MODULE 1: cell cycle Flashcards
interphase
- chromosome duplication
- cohesion of chromosomes (cohesions = proteins that hold sister chromatids together)
- centrosome duplication
prophase
- breakdown of microtubule display
- replacement by mitotic asters (centrosomes + microtubules)
- chromosome condensation
prometaphase
- nuclear envelope breaks down
- chromosomes captured, bi-orientated and bought to spindle equator
metaphase
- chromosomes aligned at metaphase plate
anaphase
- APC/C activated and cohesions degraded
- chromosome movement to poles
- spindle pole separation
telophase
- nuclear envelope reassembly
- assembly of contractile ring
cytokinesis
- reformation of interphase microtubule array
- contractile ring forms cleavage furrow
- cell separates in two
chromosome movement during mitosis
microtubules shrink (break down) on one side and grow on the other, allowing chromosome movement
kinesin-7 connects microtubules to kinetochore, pulling chromosomes along as microtubule grows and shrinks
meiosis 1
DNA replication
homologous chromosomes pair
recombination occurs between homologous chromosomes (alleles exchanged –> genetic diversity)
homologous chromosomes separate, sister chromatids in tact
cell division
daughter cells are 2n and contain different sets of chromosomes (parental or maternal)
meiosis 2
sister chromatids separate
cell division
four gametes (1n) formed
kinase
enzyme that adds phosphate to its target
phosphatase
enzyme that removes phosphate from its target
CDKs at different stages in cell cycle
M phase - M CDK G1 phase - G1 CDK + S CDK S phase - S CDK G2 phase - G2 CDK + M CDK G0 - inactive CDK
G1 CDK = CDK4 + cyclin D
G1/S CDK = CDK2 + cyclin E
S CDK = CDK2 + cyclin A
how to test for CDK activity
combine in a test tube:
- cell lysate
- control / cyclin / CDK antibodies
purify Ab protein complexes
add substrate and radioactive ATP
load reaction products on SDS page gel to see CDK activity
mechanics of regulation - ubiquitination
ubiquitination - protein ligases attach to Ub to target a protein
process repeats = polyubiquitination
proteasome recognises polyubiquitination and destroys the protein
anaohase promoting complex or cyclosome (APC/C)
involved in metaphase to anaphase + anaphase to telophase transitions
APC/C degrades securin, activating separase which splits chromosomes
phosphorylation vs ubiquitination
phosphorylation is temporary and reversible (molecular switch). phosphorylation is quick and easy
ubiquitination is permanent and irreversible (destruction)
G1 to S phase transition
very abrupt transition
PATHWAY
- CK1 bound to S cyclin/CDK
- G1/S cyclin/CDK polyubiquinates CK1, allowing SCF to recognise it
- SCF degrades CK1 and S cyclin/CDK activates
- SCF binds to S cyclind/CDK until cell is ready for S phase
- SCF is degraded by Ub-protein ligase when cell is ready
cohesin
“rubber band” around centromere on sister chromatids
temperature sensitive mutants in yeast cells
yeast cells used to examine cell cycle
foudn that cells elongate then divide
cell growth uncoupled from cell division i.e. keep growing independent of cell cycle stage
yeast cells grown cells at permissive temp then shifted to restrictive temp
cells that survived after shift contained ts mutations
mutated cells grown up an mutations characterised
- cdc2+ (wild type) = cdc is a CDK
- cdc2- (recessive) = long phenotype, no mitosis
- cdc2º (dominant) = wee phenotype, lost regulatory function, mitosis too often
- cd13 (mitotic cyclin) = mutants also give long phenotype
regulation of mitosis (cdc25 + wee1)
cdc25 (phosphate) drives mitosis
wee1 (kinase) inhibits mitosis
wee1 phosphorylates tyrosine 15, cdc25 acts on same tyrosine
elongated cells = increased G2 = deficit of cdc25 OR excess of wee1
small cells = decreased G2 = deficit of wee1 OR excess of cdc25
cascade of kinase and phosphate activity controls entry into mitosis
- mitotic cyclin forms complex with CDK
- inactive because wee1 immediately phosphorylates Y15 and blocks substrate binding site
- in G2, CAK phosphorylates T161 (near Y15) to begin activation
- when checkpoints passed, cdc25 removes phosphate from Y15 and cell can undergo mitosis
mitosis promoting factor (MPF)
i.e. mitotic cyclin/CDK
MPF drives mitosis
MPF produced in G2 phase
kept inhibited (phosphorylated) until cell is ready for mitosis
once active, MPFs phosphorylate:
- chromatin associated proteins
- nuclear envelope proteins
- microtubule associated proteins
- kinetochore proteins
- many more
mitotic checkpoint pathway
is DNA replication complete?
