Shu-Ping Lin, Ph.D.

Institute of Biomedical Engineering E-mail: splin@dragon.nchu.edu.tw

Website: http://web.nchu.edu.tw/pweb/users/splin/

Reference: The Cell Cycle: Regulation and Division by Dr.

Andrew Bieberich and Dr. Michael Goldman

Cell Division and Its Regulation

Modes of Cell Division

Cell Division in Prokaryote

Single chromosome in the form of double-stranded circular DNA (1.7mm long), cylindrical cell has diameter of ~1um and length of 4um, attach at one point to plasma membrane compacted and folded with DNA-binding protein

G1

START

M

S

DNA replication

checkpointDNA damage

checkpoint

G2

metaphase

checkpoint

Degradation

of metaphase

cyclins

Cell division

DNA Replication

Chromosome Separation

A wall forms and divides cell into 2 compartments (every 30min)

Cell Division in Eukaryote

Cells divide following DNA replication and is less frequently than prokaryotes, ~10-20percent

of cells containing condensed chromosomes undergo division (mitosis, M phase of the cell cycle): including separation of duplicated chromosomes and partition of organelles into daughter cells

Divisions of cell cycle, 4 segments: M phase (mitosis: active cell division) and interphase (S phase: Active DNA synthesis; gap phases (G1 & G2))Tangled thread of DNA corresponds to interphase(G1, S, and G2)Vertical bar of DNA in MIn most cultured cells, G1 and S phases take~10 hr each, M phase takes lessthan 1 hr, G2 ~ 4 hr

2n DNA

2n ~ 4n DNA

4n DNA

Centrosome

Cells that are not dividing appear flatter in the

culture dish with more firmly attached to the bottom

of dish and exhibit size variations.

Ultimately, cells decide whether to divide or not based

upon intrinsic information and input of information

(extrinsic (environmental)) from outside the cell.

Extrinsic factors: presence or absence of chemical

nutrients, spatial clues, differentiation inducers, and

growth factors Select outcome including staying in

G1 (G0) phase, dividing, or undergoing apoptosis

If normal G1 cells too crowded on plate, not attached to

substratum, or not appropriate nutrients and growth

factors, cells can not enter DNA-replication phase

(S).

single cells

(yeast, bacteria)

Are there

enough

nutrients?

Are toxic waste molecules

too concentrated to

proceed without cell

damage?

cells within

multi-cell organisms

Is the cell

attached to

others? Is it

too crowded?

Are the correct

growth factors

present?

G1

START

M

S

DNA replication

checkpointDNA damage

checkpoint

G2

metaphase

checkpoint

Degradation

of metaphase

cyclins

Decision-making process of an animal cell during transition from G1 to S phase. The process is represented as a Boolean (a) and neural network (b).

G0, when time stands still.

Cell cycle is regulated by biochemical steps translating external stimulus into response

Regulation of the Multi-celled

Eukaryotic Cell Cycle

1. Semi-modular control system.

2. Five major checkpoints that act

as switches in the system.

3. Cycling and Cycling and Cycling...

4. Growth factors coordinate cell cycles

across multiple cells.

5. G0, when time stands still.

6. Cancer: when switches malfunction.

G1

START

M

S

DNA replication

checkpointDNA damage

checkpoint

G2

metaphase

checkpoint

Degradation

of metaphase

cyclins

Across cell types, the cell cycle

may take minutes, months, or

arrest indefinitely.

Therefore, we know that

something sophisticated must be

controlling it.

A “clock” is not flexible. Phase

triggering is only slightly more so.

The system required is one that

uses switches controlled by

subsystems with feedback.

G1

START

M

S

DNA replication

checkpoint

DNA damage

checkpoint

G2

metaphase

checkpoint

Degradation of

metaphase cyclins

Eukaryotic Cell Cycle

G1

Chromosomes become

uncondensed. Also

called „G0‟ if cell

arrests in this state.

START

M

daughter

cells

S

DNA replication

checkpoint

DNA damage

checkpoint

DNA is replicated.

G2

Chromosomes

condense,

Topoisomerase II

helps to untangle

them.

metaphase

checkpoint

Degradation of

metaphase cyclins

(check before

entering G1)

Eukaryotic Cell Cycle

Cell-adhesion molecules regulate

actin cytoskeleton and may

thereby indirectly alter the

phosphorylation states of kinases

in the cell-cycle regulation pathways.

Signal transduction pathways common to integrins and

growth factors and protein molecules associated with

cell-division cycle.

Integrin-mediated

adhesion are

transduced into

nucleus using

pathways

downstream of

receptor tyrosine

kinases. MEK

activates cyclin D

Cyclical appearance of some cell-cycle-associated proteins during the various phase of yeast cell cycle

Abundance/activity of cell-cycle-associated proteins varies dramatically with time depending on the phase of cycle.

