. M-Cdk is
inactive until this checkpoint is approved.
Checkpoints allow the cell to decide if it should enter S phase, rest in G0 phase, or terminally differentiate
(leave the cycle).

Regulation of the cell cycle in S-phase

In G1, the cell prepares to replicate its DNA as part of S phase. It
forms a pre-replicative complex on various sites of the
chromosomes where replication will originate. The complex forms
when Cdc6 dissociates from the origin of recognition complex
(ORC) and helicase is allowed to join the ORC.

Activation of S-Cdk will

• Trigger replication (via activation of helicases)
• Prevent re-replication by phosphorylating and inducing
degradation of Cdc6 so no new origins of replication form
and DNA is replicated only once per cycle
Cytoplasm from S phase squirted into a cell in G1 will provide the
cell with activated S-Cdk to trigger replication by activating the G1
helicases. Cytoplasm form S phase squirted into a cell in G2 will
not bring the G2 cell into S phase because Cdc6 is not present in the
G2 cells. Only G1 cells have Cdc6.

DNA damage checkpoints prevent replication of

damaged DNA
• If DNA damage is detected in G1 and S,
replication is stopped
• If DNA damage is detected in G2, mitosis
is not started

Shown right, X-rays or UV light can damage

DNA by forming breaks in the strands. There is a
transcription factor p53 that is usually proteolyzed
in healthy cells. DNA damage will activate
protein kinases that phosphorylate p53, thereby
stabilizing it, activating it, and keeping it from
being degraded. The p53 transcription factor will
now bind upstream of the p21 gene, in the gene’s
regulatory region. The p21 gene is turned on,
producing the p21 protein. P21 protein is a Cdk
inhibitor protein that will bind to S-Cdk and
inactive it.
DNA damage is also capable of inactivating M-
Cdk just before the cell enters the M phase.

Cancer cells have mutated p53 genes, so p21 inhibitor is never translated.
Regulation of the cell cycle in M phase: mitosis and cytokinesis
1. Before mitosis, in late interphase, the cytoplasm of a cell contains two centrosomes, each with a pair of
centrioles. The nucleus has already replicated chromosomes (in S phase, which is when the centrioles
are also copied), but the chromosomes are still dispersed in the form of chromatin.
2. Mitosis, the division of the nucleus and its chromosomes, is divided into five phases: prophase,
prometaphase, metaphase, anaphase, and telophase. Cytokinesis follows these phases.

Prophase
• Chromosome compaction (condensation) from spaghetti strands to H-shapes
• Extended interphase chromatin (spaghetti) good for transcription, not segregation
• These events driven by phosphorylation by mitotic cylins: m-Cdk causes the chromosomes to condense
and the nuclear envelope to breakdown
• Cytoskeleton is disassembled and mitotic spindle is assembled (m-Cdk increases dynamic instability)
• Golgi complex and ER fragment into vesicles
• Nuclear envelope disperses, marking the end of prophase

Chromatids, Kinetochores, and Centromeres

During S phase, the chromosomes are duplicated. Sister chromatids are
attached to each other. Shown right is a compacted chromosome, which does
not form in S phase or G2 when chromosomes are replicated. Only in
prophase do the replicated chromosomes condense.

The centromere region of the chromosome is DNA that is never transcribed

into RNA (no active genes). The centromere is made of repetitive DNA
sequences that serve to bind to kinetochore proteins.

30 nm wide filament with histones and one long double stranded DNA
condenses in a series of 10,000 compressions.

Compression during prophase

Condensin protein complex and a
topoisomerase help compaction.

Topoisomerase helps DNA unwind a bit.

Condensin is activated when

phosphorylated by active M-Cdk.

The protein complex cohesin keeps the sister chromatids together. Chromosomes are
replicated during S phase to form sister chromatids. Cohesin rings are also formed
during S-phase to connected the two sister chromatids. In mitosis, the sister
chromatids separate into two daughter cells, so cohesin helps ensure that each daughter
cell gets one copy.
After condensin compacts the sister chromatids (in prophase), cohesin no longer spans
the length of the chromosome (as in S phase and G2). Cohesin is lost from most of the
sister chromatids and becomes localized in the centromere region.
Cohesin is proteolyzed in anaphase to separate the sister chromatids.