ATR1 (protein) detects single strands of DNA, usually associated w/ replication forks
active ATR1 activates CHK1 (kinase). CHK1 in turn inhibits cdc25 to prevent mitosis
metaphase to anaphase transition
APC/C initiates transition by inducing cohesin removal at centrosome
when all kinetochores bind MTs, cdc20 binds to APC/C
APC/C polyubiquinates securin (bound to separase, inhibiting it)
securin uninhibits separase and it cleaves scc1,
cohesins composed of Smc proteins and Scc1, cohesin falls apart when scc1 degraded
chromatids free to separate = anaphase
degredation of mitotic cyclins (APC/C)
APC/C specificity changes in late anaphase once chromatids have separated
Cdn1 becomes active and leads APC/C complex to mitotic cyclins for degredation
this is necessary for chromatin decondensation and depolymerisation of microtubules –> needed to get to telophase
necrosis definition
unplanned cell death
apoptosis
apoptosis signalling pathway
programmed cell death = common cell fate
cell chopped up and packaged for removal. DNA is fractioned, cell membranes pinch off into small structures
fragments are labelled for macrophages so that they will be cleaned up through phagocytosis
apoptosis adjusts number of nerve cells to a target. survival factor released by target cells
SIGNALLING PATHWAY
CED-9 (protein) sits on mitochondrion and binds to CED-4
EGL-1 induces release of CED-4 from CED-9, initiating cell death
CED-4 forms large octamer complex in cytoplasm
interacts with CED-3 to form capsule holoenzyme
capsule destroys vital proteins to cause cell death
once active, CED-3:
- cleaves lamins –> nuclear envelope dissolves
- activates endonucleases –> DNA digested
- attacks cytoskeletal structure
- attacks cell-cell adhesion proteins
- cleaves itself
selection of cancer cells
tumours developed through selection process
cancer cell has mutation with growth advantage
selection of cell with growth advantage= more mutations
proto-oncogenes
normally promote cell growth
once mutated (typically gain of function) or amplified they become oncogenes (Ras, myc, src)
can duplicate DNA
colon cancer
colon cancer pathways
early pollops can be identified and removed
stages I-III = benign
right before stage IV, p53 activated = cell death, cell cycle arrest, sensescence
common genes mutated along progression
APC (adenoma polyposis coli protein) ——| wnt signalling —–> myc proto-oncogene —–> proliferation advantage (benign)
Ras (growth factor) —–> MAPK (mitogen activated protein kinase) —–> proliferation
metastasis
spread of cancer cells from their site of origin and establishment of secondary growth
most cancer deaths arise from metastasised tumours
as more cells become mutated, they can detect GFs and move towards source
- penetrate basement membrane by acquiring enzymes
- migrate on ECM fibre down to blood vessel
- travel to anywhere in body (1 in 1000 land somewhere to make tumour)
retinoblastoma
diagrams of hereditary and sporadic mutations
rare childhood cancer
if caught early, 95-98% cure rate
caused by mutations in Rb gene
1) hereditary retinoblastoma
- RB+ and RB- (from parents)
- somatic mutation
- RB- and RB-
- homozygous cells give rise to tumours in retina
2) sporadic retinoblastoma
- RB+ and RB+
- two somatic mutations
- RB- and RB-
- homozygous cells give rise to tumours in retina
restriction point in cell cycle
cell is committed to another round of cell division once it passes restriction point and is no longer mitogen-sensitive
8 hours between restriction point and S phase - cell can still exit cycle within this period
Rb is a transcriptional repressor of E2F (a TF of genes required for DNA replication). Rb is also a substrate for G1 cyclin/CDK. hyper-phosphorylation of RB = restriction point passed, cell mitogen-independent and committed to cell cycle
APC/cdh1 activity in mitosis
APC/Cdh1 degrades mitotic cyclin so DNA can be decondensed = region of gene called destruction box
APC/Cdh1 can degrade many proteins required for S-phase (such as E2F) so it needs to be inactivated before cells enter S-phase
- APC inactive at G1/S boundary
- APC inactive in S phase
- APC active in mitosis
- region between inactive APC and start of DNA replication = cells cannot exit cell cycle = new committment point
G1/S CDK phosphorylates Cdh1
Emi1 leads another Ub ligase to destroy Cdh1
Cell stress pathway
Mutations in cancer cells
