How many microtubules can attach to one chromosome at max during the
mitosis and is it dependant on the organism ?"
The number of microtubules attached to one kinetochore is variable.
In the unicellular eukaryote, Saccharomyces cerevisiae only one
microtubule binds each kinetochore, whereas in mammals there can be
15–35 microtubules s bound to each kinetochore.
I have a doubt that "Is the aster's microtubules are the ones which depolymerize
during the anaphase B or it is the kinetochore microtubules from the centrosome
end which depolymerize during anaphase B?
During Anaphase the sister chromatids separate and move towards the respective
poles. This happens by shortening of the ‘kinetochore microtubules’.
The shortening can happen by shortening at the kinetochore end or at the polar
end. Experiments from different groups have shown that both possibilities exist :
in different organisms, different mechanisms are found.
The Mitotic Spindle during Metaphase
An aster is a radial array of short microtubules
Centrioles are involved in organizing the spindle microtubules
Kinetochores are protein complexes associated with centromeres
Spindle microtubules–during prometaphase, attach to kinetochore & begin to move chromosome
Metaphase plate: an imaginary structure at the midway point between the spindle poles
If two cells with G1 and M phases respectively and get fused, the cell in the G1
phase will directly go to the M phase directly without chromosome duplication?
And what will happen if this fused cell divides?
This is an artificial system, and cell with 2 nuclei will not survive. It is only
an experiment to show that signals are present for these different phases.
Can both anaphase A as well as Anaphase B happen together in the cell division, in
some organisms?
Anaphase A and Anaphase B are 2 different phases of Anaphase. In other words,
Anaphase has been split up into Anaphase A and Anaphase B. Both will happen, but
Anaphase A will occur first, followed by Anaphase B. Most books will simply refer it to as
Anaphase which is also fine.
You had mentioned that there can be 15-30 microtubules bound to each
chromosome in mammals. Does each sister chromatid have an equal number of
microtubules from the respective poles, or can they be unequal as well?
I would assume so, because otherwise it might be difficult for them to be drawn
equally. But I am not sure if any researcher has actually checked this point out.
You had mentioned that cell organelles are fragmented during prophase, and they
reassemble after cytokinesis in the daughter cells. So the daughter cells will also have
an equal number of cell organelles as the parent cell. To achieve this, do cell
organelles also undergo duplication during the synthesis phase?
Yes, cell organelles are fragmented during prophase, and they reassemble after
cytokinesis in the daughter cells.
The exact numbers may not be identical. But what happens is that the fragmented
organelles come together to grow in size, and all almost equivalent number. This
happens mostly in the G1 phase. This is still an active field of research, so hope
the answer is correct as of today.
Does a chromosome always exist with two sister chromatids? Or is it so only after replication in the
prophase and metaphase? Because in school I've learnt that a chromosome always has two sister
chromatids attached at the centromere. That was the very basis of talking about centromere
position and classifying chromosomes on that basis.
No, a chromosome does not always exist with two sister chromatids. That happens only
after the Replication of the DNA in the S Phase, when the duplicated chromosome is now
referred to as the sister chromatids.
Thus, The sister chromatids are seen Only after replication in the S phase. Then the
replicated chromosome (original + copy)are attached to each other as sister chromatids
Why it isessential for chromosomes to come on metaphase plate to split .?
The Metaphase plate is an imaginary region that lies in the centre of the cell as
it is dividing. Possibly the alignment on that plate allows it to move equally
in either direction rather than unequally.
Why spindle fibers don't connect directly to the centromeres?
The centromeres are regions of the DNA around which the cohesins also bind. Possibly the spindle
fibres don’t have access to the centromeres. So instead, what happens is that a ‘kinetochore complex of
proteins forms on either side of the two sister chromatids. This is also at the centromeric region. The
spindle
fibres can bind easily to the kinetochores that are located towards the outside of the centromeric
regions.
Are all Protein Spindle Tubule fibres correctly bound to kinetochore
?
If there are checkpoints and regulatory complexes in the cell cycle, then how chromosomal
defects happen? What really these chromosomal defects bring changes in cell cycle?
Chromosomal defects can happen either due to DNA damaging agents, chemicals. When
these damages or problems are detected by the cell, the cyclin-dependant kinases are
not able to function properly and thus do not allow the cells to move into the next phase
What happens to the organelles during cell division. You said about the nucleus membrane and
mitochondria, which I understood.
NCERT says "Cells at the end of prophase, when viewed under the microscope, do not show golgi
complexes,
endoplasmic reticulum, nucleolus and the nuclear envelope." Does some chemical degrade them? If
yes, why don't organelles get degraded during interphase?
In mammalian cells organelles cannot be viewed under the microscope during the prophase not
because they disappear, but because they fragment into smaller fragments. In other words they
become dispersed/fragmented
and during cytokinesis segregate into the two daughter cells, where they subsequently reassemble
their normal interphase configuration
What is the role of motor proteins during mitosis ? Is it only for spindle formation
orfor other functions as well ?