Protein Complexes Formed by

Cyclin-Dependent KinasesActivation of cyclin-dependent proteins kinase depends on: binding of an appropriate cyclin, state of phosphorylation, and presence of inhibitor proteins (Inh).

Phosphate group (Pi) must be added to positive(+) regulator site (Threonine side chain that activates protein).Phosphate added to negative (-) regulation site of protein (covalently bound to specific Tyrosine side chain) must be removed.

Binding of an inhibitor may halt activity of CdK even if cyclin remains bound.Only one of 3 states of CdK complex can activate target protein such as retinoblastoma protein (pRb). CdK acts like a computer chip and has binary output (activate or inactivate) based on multiple input signals (binding of cyclin, inhibitor, state of phosphorylation)

Targeted Protein Degradation

Marked by UbiquitinationCell have machinery to degrade unassembled, damaged, or misfolded proteins Affect cell cycle and other

important processes.Ubiquitination of proteins is carried out by enzymes (E1, E2, and E3).

First activated by E1 and transferred to an ubiquitin-conjugating enzyme (E2).

Activation and transfer of ubiquitin is carried out by thioester cascade.Protein complex (E3, e.g., SCF and APC) facilliates substrate-specific attachment of multiple ubiquitins to protein. Polyubiquitinated protein is then recognized by proteasome (Protein-degradation machine) existing as multiple copies in cytoplasm and nucleusUbiquitinated substrates recognized at the end of proteasome, selected proteins enter Enzymes degrade proteins to short

peptides and release from proteasome.Cylindrical in shape with a chemically active inner surface

#Role of retinoblastoma protein (pRb) in the cell cycleDephosphorylated pRb binds to and holds inactive gene-regulatory proteins such as E2F.Phosphorylated pRb detaches from the regulatory protein, freeing it to activate proliferation.Rb becomes dephosphorylated as the cell exits mitosis and is then phosphorylated late in G1 phase as the cell prepares to go past START.

Growth factors and integrin-mediated

signalling pathways both affect

production of cyclin D in the nucleus.

Cell surface

receptor

nuclear

membrane

nuclear poreGrowth factor

Integrin bound

to ECM

Rb

E2F

signal transduction pathways with

many phosphorylation steps

Growth factors and integrin-mediated

singalling pathways both affect production

of cyclin D in the nucleus.

Cyclin D: Cyclin-dependent kinase

called Cdks

cell surface

receptor

nuclear

membrane

nuclear poregrowth

factor

Integrin bound

to ECM

signal transduction

pathways with many

phosphorylation steps

RbCDK4

cyclin D

cyclin D gene

expression

RbCDK4

cyclin D

E2F

Turn on genes for DNA

replication in S phase.

Turn on gene for

cyclin E and cyclin A

RbCDK2

cyclin E / A

Progression past START is reached when

the feedback loop increases the rate at

which Rb is phosphorylated and DNA

replication genes are turned on by E2F.

RbCDK2

cyclin A

Cyclin A-CDK2, in addition to increasing the rate at

which Rb is phosphorylated, also begins phosphorylating

(and thus activating) the DNA Replication Complex.

CDK2

cyclin A

Activated DNA RC

binds to replication

fork and recruits

DNA Pol III

DNA Pol III

When activated DNA RC begins to

bind replication forks, the G1 - S phase

transition is complete.

Apoptosis can be thought of as “programmed cell death”.

A set of molecular machinery kept in reserve for this purpose

is turned on, and the cell auto-destructs by digesting all of its

components.

Loss of function of p53, or other genes that encode damage

checking proteins, can be a “pre-cancerous” condition. Cells

that divide without checking for DNA damage may replicate

mutant forms of cell cycle regulating genes. These cells may

in turn become the progenitors of tumors.

#DNA synthesis in eukaryotesDNA synthesis occurs by bidirectional growth of both strands from a single origin in prokaryotes and multiple origins in eukaryotes.DNA replication is semiconservative; that is, each daughter DNA molecule contains one old and one new strand.Arrows indicate direction of DNA.

Centrosomes play important roles in polarization and division of cells.Barrel-like structures at the center of centrosome are called centrioles.These are oriented at right angles to each other and are connected by thin fibrils.

Late in G1 phase, distance between 2 centrioles increase and centrosome duplicates during S phase, with one old and one new centriole present in both copies.

2 centrosomes remain paired until prophase of M phase , during which they migrate to opposite sides of nucleus.

Set of microtubules connects 2 centrosomes at polar ends of cell.

Another set of microtubules extending from centrosomes attach to the sites called kinetochores on centromeresof chromatide pairs, eventually pulling 2 chromatides of a single chromosome to opposing polar ends of cell.

S phase: all DNA in all chromosomes

must be replicated and checked for

damage.

DNA Pol epsilon detects

presence of replication forks-

check before exiting S phase.

Many proteins (and thus many

genes) are involved in checking

for damaged DNA at this point.

Apoptosis may occur if damage

is too great.

p53

BRCA1

Regulatory

cross-talk

ribosomes

mitochondria

histones

centrosomes/centrioles

Cell components other than DNA must

be replicated also during S phase.