22:40 in Lecture 22
 Kinetochore Structure
Kinetochores serve to link the
microtubules to the centromeric
DNA. Each chromosome has
two kinetochores, one on each
sister chromatid.

As shown in the second panel

on the left, the plus end of the
microtubule (blue) does not
touch the kinetochore. This
indirect attachment allows the
microtubule to add and subtract
subunits.

Along the lateral edge of the microtubule (near the end), there are fibers that are
linked to the kinetochore by kinesin and dynein. CENP-E and Depolymerase
are kinesins. In anaphase, when the microtubules rapidly shorten, kinesin and
dynein allow the kinetochore to remain associated to the microtubule.
Depolymerase does not move any cargo; it induces depolymerization by
hydrolyzing ATP.
Shown right, at the minus ends of the microtubules are centrioles. Centrioles
replicate during S phase. When the cell enters M phase, the centrioles separate
to the poles of the cell and form mitotic spindles.
Same S-Cdk that drives DNA replication also triggers centrosome replication.
m-Cdk induces dynamic instability in the pre-existing microtubule network in
or to rearrange the microtubules into mitotic spindles.

M-Cdk (mitotic cyclin-dependent kinase)

• Mitotic cyclins: synthesized during G2 but cdk is inhibited by
phosphorylation
• Phosphorylate chromatin-associated proteins for condensation
(condensin), nuclear envelope breakdown, and microtubule
reorganization
• Recall, the cytoplasm of a cell in M phase squirted into a cell in G1 will
induce mitosis in the G1 cell (even without DNA replication) because
the M phase cytoplasm has active M-cdk in it (active means the
inhibitory phosphate was removed by Cdc25).

As soon as the cell

begins anaphase,
mitotic cyclin is degraded and M-Cdk is turned off.
Nuclear envelope breakdown marks the end of prophase
Disassembly of the nuclear lamina
Nuclear lamina: intermediate filament protein just
underneath the nuclear envelope; gives shape to the
nucleus.
Activated M-Cdk will phosphorylate lamin, causing
depolymerization of lamin and breakdown of envelope.

Nuclear envelope may vesiculate or may join ER

Prometaphase
1. Begins with disintegration of nuclear envelope
2. Chromosomal microtubules attach kinetochores of chromosomes. Nine kinetochore microtubules
overlap with those coming from the opposite pole.
3. Chromosomes are moved to spindle equator

Mitotic Spindle Formation

• Dynamic instability of microtubules increases; M-Cdk
phosphorylates proteins that normally help stabilize
microtubules. Non-centrosomal microtubules breakdown.
• Bipolar spindle forms by selective stabilization of interacting
microtubules. Shown right are these interpolar microtubules
that overlap and become stabilized due to their interaction with
each other.
• Aster microtubules extend outward and bind to the surface of
the cell to become stabilized.
• Microtubules also bind to the kinetochores and become
stabilized that way.
• Microtubules not bound to anything will fall victim to dynamic
instability and depolymerize.

Motor proteins and chromosomes

can form a functional bipolar
spindle in the absence of
centrosomes (normal in plants and
some animal cells).

Shown left, microtubules are in

green and chromosomes are in red.
Centrioles are at each end. The
lower image shows how some cells can form bipolar mitotic spindles even
without centrosomes at each pole. These mitotic spindles have
microtubules, dynein, and kinesin.
Normally, centrioles at the centrosomes nucleate microtubules. In the lower image, the kinetochores and
chromosomes nucleate the microtubules. Chromosomes are then able to segregate.
Note the lack of aster microtubules when centrosomes are not present.

Early prometaphase
DNA is stained blue and microtubules are stained green.
The nuclear envelope has just broken down, so the
condensed chromosomes have not begun to move about.

The two starburst patterns of microtubules are called asters

(stars). The middle of each one contains the centrioles.

Aside from asters, the microtubules can overlap with each

other, or find a kinetochore to bind to.