The motor proteins include the kinesins and dyneins Motor proteins seem to have
a significant role not only in the spindle formation but in different stages
ofmitosis..
The role of kinesins is a very active area of research. I give below some of the
roles that kinesins participate in from a review on the subject:
Kinesin-4Connects chromosomes to spindle,
Kinesin-5 Establishes spindle bipolarity
Kinesin-7 Links kinetochores to microtubules
Kinesin-8 Subdues chromosome oscillation
Kinesin-10 Connects chromosomes to spindle
Kinesin-12 Maintains spindle bipolarity
Kinesin-13 Depolymerises microtubules
Kinesin-14 Restricts spindle length21
Movement of Chromosomes during Anaphase
Nonkinetochore tubules from opposite poles
overlap and push against each other
elongating the cell
In telophase: daughter nuclei
form at opposite end
Cytokinesis begins during anaphase
or telophase, and spindle eventually disssambles
"Signals relate not to single cell, but to the function of entire organism" what
does this mean?
Unlike unicellular eukaryotes like yeasts, most eukaryotic cells play distinct
roles in the organism, and thus they do not have the freedom of unicellular
cells to grow uncontrollably if the nutrient conditions are adequate.
In Campbell at pg231 it is mentioned that "G1 is the most variable in length
in different types of cell" what does this statement mean?
It means that the time spent in G1 can be different for different cell types. In some
cases it can be about 10 hours, but in other’s it might be less or more.
In NCERT Book there are no prometaphase state... What is the actual
significance of thisstate?
Yes, many books do not refer to the protometaphase. Since the protometaphase can
be distinguished under the microscope it has been designated as a separate phase.
In this phase the sister chromatids are not yet aligned on the metaphase plate
although they have begun to get linked to the microtubules.
What is difference between sister chroamtid cohesion, centromere and
kinetochore and what is actual positioning of these structure?
Centromeric region of the DNA in the sister chromatids is where the
cohesin protein forms a ring to keep the 2 chromatids together. The
kinetochore is a protein complex that also binds to the centromeric region
towards the outside of each of the sister chromatids and links each of the
chromatids through microtubules to each of thespindle poles.
Are all Protein Spindle Tubule fibres correctly bound to kinetochore
?
How can an external signal cause the initiation of cell division in the
eukaryotic cells except for the example, when we go to higher altitudes the
rate of formation of RBCs increases, adding to it how do RBCs receive the
signal? they don't even have a Nucleus.
We will talk about signals in this class
Where do the nucleolus, mitochondria and other organelles disappear
while cell division, why is the need to do so since DNA has already
been replicated and it has the sequence to form organelles itself
except mitochondria?
They don’t disappear, they fragment in prophase,
and then grow in size again after cytokinesis
Signals for Mitotic cell division ? Both external /Internal signals
What are the Internal signals ?
Signal:
At G1 to S phase
At G2 to M phase
In M (SAC)
Replication of DNA :
S phase
Segregation :
M phase ( Mitosis)
Cytokinesis :
External Signals for a cell : Come from the surrounding
tissues
All cells do not constantly divide ( even when environmental conditions
adequate)
Most eukaryote cells : part of a multicellular organism & specialized
Signals relate not to a single cell, but to the function of entire
organisms
Examples of differential behaviour of cells
Human skin cells divide frequently throughout life
Liver cells: Keep capability in reserve. Divide only when it is required. Injury
Brain cells, Muscle cells : Do not divide
Thus : Frequency varies with the type of cell
Why, what determines the control ?
Regulation occurs at the molecular level
Cancer cells manage to escape the usual controls of the cell cycle
Cancer Cells showed uncontrolled growth: Loss of control signals
How can we demonstrate the presence of signals ?
Are there really any internal signals that should tell the cell to divide ?
How can we demonstrate this ?
An experiment done to ask the question :
is there a cytoplasmic signal that drives the cell signal ?
Experiment :
Take G1 phase cells : no DNA synthesis occurs here
Fuse these G1 cells with S phase cells : make a single cell with 2 nuclei
What happens ?
How do you interpret the results of these experiments ?
Nature, 1970
A second Experiment:
Take G1 phase cells : no DNA synthesis occurs here
Fuse these G1 cells with M phase cells : make a single cell with 2 nuclei
What results can emerge ?
What would be the interpretations ?
?
The Cell Cycle has “Stop” and “Go” Signals
- Called
“Checkpoints”
The 3 Important checkpoints are :
- G1/S
- G2/M
- In M : SAC checkpoint (metaphase to anaphase)
What happens at these checkpoints ?
