Week 12: Cell cycle Flashcards
2 basic functions of the cell cycle
- Accurately duplicate the DNA of the chromosomes
- Precisely segregate the sister chromatids into 2 daughter cells
cell cycle control system
comprised of regulatory proteins that govern progression through the cell cycle
2 phases of the cell cycle and what is in them?
- S phase: chromosome duplication
- M phase: separation of sister chromatids within one cell
- Mitotic spindles (MT based): molecular muscles that pull chromatids apart
- Cytokinesis: division of the cytoplasm
- Contractile ring: actin/myosin based
what does M phase consist of? (2)
- nuclear (mitosis)
- cytoplasmic (cytokinesis) division
what does interphase contain? (3)
- G1: cell growth and monitoring
- S phase: chromosome duplication
- G2: cell growth and monitoring
how can DNA content of proliferating cells be determined?
by flow cytometry
- look at different content of DNA by labeling cells with fluorescent dyes
- each drop of medium contains 1 cell
what do the first and second peaks show in flow cytometry?
- can assume the DNA is normal in the first peak
- Unreplicated complement of DNa (G1) - the second peak shows the duplication for M phase
- Fully replicated complement of DNa (G2 and M) - there are low amounts of cells in between the two peaks
- Intermediate amount of DNA (S)
cyclin dependent kinases (Cdks)
phosphorylate substrates which control major cell cycle events ⇒ binds to cyclin as a heterodimer (active form)
- Activity of Cdks oscillates throughout the cell cycle ⇒ makes sure they are only active at the appropriate time
cyclins
bind Cdks and act as their major regulators ⇒ required for activation
- Cdks are active only when a cyclin Cdk complex is formed
- Cyclical changes in cyclin levels control Cdk activity
how does cyclin change over the cel cycle?
cyclins undergo a cycle of synthesis and degradation over the cell cycle, while levels of Cdks remain constant
G1/S-cyclins
help trigger cell division commitment
S-cyclins
trigger chromosome duplication and early mitotic events
M-cyclins
trigger entry into early mitosis
- Gradually increases amounts in G2 phase and peaks in M phase before it goes down in the middle of M phase
- Means that the complex (M-Cdk) activity should go up and down during certain phases of the cell cycle
each cyclin-Cdk complex phosphorylates what/
a different set of target proteins
cyclins not only activate Cdks but also ___
direct Cdks to their target proteins
G1-Cdk related to which cyclin?
cyclin D
G1/S-Cdk related to which cyclin?
Cyclin E
S-Cdk related to which cyclin?
cyclin A
M-Cdk related to which cyclin?
cyclin B
inactive state of Cdks
the active site is blocked by the T-loop
partial activation of Cdks
cyclin binding causes the T-loop to move out of the active site
- Not active yet because the protein conformation has been changed but there is no phosphate group
full activation of Cdks (what phosphorylates it?)
Cdk-activating kinase (CAK) phosphorylates a Thr residue near active sites
- The T loop site has been phosphorylated to make it fully active
what does phosphorylation at the active site do to cyclin-cdk activity inhibitory phosphates?
it inhibits them
- Inhibitory phosphorylation is dominant ⇒ if you have both phosphate groups added then it is inactivated vs only 1
Wee 1 kinase
cyclin-Cdk activity is turned off by this ⇒ adds the inhibitory phosphate (1 phosphate group is already present for activation)
Cdc25 phosphatase
cyclin-Cdk is activated by this ⇒ “good” enzyme for activating (removes the 2nd phosphate group)
Cdk inhibitor proteins (CKIs)
binding interferes with the active site and or the ATP binding site of Cdk
- Ex of CKIs: p21, p27
- inactivates the cyclin-Cdk complex
level
refers to amount of proteins
what is the relationship between M-Cdk levels and Cdk1 activity
Increases gradually and then drops
A- ctivity levels are different because they peak at M phase, but these are sharper
- Suggests that there is a feedback system => If you have small amounts of active molecule they feed in to form a positive feedback loop making more and more of the active molecules
what does “activity” not refer to?
the amount of protein present
what does activation of M-Cdk trigger?
