Dr.

Ghulam Raza
Principal Scientist

The Cell Cycle, Apotosis and

Cytoskelton

Books
1.5th Edition Molecular Biology
of the Cell (Chapters 16,
17,18)
2.Essential Biology 4th Edition
(chapters 17 and 18)
Lecture
No. 1
(cell
cycle)
 Contents

• Overview of the cell cycle

• The cell-cycle control
system
• G1 Phase
• S Phase
• M Phase
• Mitosis
• Cytokinesis
• Control of cell numbers and
cell size
“Where a cell arises, there must be a previous
cell, just as animals can only arise from
animals and plants from plants”
(Book, German pathologist Rudolf Virchow in
 All living organisms—from a unicellular
bacterium to a multicellular mammal—are
products of repeated rounds of
cell growth and division

explain how cells reproduce?

(1)How do cells duplicate their contents

—including the chromosomes, which carry
the genetic information?

(2) How do they partition the duplicated

contents and split in two?

(3) How do they coordinate all the

steps and machinery required for these
Overview
An orderly sequence of events in which
cell duplicates its contents and then
divides in two. This cycle of duplication
and division is known as cell cycle.

Uni-cellular

Multi-cellular
complex, millions of cells

• Vary from Organism to organism, certain

characteristics are universal
• Eukaryotic cells
Cell-cycle control system (central
role)
inside the cell
out side the cell
Basic function: Duplicate and segregate DNA
accurately
Four Phases Two events

Longe
r
time

During G phases, cell

monitors both external and
A cell-cycle control system triggers the
Processes
of the cell
Complex cycle
network of
regulatory proteins

• Ensure all events in

sequence
• Feedback from previous
events
• Molecular breaks to
pause the cycle at
certain transition
points.

Three main transition

points
1.G1to S phase
2.G2 to M Phase Cell
proliferation
3.During Mitosis Need nutrient
and signals
If unfavorable then G0
Cell-cycle control is similar in
all eukaryotes
• Time vary
• Basic organization is similar
• Similar machinery and control
mechanism to drive/regulate
events
• Proteins of the cell-cycle
(billion years ago) conserved
•Unified picture
cell-cycle control system

Two types of machinery -cell division:

1. Manufactures new components
2. Correct places and partitions
•On/off

•Series of molecular switches that operate in a

defined sequence and orchestrate the main
events of the cycle, including DNA replication
and the segregation of duplicated chromosomes

•Depends on cyclically Activated Protein kinases

(cdks)
•Phosphorylation - protein kinases
•Dephosphorylation - phosphatases
 A cdk must bind a
regulatory protein called
a cyclin before it can
become enzymatically
activeLeland H. Hartwell,
R. Timothy Hunt, and
Paul M. Nurse won the 2001
Nobel Prize in Physiology or Medi
cine
for discovery of these central
• Several types of cyclins
molecules.cdks

• M cyclin, active complex M-Cdk

• S cyclins and S-Cdk
• G1/S cyclins and G1/S-Cdk

•Different complexes triggers different

steps
The formation of active cyclin–cdk complexes
drives various cell-cycle events, including
entry into S phase or M phase.

M cyclin direct the formation of the active

cyclin–cdk complex (M–cdk) that drives entry
into M phase
Distinct Cdks associate with different cyclins to
trigger the different events of the cell cycle.
Two types of cyclin–cdk complexes -triggers S and
M phase
Lecture
No. 2
Re-Cap
• Cell cycle?
• Phases?
• Cell control system
• Check points? How many? Where?
Regulatory complex?
Specificity?,
Concentration?
Phosphorylation and De-
phosphorylation
Cyclin concentrations are regulated by
transcription and by proteolysis

The activity of some Cdks is regulated by cyclin

degradation. Ubiquitylation of S or M cyclin by
anaphase-promoting complex (APC) marks the
protein for destruction in proteosomes. The loss
of cyclin renders its cdk partner inactive
The Cell-Cycle Control System Depends on
Cyclical Proteolysis
The Activity of cyclin–cdk complexes depends on
Phosphorylation and dephosphorylation

M-Cdk to be active, inhibitory phosphates must

be removed
When M cyclin–cdk complex is formed,
it is phosphorylated at two adjacent sites by
an inhibitory protein kinase called Wee1.
The activity of a Cdk can be blocked by the
binding of a Cdk inhibitor

inhibitor protein (p27) binds to an activated

cyclin–cdk
complex.
• Attachment prevents the cdk from
phosphorylating target proteins required
for progress through G1 into
The cell-cycle control system can Pause
the cycle in various ways
G1 Phase

Metabolic activity
Cell growth
Repair,
Point of decision-making
Intracellular signals (cell
size)
Extracellular signals
(environment)
How the cell-cycle
control system
decides between
these options?

