Professional Documents
Culture Documents
Hui Zhang, Ph. D.
Department of Genetics
Yale University School of Medicine
New Haven, CT 06520, USA
Telephone: (203)7371922
Fax: (203)7857023
Email: hui.zhang@yale.edu
http://info.med.yale.edu/genetics/fac/HuiZhang.php
Cell Cycle Regulation:
the mechanisms controlling cell growth, cell duplication,
cell division, and their roles in development and diseases
Multicellular organisms
cells in an onion root
QuickTimeª and a
GIF decompressor
are needed to see this picture.
Involving complex programs to coordinate proliferation, differentiation,
and functional specialization of various cells in an organism
Cell cycle under microscope
telophase
interphase metaphase
prophase anaphase
Life Cycles
Mitosis
Reproduction
systems
Somatic cell cycle can be divided
into G1, S, G2, M phases
Mitosis (M): chromosome condensation
Gap1 Gap2
(G1) (G2)
DNA synthesis (S):
3
Hthymidine incorporation
Embryonic cell division
Many embryonic cell divisions are rapid (1015 min)
Fertilized eggs from Xenopus, Drosophila, sea urchin, clam, etc.
Alternating S phase (S) and mitosis (M).
Uses maternally stored proteins for mitotic divisions.
Growth factor
signaling
Cell Cycle Regulation/Differentiation
Developmental cell divisions
cells divide asymmetrically
Zygote (fertilized egg)
Mitosis
Continued cell divisions with different cell fate
Nerve, muscle, blood, endocrine, epithelial, stem cells, etc.
How cell cycle regulation
can be studied?
Different systems offer various ways to
identify and isolate cell cycle regulators
Cultured mammalian cells (Rao and Johnson)
Budding yeast (Saccharomyces cerevisiaeLeland Hartwell)
Fission yeast (Schizosaccharomyces pombePaul Nurse)
Frog Oocytes (Xenopus laevis, Rana pipiensYoshio Matsui)
Sea urchin/clam eggs (Joan Ruderman and Tim Hunt)
Xenopus Oocyte Maturation and Activation
Maturation Activation
(Meiosis) (Mitosis)
Assay for MPF (Yoshio Masui)
Maturation Promoting (MPF) Factor is
Transferable and Autocatalytically propagated
MPF is an universal regulator of
mitosis and meiosis
MPF from mitotic embryonic or somatic cells
and mature eggs behave the same.
Properties of MPF:
promotes oocyte maturation
common in all mitotic or meiotic cells from
different sources
activity oscillates in the cell cycle, low in
interphase and high in mitotic/meiotic cells
Nobel Prize Winners
for Cell Cycle Regulation
in Physiology or Medicine, 2001
Isolated various conditional
budding yeast mutants, mostly
temperature sensitive (ts) mutants that
arrest the cell cycle with growth or
morphological defects
Isolate yeast conditional mutants
High temperature mutants: Grow at 25 oC but not at 37 oC
Cultured yeast cells
Mutagenize with EMS or other chemicals
Classify
mutant
Replica plating
terminal
phenotypes:
small bud,
dumbbell,
Grow at permissive nongrowing mutants at no DNA
25 oC 37 oC. synthesis,
(permissive temperature)
(nonpermissive temperature)
etc.
Isolation of cdc mutants
cdc mutants arrest with a single cell morphology
at a defined cell cycle stage
Cdc mutants in budding yeast
G1 (Start) mutants: cdc28, cdc4, cdc6, cdc7, etc.
S phase mutants: cdc21, cdc46, etc.
Mitosis: cdc5, cdc14, cdc15, etc.
Isolate the genes encoding
yeast cdc mutants
Yeast genomic DNA in a plasmid library (yeast selection and E. coli
drug resistant markers)
Transfromation (Li acetate, etc)
Introduce into interested cdc mutant yeast cells
37 oC
Isolate
plasmids
Grow at permissive temperature
(25
oC) and yeast selection media
25 oC
Paul Nurse: isolate cdc mutants from distant fission yeast
Schizosaccharomyces pombe.
S. pombe has
a short G1 but a long G2
Cdc mutants arrest in
G2/M were isolated:
cdc2, cdc25, cdc13, etc.