40:30 in Lecture 22
Capturing of Chromosome onto
spindle microtubules
After the chromosomes have
condensed and the nuclear
envelope has broken down, the
microtubules in the cytoplasm
seek a kinetochore to stabilize
their instability. Once bound to
a kinetochore, the chromosome
uses dynein motors to migrate
toward the centriole pole. The
lateral attachment of
microtubule to the chromosome
converts to an end-on
attachment. Another free
microtubule from the opposite
spindle pole is then able to
capture the kinetochore on the
second sister chromatid. The
chromosome links the two poles
via a microtubule attachment to each kinetochore of the sister chromatids.

Once bound to both microtubules, the cell undergoes

regulated shortening and lengthening of the
microtubules to bring the chromosome directly in the
middle of the cell, called the metaphase plate.
Microtubules essentially become the same length on
both sides.

In yeast, there is a single microtubule bound to each

kinetochore side. In humans, there are about 20-40
microtubules bound to each kinetochore.

This is a tug-of-war pulling on each kinetochore.

Once the chromosome is near the center, there is very

slow polymerization of dimers to the microtubules to
ensure precision (cell measures this with tension).

When the sister chromatids are told to separate, there

will be rapid depolymerization of the plus ends to bring
the chromosomes to each pole.
There is an aurora kinase associated with the kinetochore that senses
tension. The sister chromatids must be pulled equally from the left and the
right. If pulling is unequal, mitosis will be halted.
Three classes of microtubules make up the mitotic spindle
1. Kinetochore: link the kinetochores to the poles (blue)
2. Interpolar: microtubules that overlap from each pole (magenta).
They are not bound to chromosomes, but rather help the cell separate
by pushing the poles apart.
3. Aster: radiate out from each pole to the cell surface (green). Help
position mitotic spindle in the middle of the cell.
 Metaphase – microtubules have captured the chromosomes and
have aligned them at the metaphase plate
Chromosomes are aligned along metaphase plate, attached by
chromosomal microtubules to both poles. It is called the
metaphase plate because the cell is three-dimensional.
As shown left , the microtubules are still in flux: there is net
addition on the plus end (near kinetochore) and net
depolymerization on the other end.

Anaphase
1. Centrosomes split, and sister chromatids separate (cohesin is cut by separase)
2. Chromosomes move to opposite spindle poles
3. Spindle poles move farther apart to ensure that two sister chromatids are not trapped
on the same side of the cell and accidentally end up in the same daughter cell

The Anaphase Promoting Complex (APC)

• APC is a ubiquitin ligase
• APC is activated by cdc20 and triggers chromatid separation by promoting the destruction of cohesins
• APC next is activated to degrade mitotic cyclins

Separase is not always active in the cell. It is normally

inhibited by securin. In anaphase, cdc-20 will
phosphorylate APC, making it active. Active APC will add
ubiquitin to securin, causing it to be degraded. Separase is
now free from securin, and is allowed to chew cohesin and
separate the sister chromatids.

cdc-20 will only phosphorylate APC if all chromosomes

are correctly lined at the metaphase plate.

After APC has caused the degradation of securin, it will

begin chewing M-Cdk.

How does the cell (cdc-20) know if all chromosomes are

aligned at the metaphase plate? This is the spindle
checkpoint. Spindle checkpoint is absent from many
tumor cells; therefore, chromosomal abnormalities survive.

Spindle checkpoint controlled by kinetochore tension and Mad2 on unattached chromosomes

• Mad2 is associated with kinetochores and provides a “wait” signal
• This signal blocks proteolysis (APC/cdc20) needed to start anaphase
• Immunofluorescence shows Mad2 present in chromosomes that never aligned properly at the metaphase
plate. Chromosomes with Mad2 still present on their kinetochore can stop anaphase.
• Cdc20 is bound to activated soluble Mad2, keeping it from activating APC.
• Mad2 falls off the chromosome after it has aligned properly.

