Topic 8: Cell Cycle Flashcards
What are the key points about the cell cycle?
undergoes cell division only when asked to
makes sure cell division steps proceeds with quality control
makes two daughter cells with exact same chromosomes and chromosome number
What are the four distinct phases of the cell cycle?
G1 - interphase
S phase - synthesis phase
G2 - continuum of interphase
M phase - mitosis
What is the G1 phase in the cell cycle?
cell growth but no division
awaiting signals to divide
DNA is not yet replicated but potentially ready for division
What is the first checkpoint in the cell cycle?
between G1 and S phase
restriction point
are there signals to divide?
is there enough space?
is there enough nutrients?
must answer yes to all three
What is the S phase in the cell cycle?
DNA replication
MTOC replication
What is the G2 phase in cell division?
cell is growing but not dividing
cell is committed to eventual cell division
What is checkpoint two in the cell cycle?
G2-M checkpoint
is DNA replication complete?
What is checkpoint three in the cell cycle?
“metaphase-anaphase” checkpoint
within the M phase
are all chromatids attached by MT?
What are centromeres?
constitutive heterochromatin region of chromosomes where chromatids attach and the kinetochore assembles
What are kinetochores?
protein complex that assembles at centromere to allow MT attachment
What are chromosomes?
composed of 2 sister chromatids upon entry into mitosis
What is the structure of the microtubules in mitosis?
mitotic spindle contains three classes of microtubules (all have negative ends embedded in MTOC)
upon entry into mitosis, MT network reorganizes
increased catastrophe and reduced rescue
many MT-associated proteins regulate this
What is the function of kinetochore microtubules in mitosis?
attach at kinetochore to sister chromatids
function: more chromosomes around cell
What is the function of polar microtubules and overlapping microtubules in mitosis?
extend from opposite MTOC and overlap in antiparallel manner
function: elongate cell and stabilize spindle
What is the function of astral microtubules in mitosis?
extend from MTOC to cell cortex (periphery)
function: anchor MTOC near poles
Why are microtubules more dynamic in mitosis?
MT network is regulated by MAP’s (MT associated proteins)
MAPs are regulated by cell cycle regulators (e.g. CDK/cyclin)
What is kinesin 13?
“special” kinesin that does not act as a motor but instead facilitates depolymerization of MT at positive end
What is prophase in mitosis?
interphase MTs are replaced by astral MTs
chromosomal condensation
MTOC move to opposite poles
What is prometaphase in mitosis?
NEBD from the phosphorylation of lamins to disassemble the nuclear lamina
“search and capture” of MTs: MTs from spindle poles (centrosomes) polymerize and radiate outwards and capture chromosomes at specialized structures called kinetochores
NEBD: nuclear envelope breakdown
nuclear membrane vesiculates
after NEBD, MT rapidly polymerize and get stabilized by kinetochore attachment
prometaphase completes when all chromosomes are attached by MT, then move chromosomes to metaphase plate
How are chromosomes moved toward the metaphase plate?
coordinated polymerization and depolymerization
motor proteins: dynein, kinesin 7, kinesin 13
kinesin 13: depolymerizes MT at positive end
dynein: pulls chromosomes toward MTOC, attaches MT to kinetochore
kinesin 7: pushes chromosomes towards metaphase plate
What is metaphase in mitosis?
chromosomes are all aligned at the metaphase plate
all chromatids are attached to spindle MTs (checkpoint)
signals originating at cell cortex dictates metaphase plate
What is anaphase in mitosis?
separation of sister chromatids
degradation of cohesin that is holding chromatids together at the centromere
occurs mainly by depolymerization of MT, some contribution of force from kinesin 13 and dynein
signal promoting entry into anaphase occurs at the APC (anaphase promoting complex)
What facilitates the chromosome movement in anaphase?
kinetochore MT depolymerizes to generate force
kinesin 13 facilitates depolymerization at kinetochore
polar microtubules: elongate the cell to create more space between separated chromosomes
astral microtubules: anchor MTOC to ensure chromosomes move to poles (MTOC doesn’t move to middle)
depolymerization of astral MT contributes to pulling force
What is the telophase in mitosis?
nuclear envelope reformation
chromosome de-condensation
restructure MT network
assembly of cytokinetic furrow
each daughter cell gets MTOC and copy of each chromosome
What is cytokinesis in mitosis?
physical separation of cytoplasm
furrow: a ring of MFs and myosin II that forms to bisect the mitotic spindle
actin filaments form a ring
force is generated by bipolar non-muscle myosin
What are the cell signals that control cytokinesis?
Rho –> ROCK –> phosphorylation of rMLC –> contraction
myosin contraction is controlled by phosphorylation of MLC
What are Rho proteins?
small G proteins that can be activated by GEFs (GTP/GDP exchange factors) and inhibited by GAPs (GTPase activating proteins)
Rho is tightly controlled by signals from spindle
How did the Heterokaryon experiments show cell cycle regulation?
two cells at different points were fused together
when cells in G1 and S phase were fused then DNA synthesis started in G1 nucleus, therefore there are signals that promote S phase
when cells in M phase and G1 phase were fused, G1 cells entered M phase, therefore there are signals that promote M phase
How did the discovery of MPF (maturation promoting factor) show cell cycle regulation?
used an unfertilized frog oocyte arrested at G2
injected either mitotic cell cytoplasm (which caused cell to enter M phase) or interphase cell cytoplasm (no M phase)
therefore there must be a soluble factor in cytoplasm of mitotic cells that promotes cell division
How did the study of S. pombe (yeast) mutants show cell cycle regulation?