entry into mitosis
- M-Cdk accumulates during G2 and M phase due to increased synthesis of M-cyclin
what does CAK do?
adds activating phosphate and Wee1 adds inhibitory phosphate ⇒ M-Cdk is initially inactive
when Cdc25 is added, what does it do?
remove the inhibitory phosphate to make fully active M-CDK
- This is an active kinase which will phosphorylate key enzymes to feed back into the system
- The active M-Cdk inhibits Wee1 and phosphorylates CAK to become active
positive feedback loop
rapidly promote the complete and irreversible activation of M-CDK
synthesis of M-cyclin during G2 increase what? rapid degeneration does what? When?
increase the level Cdk/cyclin B ⇒ entry into mitosis
- rapid degradation of cyclin at anaphase inactivates Cdk1 ⇒ exits from mitosis
Note: this is called metaphase anaphase transition => From an active controlled degradation of cyclin
Anaphase-promoting complex/cyclosome (APC/C)
controls the metaphase to anaphase transition
- APC is a ubiquitin ligase, which transfers poly-ubiquitin to target proteins, promoting their degradation in proteasomes
- M-Cdk phosphorylates APC
- M-Cdk should only be activated once throughout the cycle and then shut down until the next cell cycle
Cdc20
binds to APC to activate it during mid-mitosis
Cdh1
binds to APC to activate it during late mitosis
2 targets of APC
- Cyclin B (M-cyclin) ⇒ inactivation of M-Cdk
- Securin ⇒ activation of separase
- both have the same activity and form a complex with APC to activate it which inactivates M-Cdk
Phosphorylated APC binds to Cdc20 to what?
inactivate M-Cdk
- M-Cdk both activates APC/C and gets inactivated by APC/C
Phosphorylated Cdh-1 cannot bind to what?
APC
- M-Cdk can phosphorylate Cdh-1 which cannot bind to APC and inhibits the complex
- once M-Cdk is inactivated by Cdc20-APC complex will allow Cdh1-APC complex to become active which will further inactivate M-Cdk
what is the sequential activation of APC/C? (6)
- M-Cdk phosphorylates APC/C enhancing its binding to Cdc20
- APC/C-Cdc20 triggers anaphase
- APC/C-Cdc20 inactivates M-Cdk
- APC/C-Cdc20 is thereby inactivated in anaphase
- In anaphase, Cdk inactivation allow Cdh1 dephosphorylation, which stimulates formation of APC/C-Cdh1 => Cdh1 phosphorylation by Cdks inhibits its binding to the APC/C
- APC/C-Cdh1 remains active until Cdh1 is phosphorylated by G1/S and S-Cdks at the beginning of the next cell cycle
functions of active M-Cdk (4)
- Assembly of mitotic spindles
- Control microtubule associated proteins
- Chromosome condensation
- Nuclear envelope breakdown
M phase subphases (5 + 1 extra)
- prophase
- prometaphase
- metaphase
- anaphase
- telophase
- cytokinesis (not technically M phase)
prophase events
- chromosomes have condensed and 2 sister chromatids hold together
- Centrosomes move apart to initiate formation of 2 spindle poles
why are sister chromatids condensed?
for sister chromatids to be separated without breakage they must be compacted and resolved into distinct units ⇒ DNA strands cannot be broken
chromosome condensation
chromosomes are dramatically compacted
sister chromatid resolution
2 sister chromatids are resolved into distinct separable units
condensin
in the central core of the chromosome
- Condensin forms a ring like structure which encircles DNa loops within each sister chromatid
what does condensin need?
Needs ATP activity and when active it circles around the DNa and makes a higher order
- Requires phosphorylation of 1 subunit
what phosphorylates condensin?
M-Cdk phosphorylates condensin subunits to stimulate condensin activity
mitotic spindle (it’s components that work together)
dynamic and dense microtubule (MT) array includes MAPs and MT-dependent motors (kinesins and dyneins)
what types of mitotic spindles are there?
