BIOL 2200 mid sem

Interphase

Chromosome duplication and cohesion; centrosome duplication

Prophase

Interphase microtubule display breakdown and replacement by mitotic asters; mitotic aster separation; chromosome condensation

Prometaphase

Nuclear envelope breakdown; chromosomes captured, bi-orientated, and moved to spindle equator

Metaphase

Chromosomes align at metaphase plate

Anaphase

Anaphase Promoting Complex (APC) activated to degrade cohesin

Anaphase A

Chromosomes move to poles

Anaphase B

Spindle pole separation

Telophase

Nuclear envelope reassembly; contractile ring assembled

Cytokinesis

Interphase microtubule array reformed; contractile ring forms cleavage furrow; cell pinched apart

G1

Recovery from mitosis; preparatory growth for S-Phase

S

Chromosome and centrosome duplication

G2

Pre-mitotic checkpoints

M

Chromosome segregation; cell division

G0

Temporary OR permanent exit from the cell cycle

FGF10

Growth factor for leg development. If mutated, no leg development

Chromosome segregation

Duplicated chromosomes align independently at the metaphase plate, each pair of identical sister chromatids separate, each daughter cell (2x) receives identical genetic information

Chromosomes

dsDNA and protein

Chromatin

DNA and histones

Chromatid

Single copy of a chromosome only visible after S-Phase completed and chromosome duplication has occured

Sister chromatids

Identical copies of a chromosome attached via a centromere

Cohesin

Rubber-band-like protein that hold sister chromatids together

Centromere

Repetitive DNA sequence that acts as a landing site for mitotic machinery, i.e. kinetochores

Kinetochore

Protein complex that links centromere to microtubules to allow movement

Chromosome movement

Occurs post-chromosome-condensation, and is driven by microtubule polymerisation / depolymerisation and molecular motors

Kinesin-7

Molecular motor protein that attaches to kinetochore and walks along microtubule

Centrosome

Microtubule-based structures orientated at 90 degrees from one another. Made up of 1 daughter centrosome and 1 mother centrosome. Nucleating center for microtubules

Three functions of microtubules

1. Capture chromosomes
2. Anchor to plasma membrane to aid force generation
3. Contain motor proteins that orientate microtubule array and generate tension

Meiosis vs. mitosis

1. Meiosis \= germ line cells
Mitosis \= somatic cells

2. Meiosis \= n oscillates between 2 (before duplication) and 4 (after duplication)
Mitosis \= n \= 2

3. Meiosis \= sexual reproduction (Aa x Aa)
Mitosis \= asexual reproduction (Aa --\> Aa)

4. Meiosis \= 4x genetically different daughter cells
Mitosis \= 2x genetically identical daughter cells

M1

Homologous chromosomes pair; recombination between chromosomes; homologous chromosome separation occurs diagonally, sister chromatids remain intact; each daughter cell gets different chromosomes, both 2n containing either paternal or maternal copy of each chromosome

M2

Sister chromatid separation occurs down the middle; 4x 1n (haploid) gametes form

A duplicated chromosome has...

2x centromeres, 2x kinetochores (1 for each sister chromatid)

Negative control

Variable being tested is removed

Positive control

Variable being tested is observed in an established system where the effect is already known

Sham control

All experimental conditions replicated identically but therapeutic step excluded; a type of negative control

Internal control

Ran simultaneously with the experimental in the same mixture; the best kind of negative control

Kinase

Uses ATP to add phosphate to a substrate containing an R-OH group: serine, threonine, or tyrosine

Phosphatase

Uses H20 to remove phosphate from a substrate; opposite to kinase

Phosphorylation

Adding phosphate to a protein to alter its activity

Cyclin CDK complex is...

heterodimeric, requires 2x proteins bound together for it to be activated

Cyclin

Regulatory subunit; regulated in a cell-phase-specific fashion; determines specificity

CDK

Catalytic subunit; always present

Names of major kinase classes

1. G1
2. G1/S
3. S
4. G2/M
5. M

Describe a biochemical assay that can be used to detect kinase activity in-vitro

1. Place the following into a test tube: active enzyme \-- cyclin bound to CDK; substrate \-- target for phosphorylation, ATP

2. Create cell lysate by breaking open cells with known cyclin-CDK activity

3. Isolate the complex with antibodies specific to cyclin or CDK to bring down complex and associated factors

4. Add beads coupled to the bacterial protein G \-- heavy, easily purified, and binds to constant region of the antibody

5. Add substrate, e.g. histone H1 and ATP

6. Look for phosphate transfer to substrate

7. Visualise on SDS page gel \-- separates protein by size

8. Label ATP with radioactive isotope \-- when gamma phosphate transferred to protein, can be visualised on gel

PC12 cells

Actively dividing; differentiate into neural cells if neural growth factor is present

p27

Inhibitor of S-CDK, prevents entry into S-Phase in mammals

sic1

Inhibitor of S-CDK, prevents entry into S-Phase in yeast

Polyubiquitination

Addition of ubiquitin onto lysine in a target protein to target it for degradation

E1/E2

Prepares ubiquitin for conjugation to lysine on target protein

E3

Recognises substrate to allow lysine transfer; causes lengthening, which is the recognition signal for proteasome degradation

Proteasome made up of...