The S. pombe cdc2 mutant
G2 cells Mitosis Cell division
Cdc2 mutant is defective in both G1 (start) and G2 phases
cdc2ts
cdc2ts CDC2 plasmid
WildType cdc2ts Cells
Cells cdc2ts Cells + Human CDC2 plasmid
S. Pombe CDC2 encodes an evolutionarily
conserved serine/threonin kinase
the fission yeast CDC2 gene by recovering the gene
that rescues the cdc2 G2/M arrest phenotypes.
S. pombe cdc2 mutant can be rescued by introducing
budding yeast cdc28 (S. cerevisiae) which is primarily
defective in G1 (Start) in budding yeast.
human CDC2 was isolated using the same rescue strategy.
CDC2=CDC28 and regulates both G1 and G2/M.
CDC2 is part of MPF activity
Xenopus MPF Inject Oocyte Oocyte matuation
(meiosis)
Absorb by p13SUC1 beads
Bound p34 = CDC2; another protein ~ 5060 kDa?
(yeast p13SUC1
binds yeast and human CDC2)
Discovery of cyclins (Tim Hunt)
sperm
MPF activity fluctuates; cyclin proteins fluctuate
MPF has a kinase activity
MPF contains CDC2 and another component
MPF= cyclin B (cyclin A) + CDC2
Cyclin dependent kinases
(CDKs)
Replication
Cyclin A/B Chromatin condensation
Nuclear envelop breakdown
CDC2
Mitotic spindle formation
Active CDC2 Kinase Activation of Anaphase Promoting
Complex/Cyclosome (APC/C)
Chromosome cohesion, etc.
Proteolysis of cyclin A and cyclin B in late mitosis
inactivates CDC2 and causes the exit of mitosis.
Wee1 and CDC25
Cell size
A. wildtype S. pombe
B. cdc25, cdc13, cdc2 loss of function mutants
C. wee1 and certain gain of function cdc2 mutants
CDC13
CDC25
Inactive CDC2 Active CDC2/CDC13
WEE1
CDC13=cyclin B
Y=tyrosine; T=threonine
CDC25 and WEE1 couples CDC2
activation to S phase in S. pombe
WEE1 kinase
Y15PO4 Y15
CDC25 pptase Cells divide only
WT CDC2 CDC2 in mitosis
Inactive CDC2 Active CDC2
S phase cells G2 and mitotic cells
CDC25 is regulated by phosphorylation and protein degradation
Budding yeast S. cerevisiae
CDC28 regulates G1 and G2
but most mutants arrest before G1 start
CDC28
G1 G2/M
S
How?
Budding yeast S. cerevisiae
CDC28 regulates G1, S, and G2/M by synthesize
distinct cyclins in each phase of the cell cycle
CDC28
G1 G2/M
S
Cln1 Clb5 Clb1
Cln2 Clb6 Clb2
Cln3 Clb3
G1 progression S phase progression Clb4
Nutrient & cell S phase checkpoint G2 and mitosis
size
a/α factor
Yeast G1 cell cycle regulation
α/a mating factors
Cln1 Nutrients (carbon, nitrogen, lipid, etc.)
Cln2 Expression:
G1 G1/S S G2/M
Cln3 constant
Clb56
Cln13 accumulation leads to the progression of G1 phase
Overexpression of Cln3 promotes S phase entry at smaller size
Clb56 starts to synthesize at late G1
How Sphase starts in yeast
α/a mating factors
Cln1 Nutrients (carbon, nitrogen, lipid, etc.)
Cln2 Expression:
G1 G1/S S
Cln3 constant
Clb56
p40Sic1 cdc28
Clb56
p40Sic1 is a CDK inhibitor
Inactive Clb56/CDC28 kinase
How Sphase starts in yeast
α/a mating factors
Cln1
Cln2 Expression:
G1 G1/S S
Cln3 constant
Clb56
p40Sic1 cdc28
PO4 p40Sic1
Clb56 PO4
SCFCDC4
SCF Ubiquitin E3 Ligase
(SKP1, CUL1/CDC53, Fbox proteins)
ub ub
E1 & E2 enzymes ub ub
Ubiquitin(ub) ub ub
ub
26S proteosome
Substrate
proteolysis
> 10 Fbox proteins found in yeast
26S proteasome proteolyzes
polyubiquitinated proteins
Clb56/CDC28 in S phase
Cdc28
For S
Phosphorylate Cln1/Cln2 Clb14 expression
Regulate replication Other events for G2/M
SCF binding initiation at origins
Degradation of Cln1/Cln2
Turnoff G1 events