G1/S
DNA Intact
Favourable environmental conditions
Provides time to repair damaged DNA
G2/M
DNA Intact
DNA synthesis complete
Cell size ?
Prevents cells from dividing with damaged DNA
Spindle Assembly Checkpoint : metaphase to anaphase)
microtubule defects
erroneous kinetochore attachment
enter mitosis with damaged DNA
Inactivation of the SAC can lead to chromosome mis-segregation and aneuploidy
G1/S Checkpoint:
For many cells, G1 checkpoint seems to be the most important
If cells receive a ‘Go-ahead’ signal at the G1 checkpoint
it will usually complete the S,G2 and M phases and
divide
If cells do not receive the Go-ahead signal, it will exit the cell cycle
it will switch into a non-dividing state called the Go
phase.
Cell Cycle Controls
Internal Signals
Cyclins
(proteins)
Cyclin-dependant
kinases (CDK)
Proteins
whose
Conc
Vary within
Cell cycle
CDKs activities
Fluctuate
because it is
Controlled by
cyclins
Example:
MPF: Cyclin-CDK that triggers
cell’s Passage past G2 into M
External Signals
Growth
Factors
Density dependant
inhibition
Anchorage
dependance
Cyclins
What is a cyclin ?
A protein that ‘cycles’ during the cell cycle
There are many different Cyclins :
the protein levels go up and down with the
cell cycle
M Cyclins ( or B cyclins ) are maximum in M phase
G1/S Cyclins are maximum in G1/S Boundary
Different Cyclins reach Optimum Levels at different time
How are the protein levels regulated ?
G1/S = Cyclin E2
S = Cyclin A
M = cyclin B
- At the transcriptional level
- By protein degradation
Kinases
What is a kinase ?
A kinase is an enzyme that phosphorylates
a protein target
There are many different kinases in the cell
Phosphorylation of a protein can change its function
Cyclin Dependant Kinases (CDK)
A special type of kinase whose activity depends on the binding to a specific cyclin
It is a kinase that gets active
when it binds to a specific cyclin
Cdk protein levels
Cyclin levels : up /down
CDK levels : constant
CDK activity : up/down
Let us see how the G1/ S cyclin acts as a checkpoint Signal
G1/S = Cyclin E2
The G1/S cyclin binds to its kinase, and the kinase becomes active
The active ‘CDK Kinase’ then phosphorylates its targets, leading to signals
How does DNA damage affect G1/S cyclin, and act as a checkpoint regulator ?
MPF : Mitosis Promoting Factor is a Cyclin-dependant Kinase
M (or B) Cyclin levels vary with the cell cycle. Most abundant at Mitosis
MPF-dependant Kinase (MPF Cdk) protein levels remain constant
MPF-dependant kinase (MPF CdK) activity changes during the cell cycle
Molecular Mechanisms that help regulate the Cell cycle: G2/M
G2/M : Checkpoint
Cyclin B
or
Cyclin M
Permitting entry into Mitosis
CDK Levels remain constant ,
but gets activated by the Cyclin M ( or Cyclin B)
M-cyclin bound to CDK activates this CDK.
Activated CDK phosphorylates proteins, leads to entry into mitosis
What about the Spindle Activation Checkpoint ?
It allows cells to shift from Metaphase to Anaphase
Does not
Involve a
CDK
Ensures All the microtubules are bound to the kinetochores
A “straggler” chromosome will lead to mitosis pausing
Provides time for chromosomes to align before proceeding further
Spindle checkpoint ensures that all is ‘OK’ before proceeding for
Anaphase
Metaphase
Anaphase
Are all Protein Spindle Tubule fibres correctly bound to kinetochore ?
Anaphase Promoting Complex (Cyclosome)
Ub= ubiquitin
Cell Cycle Controls
Internal Signals
Cyclins
(proteins)
Cyclin-dependant
kinases (CDK)
External Signals
Growth
Factors
Proteins
Released by
cells that
Stimulate
other cells
to grow
Density dependant
inhibition
After a certain
density is reached
cells stop growing
Epidermal Growth Factor (EGF),
Platelet derived growth factor (PDGF)
Anchorage
dependance
Must be
attached to
a substratum
in order
to divide
Platelet Derived Growth Factor (PDGF) allows growth of fibroblasts
Anchorage-dependance Growth
Density Dependant Inhibition
Loss of Cell Cycle Controls in Cancer Cells
Cancer Cells do not respond normally to the body’s control mechanism
Cancer Cells may not need growth factors to grow and divide
- They make their own growth factor
- They may convey a growth factors signal without the presence
of
growth factor
They may have an abnormal cell cycle control system
Types of Cancer Cells
A normal cell is converted to a cancerous cell by a process called transformation
Cancer cells that are not eliminated by the immune system, form tumours, masses of
abnormal cells within otherwise normal tissue
Benign tumour: Abnormal cells remain at original site.