1:06:00 in Lecture 22
Tension is required on both kinetochores to release Mad2

Shown right, when a kinetochore is only attached on one side,

the left kinetochore feels tension, but the right kinetochore does
not. The cell is arrested at metaphase. When the kinetochore
tension is applied with a needle, both kinetochores feel tension.
Mad2 is released and APC adds ubiquitin to securin. Separase is
allowed to cut cohesins from the sister chromatids. The cell
proceeds into anaphase.

Anaphase as shown in the image below.

Anaphase A: Beings when the two centromeres of each
chromosome come apart to separate the sister chromatids.
Motor proteins walk the daughter chromosomes along the
spindle microtubules as the kinetochore microtubules shorten.
Anaphase B: The spindle fibers not attached to chromosomes
(interpolar microtubules) lengthen, thereby pushing the poles
farther apart and elongating the cell.

Anaphase A and B occur simultaneously. GFP-labeled kinetochores seem to disassemble after the
chromosomes have migrated. The centromere region of the chromosome is unaffected.

Proposed roles of motor proteins during mitosis

• Motors required to bring chromosomes to metaphase plate: microtubules seek out the kinetochore, attach
to the side of the chromosome, the chromosome heads toward the minus end of the microtubule, the
attachment is converted to an end-on attachment, the chromosome re-migrates toward metaphase plate
• Motors required for kinetochore microtubule shortening (kinesin called depolymerase uses ATP
hydrolysis to chew the plus ends of the microtubule)
• Motors push poles apart (Anaphase B)
• Plus ends of the microtubules associated with the cell surface will use motors to further aid movement of
the poles farther from each other

10:55 in Lecture 23
Chromosomes can only migrate as fast as the
kinetochore microtubules shorten.

The drug taxol will prevent microtubules from

depolymerizing. When treated with taxol, the
cell will keep chromosomes at the metaphase
plate. Chromosomes are never able to migrate
since the microtubules never shorten.

Taxol is excellent for cancer treatment

because it blocks cell division.

Shown right are two models of force

production during Anaphase A. The left panel
shows an ATP-driven motor protein
(Depolymerase) that induces the microtubule depolymerization. The right panel shows dynein’s affinity to
polymerized tubulin, so it stays just ahead of the depolymerization site.
Telophase
• Chromosomes cluster at opposite spindle poles
• Chromosomes become dispersed (spaghetti strands again)
• Nuclear envelope assembles around chromosome clusters
• Golgi complex and ER reforms
• Daughter cells formed by cytokinesis
Cells without degraded m-Cdk never finish mitosis.

After the phases of karyokinesis are complete, cytokinesis begins.

Shown left, a contractile ring of actin and myosin will form in

between the two nuclei. The ring separates the cytoplasm of the two
daughter cells by squeezing them apart.

There are no clear checkpoints to ensure that each daughter cell

receives the same number of each organelle.

On the surface of the plasma membrane forms a cleavage furrow.

Directly beneath the plasma membrane’s cleavage furrow is the ring
of actin and myosin.

Shown right is the mid-body, used to

describe the leftover interpolar
microtubules that remain connecting
the poles as cytokinesis occurs.

An experiment that used an antibody

to inhibit myosin (such that it would
not interact with actin) showed that
the cell continued to undergo mitosis, but was never able to undergo
cytokinesis. The resulting cell will have multiple spindles and multiple copies of DNA that were never
segregated into separate cells.
Astral microtubules that arrive at
the plasma membrane send signals
to determine the cleavage plane (the
placement of the contractile ring).

The region where the astral

microtubules overlap (e.g., the
center of the cell), more
microtubules are present and therefore more signals radiate toward the plasma membrane. This high
concentration of signal notifies the cell to form the contractile ring in that area.

Two adjacent cells were undergoing cytokinesis. A scientist used a

pipette to remove one of the cells. Placement in the pipette elongated
the cell that was removed. Originally, aster microtubules radiating
outward from each centriole (black dots) would have told the dividing
cell to form a cleavage furrow between the two new nuclei. Now,
aster microtubules radiating outward from the centrioles of the
daughter cells will split the daughter cells (inappropriate), as shown
below.