S. pombe is a unicellular eukaryotic yeast
used to characterize enzymes that modify Cdks by phosphorylation
isolated 2 types of mutants:
elongated cells (deficit of Cdc25 or excess of Wee1): cells were too slow in cell division, therefore Cdc25 normally drives cell division forward
small cells (deficit in Wee1 or excess of Cdc25): cells divided too quickly therefore Wee1 normal function is to slow cell division)
What is Wee-1?
inhibitory kinase
adds a PO4
What is Cdc25?
activating phosphatase
removes PO4
What is a Fluorescence Activated Cell Sorter (FACS)?
a culture of growing cells are exposed to a fluorescence dye for DNA
as cells pass through a small opening the relative fluorescence is counted by the machine
the amount of DNA can be determined thus the phase of the cell cycle can be determined (have they replicated DNA?)
G1 = 1xDNA
G2 = 2xDNA
What is the G1 checkpoint?
controls entry into S phase
activated G1-cyclin/CDK promotes entry into S phase
targets –> Rb (retinoblastoma)
What is the G2 checkpoint?
controls entry into mitosis
activated by MPF (Mcyclin/CDK)
target of MPF (lamins, APC, and histone H1 activate mitosis)
What is the anaphase checkpoint?
controls entry into anaphase
activated by APC (anaphase promoting complex) not a cyclin/CDK
How is the cell cycle regulated?
MPF was later refined to show that it was a dimer of a cyclin and a cyclin-dependent protein kinase (cyclin/CDK complex)
cell cycle progression governed by key protein kinases in a cyclical manner
these cyclical proteins (called cyclins) regulate passage through the various checkpoints
involves a series of proteins being phosphorylated and dephosphorylated
activation and deactivation are under the control of cyclins
cyclins have cyclical expression but Cdks are always present
What is MPF?
dimer of cyclin/CDK
association of CDK with cyclin is necessary for activation of MPF, but it is not sufficient
What is CDK?
cyclin-dependent kinase
levels are consistent in cell cycle
What is cyclin?
levels vary throughout cell cycle
regulators of CDK activity
levels peak just before entry into mitosis
What is the relationship between cyclin and cyclin-dependent kinases?
cyclins do not have enzymatic activity but must bind to kinases before they can become active
heterodimer is inactive
dimer only forms when a threshold of cyclin accumulates in the cell
How is CDK activity activated?
association of CDK with cyclin is necessary for activity but not sufficient
phosphorylation of key resides must be accomplished in order for activation of the CDK-cyclin complex
What are the steps in CDK activation?
CDK and cyclin come together to form an inactive heterodimer
cyclin activated kinase (CAK) adds one phosphate
Wee1 adds two inhibitory phosphates
Cdc25 removes inhibition phosphorylation by Wee1
What is the restriction point?
passing through the restriction point commits a cell for eventual division
senses the availability of nutrients, presence of growth factors, and cell size
activation of G1 CDK/cyclin
DNA damage, loss of adhesin and growth factor signaling all slow cell cycle
How do Rb (retinoblastoma associated gene) proteins control the restriction point?
normally binds and inhibits E2F
E2F is a transcription factor that regulates DNA synthesis
Rb is a target of G1 cyclin/CDK
cyclin/CDK promotes phosphorylation of Rb which inactivates it
E2F is then available to trigger gene expression
What controls the G2/M checkpoint?
CDK/cyclin complex - MPF
M-CDK
What are the functions of active M-CDK during the G2/M checkpoint?
phosphorylation of lamin causes NEBD in prometaphase
phosphorylation of nuclear material causes nucleoli dismantling
phosphorylation of condensin cause chromosomal condensation in prophase
phosphorylation of Histone H1 causes chromosomal condensation
phosphorylation of MAPs decreases MT stability and increases MT dynamics
indirect activation of APC
What controls the metaphase/anaphase checkpoint?
M-CDK activates APC (anaphase promoting complex)
APC acts at the metaphase-anaphase transition
cohesin (complex of Smc1/Smc3/Klesin) which holds sister chromatids together needs to be broken down
How is cohesin broken down during anaphase?
APC induces polyubiquitination of securin (signal that causes protein degradation by proteasome)
securin normally blocks separase, but now separase can cleave kleisin and allow separation
APC is regulated by Mad/Bub
Mad/Bub is produced by unattached kinetochores, therefore Mad/Bub inhibits APC to prevent passage in anaphase if chromatids are not all bound by MTs
MT attachment inhibits Mad/Bub
What if the signals regulating the cell cycle don’t work?
if the signals for cell cycle progression are ignored or don’t work, this can result in cancer and uncontrolled cell proliferation
