Shu-Ping Lin, Ph.D.
Institute of Biomedical Engineering E-mail: [email protected]
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.