By moving the spindle fibers around, the placement

of the cleavage furrow can change. Constant
movement of the spindle fibers will form several
cleavage furrows throughout the dividing cell.

Scientists can also keep the aster microtubules from

touching the plasma membrane, thereby preventing
a cleavage furrow from forming altogether.

Animal cells round up in mitosis. Their morphology transitions from spread out (nicely formed cytoskeleton) to
rounding up into a ball (smaller surface area). Daughter cells are noticeable smaller than parent.

Cytokinesis in plant cells (have cell wall)

• Interpolar microtubules remaining at
telophase form a phragmoplast
• Golgi vesicles migrate toward the
middle; in between the two daughter
cells
• The membrane vesicles are guided to
build a cell wall (cellulose included)

Wall is built between cells instead of cells

pinching apart.

Video on plant cell division shown in lecture:

http://www.youtube.com/watch?v=SlgV_zoHQxE

Control of cell number and size

• A fertilized mouse egg and a fertilized human egg are similar in size, but an adult human is much bigger
than an adult mouse
• 1010 – 1011 cells in a human body die everyday by apoptosis
Programmed cell death (apoptosis) – helps regulate animal cell numbers
• “Cell suicide” occurs during development. Shown right is the
transition from a tadpole to frog. The tail of the tadpole is lost
because its cells die by apoptosis. Humans have cells in between
the digits of our fingers during development. These cells are
eventually removed by apoptosis.
• In adults, apoptosis removes unnecessary cells (e.g., immune
cells after infection is gone)
• Main defense against cancer development
• Signal activation phase followed by execution phase

Caenorhabditis elegans (C. elegans) – nematode worm with clear cuticle: the development from a fertilized egg
to a 959-cell body has been observed and traced extensively.
• Provided insight into the molecular basis of apoptosis
• The fate and location of all cells in the worm are known
• During development, 1090 cells are produced and 131 die by apoptosis
• CED-3 gene (a caspase) identified first in C. elegans but present in our cells as well: cell death gene

Caspases induce apoptosis by proteolyzing:

• Protein kinases (e.g., FAK, PKA, PKC)
• Nuclear envelope proteins (lamins)
• Cell structure proteins (e.g., actin, IF)
• DNA repair enzymes
• DNase inhibitory protein (now is able to cleave DNA)

Apoptosis is a clean death. Cell contents remain within the cell membrane, rather than spilling out all over.
Phagocytic cells clean up the fragments after the cell is degraded.

Execution phase is best understood (after caspase activation)

• Cytoplasm and nucleus shrink

• Cell rounds up and loses cell-cell contacts
• DNA fragments; cell may fragment as well
• Phosphatidylserine (phosphoglyceride normally on inner leaflet of PM) flips to outer plasma membrane
leaflet. These are “eat me” signals to phagocytic cells

Apoptotic cells die quickly and cleanly. Compared to necrotic cells, whose cytoplasm and organelles spill out
all over the place, apoptotic cells are always enclosed in membranes. Death occurs by clean fragmentation.

We know much about the enzymes involved in apoptosis, but we are unclear about what induces apoptosis.
Signal Activation Phase
• External signals can activate
plasma membrane receptors
Killer lymphocytes with a three-
part Fas ligand on their surface
can bind to the Fas death
receptor. Pro-caspases on DISC
(death inducing signal
complex) are activated to
become caspases.
• Internal damage signals can
activate via mitochondria
• Caspases: proteases that induce changes during execution phase

Caspase is a protease that is referred to as procaspase in its

inactivated form. Shown left, activation will cause procaspase to
cleave two sites on its adjacent procaspase, and vice versa. The
two cleaved procaspases will assemble to form one activated
caspase molecule.

One activated initiator caspase will activate several other

Caspases, which will each activate several Caspases. Activation
of apoptotic program is “all-or-none,” meaning once it is started,
it usually leads to cell destruction.