can be removed by surgery
Malignant tumours: Invade surrounding tissues and can metastatize
Export cancer cells to other parts of the body where they
form
additional tumours
How a Breast cancer can become metastatic
Action of anticancer drugs in the cell cycle
• restriction point: (G1 checkpoint) a
point in the animal cell cycle at
which the cell becomes
“committed” to the cell cycle, which
is determined by external factors
and signals
Cell-Cycle Checkpoints
Cell-cycle checkpoints enable a cell to ensure that important processes, such as DNA
replication, are complete [18]. Cell-cycle checkpoints prevent the transmission of genetic
errors to daughter cells. There exist three major cell-cycle checkpoints; the G1/S
checkpoint, the G2/M checkpoint, and the spindle assembly checkpoint (SAC).
The SAC ensures that chromosome segregation occurs correctly and is activated at the
metaphase to anaphase transition in mitosis, in response to microtubule defects [19] or an
erroneous kinetochore attachment [20]. Cells also arrest at the SAC when they enter
mitosis with damaged DNA [21]. Inactivation of the SAC can lead to chromosome missegregation and aneuploidy [22].
The G1/S and G2/M checkpoints are initiated in response to DNA damage to prevent the
transmission of damaged or incomplete chromosomes to daughter cells. The DNA-damage
checkpoints provide cells with time to repair damaged DNA. If the DNA damage is
irreparable, cells may initiate senescence (growth arrest) or cell death. The G1/S
checkpoint prevents cells from replicating damaged DNA, whereas the G2/M checkpoint
prevents cells from dividing with damaged DNA [18]. The G1/S checkpoint does not
function when p53 or p21 are either absent or not functional [23]. This checkpoint is often
defective in cancer cells because many of them have mutations in the genes that encode
either p53, pRb, or p21 [2,19]. This means that the only DNA-damage checkpoint available
to these cancer cells is the G2/M checkpoint [18].
3 The kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3 related (ATR) are
both involved in the initiation of the G2/M checkpoint, however ATR is the main effector
kinase associated with G2/M arrest [24]. When single-stranded DNA (ssDNA) is present, it
is bound by replication protein A (RPA) [25]. RPA recruits the ATR-interacting protein
(ATRIP) in complex with ATR and the Rad9–Rad1–Hus1 (9-1-1) complex to ssDNA [26]. The
9-1-1 complex then recruits DNA topoisomerase-binding protein 1 (TOPBP1) which triggers
the ATR-mediated phosphorylation of checkpoint kinase 1 (Chk1) [26]. The Rad17–
replication factor C complex, the 9-1-1 complex, and the adaptor protein claspin are also
required for Chk1 activation [25,27]. The Rad17–replication factor C complex acts as a
clamp loader for the 9-1-1 complex [25] and claspin links ATR and Chk1, allowing for the
phosphorylation of Chk1 on serine 317 and serine 345 [28]. Of these phosphorylation sites,
serine 345 is essential for Chk1 activation, while serine 317 plays a contributory role [29].
Once active, Chk1 prevents the activation of Cdk1 by phosphorylating Cdc25A and Cdc25C,
targeting them for cytoplasmic sequestration by the 14-3-3 proteins [30] or
for ubiquitination and degradation by the proteasome [31]. This prevents the removal of
inhibitory phosphates on threonine 14 and tyrosine 15 of Cdk1, preventing Cdk1 activity.
Active Chk1 also stabilizes the Wee1 kinase, which is responsible for phosphorylating
tyrosine 15 of Cdk1 [32].
It has been reported that Chk1, but not Chk2, is essential for the activation of the G2/M
checkpoint. In 2000, Liu et al. generated an inducible Chk1 deficient line of
murine embryonic stem cells [28]. They found that when this cell line was irradiated and
Chk1 depleted, these cells abrogated the G2/M checkpoint [28]. It has also been
demonstrated that H1299 human lung carcinoma cells treated with doxorubicin (a
What were the first experiments done to know that there
is there a cytoplasmic signal that drives the cell signal ?
Experiment:
Take G1 phase cells : no DNA synthesis occurs here
Fuse these G1 cells with S phase cells : make a single cell with 2 nuclei
Even the G1 cells now enter S phase
Experiment:
Take G1 phase cells : no DNA synthesis occurs here
Fuse these G1 cells with M phase cells : make a single cell with 2 nuclei
Even the G1 cells now enter M phase
The G1 Cyclin and the G1 CDK activity
The Relationship between Cyclin levels and CDK activity
The Cell Cycle has “Stop” and “Go” Signals
- Called
“Checkpoints”
The Cell cycle control system is regulated by both”Internal” and External controls
The 3 Important checkpoints are :
- G1/S
- G2/M
- M (spindle checkpoint: metaphase to anaphase)
Cyclin D
CyclinE
Cyclin A
Cyclin B