Microtubule:13 cylindrical fibers in parallel, but staggered protofilaments containing α and β-tubulin subunits.

Form tracks for transport of organelles and movement of chromatin toward opposing poles in a dividing cell.

Schematic showing in-vitro motility involving ATP-driven protein motor kinesin and microtubule tracks (a).Globular motor head regions of kinesin interact with microtubular tracks, whereas tail domains bind to organelles to be transported (b).Kinesin dimers can walk on microtubule without losing contact for several microns.

John Kyrk‟s webpage has an illustration of primary DNA

packaging with histones, and this serves as a powerful

supplement to our discussion of G2 phase.

You may see this by clicking this link:

http://www.johnkyrk.com/chromosomestructure.html

As is mentioned in Tozeren and Byers, not much is known about

the molecular signalling that controls timing of G2.

We know that S phase ends when the DNA replication and DNA

damage checkpoints are passed, and mitosis (M phase) is

considered to have begun when chromosomes are fully

condensed. During G2, in between S and M, DNA topoisomerase

II works to untangle the uncondensed chromosomes so that they

may condense separately.

Complex of DNA and associated proteins in eukaryotic cell is referred to as chromatinDNA carries genetic information, and associated proteins organize chromosomephysically and regulate activities of DNA.DNA double helix whose diameter is about 2 nm wraps around bead-like structures called nucleosomes.

Nucleosomes are composed of proteins of histone family and have a diameter of about 11 nm, still not visible under light microscope.

Histone H1 clamps DNA on to surface of nucleosome.

During M phase, nucleosomes pack into coils and loops, eventually forming supercoiled chromatin fibers.

#Schematic of mitotic division in eukaryotic cellsDuring interphase, cell integrates external signals for growth and adhesion and replicates its chromosomes and centrosome.After DNA replication, each chromosome consists of identical, paired chromatids.M phase: prophase, metaphase, anaphase, and telophase

At the beginning of the M phase, chromosomes condense.

Nuclear envelope breaks down, duplicated centrosomes move to opposite poles, and paired chromosomes become aligned in a plane at the equator of the cell.

Chromatids separate from each other and begin to move toward the poles, the nuclear envelop reforms, and chromosomes decondense and are no longer visible.

Mitosis: Tozeren and Byers explain the separate phases of mitosis

completely, so it is not necessary to simply repeat what they said.

However, you should make sure that you are familiar with what happens

during each of these phases:

Prophase

Metaphase

Anaphase

Telophase

You will notice that not everyone describes these in exactly the same

way. (For instance, John Kyrk lists „Prometaphase‟ as an extra step.

I‟ve never seen that before.) The basic events that take place are as

described in Chapter 7 of Tozeren and Byers, so you may go by what

they have written. To see John Kyrk‟s mitosis animation:

http://www.johnkyrk.com/mitosis.html

centrosome

microtubule

paired sister chromatids

aligned along metaphase plate

There is a checkpoint at metaphase of mitosis.

The sensor proteins detect whether tension is exerted

on centromeres of all chromatid pairs.

centromere

#Spindle structure and chromosome behaviorIn vertebrate cells, mitotic spindle consists of 2 overlapping arrays of microtubules oriented with “+” ends to distal to “-” ends proximal to the poles. (a)One kinetochore (of the 2 kinetochores) becomes attached to single microtubule and moves rapidly to pole (long arrow)(b)During this movement, additional MTs become attached to outer plate of same kinetochore.(c)Chromosome oscillates to and from the pole until another MT from opposite spindle pole attaches to remaining kinetochore.

(d)Opposing MT-generated tension on 2 kinetochores results in chromosome adopting an average position around equator.(e)Assuming that all checkpoints are passed the action of APC (anaphase promoting complex) during anaphase allows 2 chromatids to separate and there is a net movement toward spindle poles.

securin proteins binding

centromeres of sister chromatids

centromere

kinetochore

microtubule made of

alpha and beta tubulin

subunits

MT motor

Model of the Role of Tension in Kinetochore-MT Interactions

Attached kinetochore moves poleward at a rate of approximately 2um/min.During this process, it pulls on its associated MTs to stretch centromere region.Force for poleward motion is provided by MT motors (a dynein family member), which are attached to the kinetochore.During poleward movement, kinetochore MTs shorten by disassembly at the kinetochore.

After the metaphase checkpoint sensory system

detects perfect alignment, anaphase begins. The MT

motors pull each chromatid along its microtubule,

and the microtubule disassembles as it is pulled

toward the kinetochore.

Growing animal cells under artificial culture conditions:

Normal cells will usually not divide more than ~10

times under culture conditions, so primary cultures of

recently isolated cells do not last long.

Alternatively, cell lines that are immortal may be used-

these are often cells containing genetic mutations that

cause cancer-like growth, and so are less accurate

model systems for “normal” cell activity.