Bcl2 family (many amino acids in common) regulates apoptosis

• Animal cells have inactive procaspases waiting for a signal to destroy the cell; thus, it is important that
the death program is not activated until needed
• Activation is controlled by Bcl2 proteins
• Some Bcl2 proteins promote apoptosis, other Bcl2 proteins inhibit apoptosis

The Bax and Bak members of the

Bcl2 family lead to activation of
caspases
• Death-promoting members of
the Blc2 family
• Activated by other Bcl2
proteins, which are activated
by such things as DNA
damage
• Trigger apoptosis by
releasing cytochrome c from
mitochondria (shown right)

Activated Bax and Bak are membrane pores on mitochondria, which allow the release of cytochrome C.
DNA damage ! Bax and Bak activated ! cytochrome C release into cytoplasm ! apoptosome of activated
Caspases forms ! apoptosis begins
Note: Bax and Bak do not begin in the mitochondria membrane. They are not the name of the pore in the
mitochondria. Their activation facilitates release of cytochrome C from the pore.
52:10 in Lecture 23
Animal cells need extracellular signals to survive, grow, and divide. When cell stops receiving signals,
apoptosis is induced
1. Survival factors: suppress apoptosis
2. Mitogens: stimulate cell division by overcoming cell cycle braking
3. Growth factors: stimulate growth by promoting protein synthesis and inhibiting degradation

Cancer cells do not need these signals to survive.

Survival factors
Cell death helps adjust the number of
nerve cells to the number of target
cells they contact. Body eliminates the
unnecessary neurons.

Survival factors are retrotransported to

the nerve cells, telling the nerve cells
to stay alive. Neurons that do not
receive these undergo apoptosis.

A survival factor that does this is called neurotropin (e.g., NGF, nerve growth factor), which binds to receptors
at the end of the axon and is transported back to the neuron’s cell body.

Experiment on chick embryo shows that the survival of motor neurons depends on the size of the muscle target
field that the nerve cells innervate. When the muscle target field is increased, the number of motor neurons
increases, and vice versa.

Animal cells need signals from other cells to survive. Survival factors increase the synthesis of Bcl2 protein,
which inhibits apoptosis. Survival factor binds to surface of neuron ! transcription regulator activated ! Bcl2
gene expression is turned on ! Bcl2 protein synthesized to inhibit apoptosis
Bcl2 in normally inhibited, but survival signals will block the inhibition of Bcl2.

1:01:57 in Lecture 23

Shown right, activated Akt

(Protein Kinase B) promotes
cell survival.
The Bad protein is a Bcl2
member that promotes
apoptosis.
Active Akt will
phosphorylate Bad, and lift
the inhibition off of Bcl2.
Bad is now inactivated and
Bcl2 is now active. Active
Bcl2 will promote cell
survival.
Mitogens
• Most mitogens are secreted signal proteins that bind to cell surface receptors
• Activate signaling pathways that release block of G1 and S transition

Example of Mitogen: Retinoblastoma (Rb) protein – binds to and inhibits many transcription factors for genes
involved in cell division. Mitogens binding to the cell surface will release the retinoblastoma brake by
activating S-Cdk, which phosphorylates Rb, which releases the transcription factors needed for cell division.
People with retinoblastoma mutations are more likely to get cancer.

Growth Factors
• If cells divide without growing, they would become progressively smaller
• Animal cell growth requires signals from other cells
• Growth does not require cell-cycle control, many nondividing cells grow
• Little is known about control of growth

Example: the mouse nerve cell and lymphocyte are compared side-by-side.
Neurons are much, much larger because they grow greatly after division stops.

Shown right, cells get larger:

1) Increase in rate of macromolecule synthesis
2) Decrease rate of macromolecule degradation

Some proteins (e.g., PDGF, platelet division growth factor) can act as mitogens
and growth factors. Cells divide and grow using the same protein.

Growth factors have been shown to activate protein kinase B, which

phosphorylates and activates a protein called Tor. Tor is a ser/thr kinase that
stimulates cells to grow.

Anticancer drug rapamycin inactivates Tor.

Some extracellular signals can inhibit growth:

Myostatin normally inhibits growth and division of the myoblasts that fuse to
form muscle cells. Mutants of myostatin have larger muscles.
There is a breed of cattle with mutated myostatin gene.