What it does once

the decision is
made?

What are molecular

mechanisms
involved? Which
helped to avoid
cancer. The transition from G1 to S
phase offers the cell a
crossroad.
s are stably inactivated in G1
• Must inactivate its inventory of S-Cdk
and M-Cdk if not????
• How?
Eliminating existing cyclins
Blocking the synthesis of new
ones
Deploying Cdk inhibitor
The multiple mechanisms makes this system-
suppression robust

Resulted in Stable G1 phase

Mitogens promote the production of the
cyclins that stimulate
One cell
way indivision
which mitogens
stimulate cell proliferation is by
• Extracellular inhibiting the rb protein.
signals produced by
other cells.

• If no signals, the
cell cycle arrests
in G1
• If this period is
long
withdraw from the
cell cycle and
enter a non-
proliferating
state which
remain for (days,
weeks, months, or
even for the life
time)

• For Escaping
DNA damage can temporarily halt
progression through G1
istinct Mechanism
Increased concentration
and activity (p53
transcription
regulator)

Activation a gene (p21)

Binds to G1/S-Cdk and

S-Cdk

Preventing cell to
If no prevention then
enter into S phase
what happen?
Cells can delay division for prolonged
periods by entering
specialized non-dividing states
Special importance in multi-
cellular organisms
Permanently -terminally differentiated cells
(nerve or muscle cells
The cell-cycle control system –
completely stopped
Genes encoding the relevant cyclins
and Cdk- irreversibly shut down

Temporarily - G0.
Reassemble the cell-cycle control
system quickly
(Liver cells)
Cell-division rates proliferation
Stimulate (liver is
damaged)
Depends on time spend in G0 or in G1
S Phase

Before cell division

• DNA replication is must

• Need extreme accuracy, why?

Elegant molecular mechanisms cell-

cycle control system

initiates DNA replication

prevents replication
s-Cdk Initiates DNA Replication Once per Cycle
S-CDK initiates DNA replication and blocks
re-replication
DNA replication
two steps

During
G1 phase

S phase
sins Help Hold sister Chromatids Together

Condensin

Two Protein
Complexes- similar
structure
Cohesins and condensins help to configure
duplicated chromosomes for separation

Condensins
Cohesions •Chromosome condensation
•Sister chromatids are held •assemble on each
together individual sister chromatid
•Assemble along the length •triggered by
of each chromatid as the phosphorylation of
DNA is replicated condensins by M-Cdk
•Proper chromosome
segregation

If defects?
Chromosome Duplication Requires Duplication of
Chromatin Structure

Chromosome duplication- is not simply duplicating

the DNA, but also require duplication of Chromatin
proteins

S-Cdks stimulate-synthesis four histone subunits-

histone octamers

•Assembled into nucleosomes on the DNA by nucleosome

assembly factors
•Associate with the replication fork
•Distribute nucleosomes on both strands of the
DNA as it emerge from the DNA synthesis machinery

•Chromatin structure (like DNA), accurately

reproduced in S phase.
Lecture
No. 4
M Phase
How the mitotic spindle assembles and
functions?

How the dynamic instability of microtubules

and the activity of microtubule-associated
motor proteins contribute to both the assembly
of the spindle and its ability to segregate
the duplicated chromosomes?

What are the mechanism that operates during

mitosis to ensure the synchronous separation
of these chromosomes?

How the daughter nuclei form?

M Phase

• Mitosis plus Cytokinesis

• Short amount of time

Central issue; accurately segregate the

chromosomes

Two specialized cytoskeletal machines

1)One pulls the duplicated chromosomes apart

2)Other divides the cytoplasm into two
M-phase
(Stages:PMAT)
M-CDK drives entry into m-phase and
mitosis
In-spite of entry-M-phase
•Prepare duplicated chromosomes for segregation
•Induces the assembly of the mitotic spindle
• Activation is self-reinforcing
activating more Cdc25
shuts down the inhibitory kinase Wee1

indirectly activates more m-Cdk creating a

positive feedback loop
The mitotic spindle starts to assemble in
prophase

• Dynamic instability- polymerize and depolymerize by

the addition and loss of their tubulin subunits,
growing and shrinking

• M-Cdk phosphorylates microtubule-associated proteins -

influence the stability
Result in rapidly growing and shrinking
Exploring interior of cell
 The centrosome, (A) Electron micrograph
of an S-phase mammalian cell in
culture, (duplicated centrosome)