- kinetochore MTs
- Non-kinetochore MTs
- Astral MTs
kinetochore MTs
connected to kinetochore which binds ⇒ comes from centrosome
Non-kinetochore MTs
dynamic interconnected meshwork not bound to the kinetochore coming from the centrosome but is segmented
- Eventually become interconnected through motor proteins
Astral MTs
radiated out from centrosomes and bind to the internal part of the cell membrane at the cellular cortex ⇒ sometimes the + end goes to the opposite side
- These control the position of the centrosome
kinesins
plus end directed (most are + end)
dyneins
minus end directed
- motors attach cell cortex to astral MTs and pull centrosomes ⇒ controls the position of the centrosomes
Kinesin 5
attach to non kinetochore MTs and push MTs to lengthen the spindle (look like an X)
- Form 2 together and each side associated with different tubules
- Each part wants to go to the plus end, but since they are connected each fragment will move the opposite ways to extend the microtubules
kinesin 12
(exception) minus end directed motors attach to non-kinetochores MTs and pull MTs shortening the spindle
the centrosome side is always + or -?
always -
Chromokinesins (Kinesin4 and 10)
associate with chromosome arms and push the chromosome away from the centrosome
prometaphase
nuclear envelope breaks down and chromosomes attach to kinetochore MTs ⇒ cannot have the envelope so the microtubules can meet the chromosomes
nuclear lamina
control shape and stability of nuclear envelope
what do nuclear lamina do in interphase?
In interphase nucleus, nuclear lamins polymerize into a 2 dimensional lattice
at pro metaphase what phosphorylates lamina?
M-Cdks
- dephosphorylation occurs between prophase and telophase
- Phosphorylated lamins depolymerize ⇒ disassemble the nuclear lamina
- Nuclear envelope breaks down into small vesicles
kinetochore
large protein complex assembling onto the centromere
- attach chromosomes to the mitotic spindles ⇒ + ends of kinetochore MTs
- MTs are attached on both sides ⇒ both sides of the kinetochore need to be connected with these
centromere
the domain on the chromosome where kinetochores bind
- This is where the chromosomes are attaching at metaphase
- these events are random because the dynamic microtubules must meet at the chromosome from each side
Ndc80
a large protein complex binds from the sides of the centromeric nucleosome which makes the + end of the microtubules free to polymerize and depolymerize
- the complex slides along the microtubules as it treadmills
how are correct attachments detected?
The kinetochore senses a correct attachment through tension
- Incorrect attachment results in low tension
- Correct bipolar attachment results in a high level of tension
- When you have a small gap between the two chromosomes from tension that is what signals it is correctly been done
aurora B kinase
connects the inner kinetochore to the outer kinetochore where it kinase domain is so it can phosphorylate target proteins
- tethered to the inner kinetochore and phosphorylates MT attachment site including Ndc80
- This reduces the affinity of MT binding
bi polar attachment (high tension) components (4)
- aurora-B unable to reach the outer kinetochore
- MT attachment site not phosphorylated (Ndc80)
- Increases the affinity of MT binding
- Stable attachment
difference between unphosphorylated and phosphorylated Ndc80?
unphosphorylated Ndc80 has a stronger affinity to the MT so it won’t release them
- it uses a spring and by pulling on the spring you can measure the distance in how much the spring extends
metaphase
Chromosomes are aligned at the equator ⇒ metaphase plate and they come to the middle and stabilize
- state is dynamic but you don’t see it
where do kinetochore Mts polymerize?
they are polymerizing at the plus end (kinetochore side) and depolymerizing at the minus end (spindle side)
dynamic equilibrium
a net addition of tubulin subunits equal to a net loss of tubulin subunits ⇒ treadmilling (MT length constant)
- The rates are equal so length doesn’t change
anaphase
sister chromatids abruptly and synchronously separate and move toward opposite poles
why is it important to start anaphase? When?