cell stress —–> p16 —–| G1 cyclin/CDK (cyclin D + CDK4) —–> Rb —–| E2F —–> transcription of genes that control entry into S-phase
- under stress, p16 sits atop G1 cyclin/CDK complex
- this prevents CDK/cyclin complex from hyper-phosphorylating Rb
- Rb sits atop E2F to prevent gene transcription
- no hyperphosphorylation = no Rb release from E2F = no gene transcription
in presence of mitogens, p16 is inactive –> G1 cyclin/CDK active –> Rb phosphorylated and removed from E2F –> E2F transcribes genes for S-phase
cancer cells frequently: - delete/inactivate p16 - over express/amplify G1 cyclin - delete/inactivate Rb tumours have only one of these mutations, indicating that regulation of this genetic pathway needs to be disabled for growth
tumour suppressor pathway
p53 = tumour supressor
detects conflicts within cell (DNA damage, telomere shortening, etc) and can activate several different pathways to resolve conflicts (cell cycle arrest, apoptosis)
ATR —| MDM2 —| p53
- p53 controls WAF1 gene - an inhibitor of CDKs
- therefore, p53 activity inhibits cell cycle progression
- MDM2 degrades p53
cancer cells have GOF mutation in MDM2 or LOF mutation in p53 = excessive proliferation
50% of tumours have mutations in p53 pathway/gene
types of gene regulation
transcriptional regulation: distinct genes turned on/off controlled via various mechanisms
translational regulation: distinct mRNAs translated or repressed and localised to different regions of cell
epigenetic regulation: marks on DNA that regulate gene expression are inherited by daughter cells but independent of DNA sequence
mechanisms of cell specification: asymmetric cell division
cells contain factors which provide unique info related to cell
factors localised to particular region of cell
cell divides to produce two identical cells in terms of DNA, but different regarding cytoplasmic contents
mechanisms of cell specification: cell-cell interactions
cell fate determined bu interactions with neighbouring cells
- back cells in blastula form back tissue
- transplant these back cells into belly regions
- these cells then go on to form belly tissue
mechanisms of cell specification: morphogen gradients
bicoid and nanos
diagram of bicoid, nanos, hunchback and caudal
morphogen gradients are typically TF’s that are “master gene regulators” i.e. determine whether it is head or tail region of animal
example: drosophila embryos
- bicoid = head and nanos = tail
- mother deposits mRNA in unfertilised egg
- egg already polarised
- mRNA localised at poles of egg
- egg fertilised, DNA replicated with no cell membranes —> gradients of cell specificity factors
- exposure to amount of factors determines cell fate
bicoid and nanos are tranlational inhibitors and transcription factors
inhibity translation of two other mRNAs involved in early patterning of embryo, hunchback and caudal
hunchback and caudal not localised
bicoid binds to caudal and prevents it being made into protein (same re. nanos and hunchback)
transcriptional regulation: molecular level
Dna contains elements that allow for cell specific gene regulation
TFs - proteins that promote or inhibit gene expression
enhancers - TF binding sites
histones - proteins that package DNA and can be modified to regulate gene expression. positively charged tails interact with negative phosphate backbone of DNA
epigenetic marks - histone acetylation and methylations as well as DNA methylation. modify histones to allow DNA translation
acetyl and methyltransferases - enzymes that add epigenetic marks to histones and DNA
in general, modifications that decrease histone tail interactions with DNA open up the chromatin
acetylated histones –> open chromatin, transcriptionally active
deacetylated histones –> close chromatin, transcriptionally silent
terminal differentiation
terminal differentiation pathway
permanent cell cycle exit
differentiated cells become refractory to proliferative signals (respond in different way)
G1/S CDK inhibitor and Rb family members play major role in cell cycle exit
terminal differentiation distinct from senescence
cell fate determining TFs *
- —-> G1/S CDK inhibitors
- —-| substrates for cycling CDKs become dephosphorylated (less phosphorylated Rb)
- —-> Rb recruits epigenetic modifying complexes that permanently repress E2F transcriptional activity
- cell fate inhibits cdc25 and activates eigenetic regulators. Rb recruits epigenetic regulators to locus in genome and mark histones and DNA to permanently silence it