Principal microtubule-organizing center in animal cells

Pericentriolar matrix
Pair of centrioles
Nucleates a radial array of microtubules (fast-growing
plus ends projecting outward minus ends associated with
the centrosome
Contains a variety of proteins
microtubule-dependent motor proteins
coiled-coil proteins link the motors to the
centrosome and structural proteins
Centrosomes duplicate to help form the two poles
of the mitotic spindle
• DNA replication/Centrosome duplication must
occur before M phase
• Cdks (G1/S-Cdk and S-Cdk)
• Duplication form the two poles of the mitotic
spindle-bipolar
• Centrosome cycle (process of centrosome
duplication and separation)
A bipolar mitotic spindle is formed by the
selective stabilization of interacting
microtubules.
New microtubules grow randomly in
all directions
plus end– unstable, suddenly switch
from uniform growth to rapid
shrinkage
minus ends –anchored

Interaction of two microtubules in

an overlap zone (Motor proteins and
other microtubule-associated
proteins cross-link- stabilizes the
plus ends)

Decreasing the probability of their

de-polymerization
Different cytoskeletal assemblies carry out
mitosis and cytokinesis

Two complex cytoskeletal

machines
Three classes of microtubules make up the
mitotic spindle
Microtubule-dependent motor proteins govern spindle
assembly and Function

• Kinesin-5 proteins contain two motor domains that interact with the plus ends
of antiparallel microtubules, Push the poles apart
• Kinesin-14 proteins, are minus-end can interact with a neighboring
microtubule, cross-link antiparallel interpolar microtubules
• Kinesin-4 and kinesin-10, (chromokinesins) Plus end-directed motors,
associate
with chromosome arms, push the attached chromosome away from the pole
• Dyneins, minus-end directed, together with associated proteins-organize
microtubules at various locations in the cell, link the plus ends of astral
microtubules to components of the actin cytoskeleton at the cell cortex, pull
the spindle poles toward the cell cortex and away from each other
kinetochores attach chromosomes to the mitotic
spindle
• Protein complexes that assemble on the centromere of each
condensed chromosome
• Two on each duplicated chromosome –facing opposite
• Bi-orientation- each duplicated chromosome becomes linked to
opposite poles
• Recognize the special sequence on centromere if sequence
altered? Then what happen?

Schematic drawing of a
canning micrograph
A fluorescence micrograph mitotic chromosome
The number of microtubules attached to each
kinetochore varies human - 20–40 microtubule
yeast - just one.
Bi-orientation Is achieved by Trial and
Error
Bi-orientation Is achieved by Trial and
Error

The capture of centrosome microtubules by

kinetochore
 Multiple forces act on Chromosomes in the
spindle

How opposing force may drive chromosome to

metaphase plate?
Chromosomes attach to the mitotic spindle
Chromosomes line up at the spindle equator at
metaphase
• A continuous balanced addition and loss
of tubulin -maintain the metaphase spindle

• colchicine (tubulin addition to the ends

of microtubules is blocked
Proteolysis triggers sister-chromatid
separation at anaphase
APC triggers-sister
chromatids separation by
promoting the destruction
of cohesins

Ubiquitylation
destruction of an
inhibitory protein called
securin.

Securin inhibits the

activity of a proteolytic
enzyme called separase
which cleaves the cohesin
complexes,

Allow the mitotic spindle

to pull the sister
chromatids apart.
Chromosomes segregate during Anaphase
An unattached chromosome will prevent sister-
chromatid separation
One daughter cell
incomplete set of chromosomes
other receive a surplus

Both are lethal

“stop” signal to the cell-cycle control system

blocking the activation of the APC
(spindle assembly checkpoint)
The nuclear envelope re-forms at
telophase

The nuclear envelope breaks down and re-forms

during mitosis
Cytokines
is
Process by which the cytoplasm is cleaved,
completes M phase
Begins in anaphase but not complete untill two
daughter nuclei (Telophase)
The mitotic spindle determines the plane of
cytoplasmic
• cleavage
First visible sign-
puckering and furrowing of
the plasma membrane