- the cell is in danger ⇒ too many chromosomes
- Cells need to make sure they don’t start anaphase until all chromosomes are attached to MT’s or they end up with doubles of genes
cohesion
ring holds sister chromatids together until anaphase
- when APC gets activated it breaks this glue
securin
binds to and inhibits a protease called separase
- This happens before anaphase
separase
cleaves cohesin which frees the sister chromatids
- The tension will then pull the two apart
spindle assembly checkpoint (metaphase checkpoint)
ensures that cells do not enter anaphase until all chromosomes are properly attached to the spindles
what does improperly attached kinetochores do?
sends out a diffusible signal that blocks Cdc20-APC/C activation
- Unattached kinetochores catalyze conformational changes in Mad2
Mad2
gets recruited to unattached kinetochores and binds and inhibits Cdc20-APC/C
- Mad2 is involved with blocking APC which is what signals for anaphase to occur ⇒ we don’t know mechanism yet
2 ways to separate chromosomes
- Shrink microtubules
- Pulling the centrosomes to the poles of the cell
- anaphase A and B are not completely sequential ⇒ often overlap in some cells (cells do both)
anaphase A
plus ends of kinetochore MTs depolymerize, pulling the attached chromatid toward the pole
what role do Ndc80 complexes play in anaphase A?
hold on to the disassembling MTs
anaphase B
dynein motors on the cell cortex attach to astral MTs and pull spindle poles while kinesin-5 on non-kinetochore MTs pushing spindle poles at the same time
telophase
- Daughter chromosomes arrive at the spindle poles and decondense, the mitotic spindles disassemble
- Nuclear envelope reassembles
→ cells wrap up what they have done already in mitosis (reversal of beginning of mitosis)
how does exit from mitosis work?
APC/C causes Degradation of securin ⇒ activate separase (anaphase event) and Degradation of M-cyclin ⇒ inactivate M-Cdk (telophase event)
- M-Cdk gets inactivated
what happens when M-Cdk gets inactivated when exiting mitosis?
Set of phosphatases are activated to reverse phosphorylation of M-Cdk substrates => Reversion of mitotic program
- Chromosomes disassemble
- Spindles disassemble
- Lamin proteins are dephosphorylated ⇒ reform the nuclear envelope
cytokinesis
Cytoplasm is divided in two ⇒ this is done by the contractile ring structure and cleavage furrow occurs on the cell surface
- no longer mitosis phase
contractile ring
assembles during anaphase and is composed of actin and myosin filaments to pinch the cytoplasm into 2
- Structure underlying the cytokinesis process
- Located just beneath the plasma membrane of the cleavage furrow and the actin myosin bundles contract to pull the membrane inward
midbody
the plasma membrane of the cleavage narrows ⇒ there are some structures in between the separating cells
- Daughter cells remain connected by the midbody
- Midbody contains remnants of the central spindle midzone surrounded by dense matrix material
what may residual midbody components function to do?
mark orientation of next spindles
- we don’t know completely what is in the midbody
- There may be translation going on ⇒ very new
- Some kind of spindle midzone
3 models of determining the placement of the contractile ring
- Astral stimulation model
- Central spindle stimulation model
- Astral relaxation model
Astral stimulation model
astral MTs carry signals to the cell cortex which specify the site of furrow formation
- 2 signals will meet int he middle which forms a certain position for cleavage contractile ring
Central spindle stimulation model
spindle midzone generates signals which allows the contractile ring to form here
- This model may likely be the correct model
Astral relaxation model
astral MTs promote the relaxation of actin myosin bundles except at site encircling the midzone of the spindle
- These send a negative/inhibitory signals not to assemble the contractile ring in the region
RhoA
a small GTPase that triggers assembly and contraction of contractile ring
- actin and myosin must both be activated
RhoGEF (Ect2)
is localized in the cell cortex at the future site of cell division
- Stimulates Rhoa activation to occur at the cleavage site
RhoA-GTP
activates formis and multiple protein kinases (such as Rock)
RhoA activates which 2 molecules?
- formis
- rock
formis
nucleates the growth of actin filaments ⇒ controlled by RhoA
rock
group of kinases stimulating myosin 2 filament formation and motor activity
centralspindlin
concentrated at the beginning of cytokinesis at the overlapping plus ends of antiparallel microtubules upstream of RhoGEF
- starts in the middle of the cell and goes out toward the ends somehow
what does centralspindlin bind to?
binds to the RhoA-GEF (Ect2) at the spindle midzone
- These form a complex at the spindle midzone
These complexes at the midzone will migrate to the cortex underneath the plasma membrane
what does the centralspindlin and RhoA-GEF complex activate?
RhoA