Egg surface
The cleavage furrow is formed by the
action of the contractile ring
underneath the plasma membrane

How does the mitotic spindle dictate the

position of the cleavage furrow?
Overlapping interpolar microtubules, recruit and activate
proteins - signal
Cell cortex to initiate the assembly of the contractile
ring
The contractile ring of animal cells is made of
actin and myosin filaments

• An overlapping array
of actin and myosin

•Assembled and attach to

membrane-associated
proteins

• Exert force (by

sliding)

• Transient structure

(a)Scanning electron micrograph

(b)Schematic diagram of the mid-region
of a similar cell
Animal cells change shape during M-phase

Mouse fibroblast dividing in culture, cell rounds up and

flatten out after cytokinesis
Cytokinesis in plant cells involves the formation
of a new cell wall
• Different mechanism,
why?

– Separation is not
by-action of a
contractile ring but
by formation of a
cell wall
– Guided by
phragmoplast-
specialized
microtubule-based
structure
– Remaining interpolar
microtubules-form
the Phragmoplast
– Guide vesicles – at
the equator of the
old mitotic
spindle
Mitotic Chromosomes promote Bipolar spindle assembly

• More than passive passengers

for spindle assembly
• stabilize and organize
microtubules into functional
mitotic spindles

•In cells without centrosomes—

plant cells and some
animal cell types
the chromosomes
nucleate microtubule
assembly,
motor proteins then
move and arrange the
microtubules and
chromosomes into a bipolar s
spindle

Even in animal cells (which

normally have centrosomes) if
removed
Chromosome
create favorable environment for
microtubule nucleation and stabilization
spindle formation

Depends upon Guanine nucleotide exchange

factor (GEF)- bound to chromatin
Stimulate GTPase in cytosol
Activate Ran-GTP
Release microtubule stabilizing
proteins from protein complexes in
cytosol-
stimulate local nucleation and
stabilization of microtubules
around chromosome
Membrane-enclosed organelles must be distributed
to daughter cells when a cell divide
Mitochondria, Chloroplasts, Endoplasmic reticulum, Golgi
apparatus-
arise only from the growth and division of the
preexisting organelles

large numbers, safely inherited, their numbers simply double

once in each cycle

The ER in interphase cells is continuous with the nuclear

membrane
Organized by the microtubule cytoskeleton
Upon entry into M phase
The reorganization of the microtubules
Releases the ER
In most cells, the released ER remains intact during
mitosis and is cut in two during cytokinesis
The Golgi apparatus
fragments during mitosis
associate with the spindle microtubules via motor
proteins
hitching a ride into the daughter cells as the spindle
Re-Cap
• M Phase entry, How? Other functions
• Centrosome
• Microtubules, dynamic
instability
• Condensins, Cohesion,
• Centromere, Kinetochore, stable and
unstable attachment
• Three classes of microtubules
• Mitotic spindle

• Cytokinesis in animal and plant

cells, Different?
• Mitotic spindle formation in cells
without centrosome
M Phase
How the mitotic spindle assembles and
functions?

How the dynamic instability of microtubules

and the activity of microtubule-associated
motor proteins contribute to both the assembly
of the spindle and its ability to segregate
the duplicated chromosomes?

What are the mechanism that operates during

mitosis to ensure the synchronous separation
of these chromosomes?

How the daughter nuclei form?

Meiosis
• Specialized nuclear division
• Produces haploid cells, a single copy of each
chromosome
• Differentiate into specialized reproductive cells
called
gametes—eggs and sperm in most species
• diploid zygote, the potential to form a new
individual
Meiosis Includes Two Rounds of Chromosome
Segregation
• Reduces the
chromosome number
by half-same
molecular machines
and control systems
that operate in mitosis
• Cell begins the
meiotic program by
duplicating its
chromosomes in
meiotic S phase
• Unlike mitosis,
however, two
successive rounds of
chromosome
segregation
Duplicated Homologs pair during meiotic
prophase
Synaptonemal
complex
Morphological changes that occur during homolog pairing-divide
meiotic prophase into five sequential stage
Homolog segregation depends on several Unique
Features of meiosis I
Crossing-Over Is Highly Regulated
• Two distinct functions
• Helps hold homologs together-proper
segregation to two daughter nuclei
• Contributes to the genetic diversification of the
gametes
• Highly regulated: controlled number and location
of double-strand breaks
• On average, each pair of human homologs –
about two or three crossovers
Control of cell numbers and cell size

A fertilized mouse and human egg are similar in size—about

100 μm in diameter.
But Adult? Even tissue and organ within body?

How organisms eliminate unwanted cells by a form of

programmed cell death called apoptosis?

How extracellular signals balance cell death, cell growth,

and cell division?

Three fundamental processes- determine organ and body size

cell growth
cell division
Cell death
Apoptosis helps regulate animal cell numbers

In the developing vertebrate nervous system-

half of some types of nerve cells normally
die soon after they are formed.

In a healthy adult human, billions of cells

die in the bone marrow and intestine every
hour.

Wasteful for so many cells to die, especially

as the vast majority are perfectly healthy at
the time they kill themselves.

What purposes does this massive cell suicide

serve?
Apoptosis helps regulate animal cell numbers

Some time die -make

proper structure

Apoptosis in the developing

mouse paw
Cells die when the structure
they form is no longer needed

A tadpole changes into a frog,

the cells in its tail are
induced to undergo apoptosis
In adult tissues, cell • Rat-treated with drug
phenobarbital
death usually exactly stimulates liver cell
balances cell division- Liver enlarges.
division, unless the • stopped, apoptosis
greatly increases
tissue is growing or until the organ has
The liver
shrinking.is kept at a constant size through regulation of
returned to its original
both the cell death rate and the cell birth rate
Cell necrosis
a cell that undergoes apoptosis
dies neatly

Organic components of
the apoptotic cell to be
recycled by the cell
that ingests it
Three fundamental processes- determine
organ and body size?

Difference between Apoptosis and Necrosis?

Significance of Apoptosis?
Apoptosis is mediated by an intracellular
proteolytic cascade
• Family of proteases – caspases
• Made as inactive precursors-procaspases
• Activate in response to signal
• Activation of the apoptotic program, like entry into a
new stage of the cell cycle

Caspase cascade is not only destructive and self-amplifying but

also irreversible
Once a cell reaches a critical point -destruction, it cannot turn
Cell-Surface Death Receptors Activate the Extrinsic
Pathway of Apoptosis
The intrinsic Pathway of Apoptosis Depends
on Mitochondria
• Seeds of their own destruction (Inactive pro-caspases
lie waiting for a signal to destroy)
Bcl2 Proteins Regulate the lntrinsic Pathway
of Apoptosis
Tightly regulated
Bcl2 family members
• Bax and Bak (death-inducing family members)
•Other inhibit apoptosis (Bcl2 itself)
Animal cells require extracellular signals to
survive, Grow, and divide
In a multicellular organism, the fate of individual cells
is controlled by signals from other cells,

(soluble proteins secreted by other cells or

proteins that are bound to the surface of other
cells or to the extracellular matrix ).

Nutrients are not enough

Three major categories:

1.Survival factors promote cell survival, largely by

suppressing apoptosis.

2.Mitogens stimulate cell division by triggering a wave of

G1/S-Cdk activity and overcoming the intracellular braking
mechanisms that tend to block progression through the cell
cycle

3. Growth factors stimulate cell growth (an increase in cell

size and mass) by promoting the synthesis and inhibiting the
Survival factors suppress apoptosis
• Animal cells need signals from other cells to survive
• If deprived -activate a caspase-dependent intracellular
suicide program- die by apoptosis

Cell death can help adjust the number of developing nerve cells
to the number of target cells they contact
Survival factors- activating cell-surface receptors, turn on
intracellular signaling pathways-keep the apoptotic death
program suppressed by regulating members of the Bcl2 family of
proteins.
Mitogens stimulate cell division by Promoting
entry into S-phase

releasing the molecular b

First mitogen- platelet-derived growth factor, or PDGF

(Blood clot)
Liver is lost through surgery/injury, a mitogen called
hepatocyte growth factor
Mitogens stimulate G1-Cdk and G1/s-Cdk activities
Growth factors stimulate
cells to grow
• The growth of an organism or organ depends on cell growth as
much as on cell division

If cells divided without growing then?

Some extracellular signal proteins inhibit cell
survival, division, or growth
• extracellular signal
proteins (survival factors,
mitogens, and growth
factors) This Belgian Blue was
– act positively to increase produced by cattle
the size of organs and
breeders and was only
organism
later found to have a
• Myostatin (Oppose these mutation in the
factors) myostatin gene

– Mutation- Mice
the number and the size of
muscle cells is increased