19S cap; 20S core

19S cap

Landing site for ubiquitin-conjugated proteins

20S core

Contain peptidases which degrade ubiquitin-conjugated proteins loaded into the 19S cap

Phosphorylation vs. Ubiquitination

Phosphorylation \= temporary, reversible ("molecular switch")
Ubiquitination \= permanent, irreversible (destruction)

SCF Complex

Skp1 + Cullin + F-Box; a ubiquitin-protein ligase that allows transition from G1 to S by degrading S-CDK inhibitor

APC

Anaphase Promoting Complex; a ubiquitin-protein ligase involved in the transition of metaphase to anaphase by degrading securin

G1/S Transition

Involves 3x CDK: G1-CDK, G1/S-CDK, S-CDK

Inhibitor bound to S-CDK to inactivate it and prevent entry to S-phase until cell is ready.

G1/S-CDK phosphorylates S-CDK inhibitor to target it for polyubiquitination by a ubiquitin ligase; poor substrate, requires a lot of phosphorylation for degradation to occur

ABRUPT to maintain fidelity

S-CDKs...

Promote DNA replication; prepare cell for mitosis

G1/S-CDKs...

Prepare cell for S-Phase by inducing expression of DNA replication genes

Describe an experiment used to elucidate how chromosome condensation occurs

1. Fuse mitotic and G1 phase cells and see which activity is dominant.

2. Isolate them either by:
(A) SIZE \-- mitotic cells will always be bigger than G1 cells
(B) DRUGS \-- inhibit S-Phase to arrest cells @ G1/S transition or use a microtubule inhibitor to arrest cells @ G2/M boundary

3. Found that both chromosomes condensed, therefore there must be a dominant factor in the cytoplasm of mitotic cells that induces chromosome condensation \-- MPF

Components of cell cycle progression were first identified in...

S. Pombe (yeast)

How would you screen for cell cycle genes?

Use temperature-sensitive (TS) mutants:
@ permissive temp: protein \= fine
@ non-permissive temp: protein \= non-functional due to misfolding

CDC2+ phenotype is ...

Normal

Because CDC2 is a CDK required to form activated MPF and enter mitosis

In the normal amounts, time delay is correct and an appropriate amount of pre-mitotic growth can occur prior to entry into mitosis

CDC2d phenotype is ...

Wee (short cell)

Because CDC2 is a CDK required to form activated MPF and enter mitosis

If excess, early entry into mitosis, insufficient time delay to allow for adequate growth so cells are SHORT

CDC2- phenotype is ...

Long cell

Because CDC2 is a CDK required to form activated MPF and enter mitosis

If there is less, late entry into mitosis, increased time delay allowing overgrowth so cells are LONG

CDC13+ phenotype is ...

Normal

Because CDC13 forms heterodimers with CDC2; is required to form activated MPF and enter mitosis

In the normal amounts, time delay is correct and an appropriate amount of pre-mitotic growth can occur prior to entry into mitosis

CDC13- phenotype is ...

Long cell

Because CDC13 forms heterodimers with CDC2; is required to form activated MPF and enter mitosis

If there is less, late entry into mitosis, increased time delay allowing overgrowth so cells are LONG

Loss of function in CDC25 would cause ...

Long cell

Because CDC25 drives entry into mitosis; is an activating step

Loss of function in wee1 would cause ...

Wee (short cell)

Because wee1 blocks entry into mitosis; is an inhibitory step

CDC25

Phosphatase that removes phosphate from phosphorylated Y15 on inactivated MPF; activating step that allows formation of active MPF

Wee1

Kinase that phosphorylates Y15 on inactivated MPF; inhibiting step

CAK

Phosphorylates T161 on inactivated MPF; activating phosphorylation

G2/M Checkpoint

1. Mitotic cyclin and CDK bind to form inactivated MPF

2. Wee1 phosphorylates Y15 domain on CDK (inhibitory phosphorylation)

2. CAK phosphorylates T161 domain on CDK (activating phosphorylation)

3. ATR1 detects ssDNA, if activated, CHK1 inhibits CDC25.

4. If inactivated and no ssDNA), CDC25 removes phosphate from phosphorylated Y15 domain (activating step)

5. Active MPF formed, G2/M transition occurs

Lamin

Part of the nuclear envelope; tetramer of 2 dimers bound together, forms filaments that make up lattice structure

MPF and lamin

MPF phosphorylates lamin after G2/M transition; prevents formation of tetramer structure and nuclear envelope dissolves

CDC20

Specificity factor that binds to APC; involved in metaphase to anaphase transition; allows sister chromatid separation

1. APC binds to CDC20
2. Securin ubiquitinated, separase activated
3. Separase cleaves scc from cohesin
4. Sister chromatids separate

CDH1

Specificity factor that binds to APC; involved in exit of mitosis; allows degradation of mitotic cyclin

Degradation of mitotic cyclin allows chromosome de-condensation and microtubule re-organisation required for re-entry into G1

Metaphase / Anaphase Transition

1. Cohesins loaded onto sister chromatids prior to duplication in G1.
2. During mitosis, distal cohesins removed by phosphorylation via kinases
3. Innermost cohesins proximal to kinetochores which are associated with phosphatase PP2A, prevent cohesin removal via phosphorylation.
4. APC binds to specificity factor CDC20
5. APC/CDC20 dooms securin, activating separase
6. Separase cleaves scc on innermost cohesin, allowing sister chromatid separation

PP2A

Phosphatase associated with kinetochores that prevents phosphorylation of proximal cohesin structures binding sister chromatids together

Necrosis

Unplanned cell death; bad for proximal cells due to release of toxic cytoplasmic contents e.g. acidic lysosomes

Apoptosis

Programmed cell death; first discovered in C. elegans; cells are compartmentalized into vesicles and marked for phagocytosis by macrophages of the immune system

Apoptosis Signalling Cascade

1. Apoptosis signal
2. EGL1 produced
3. EGL1 binds to CED-9
4. CED-4 dimer released from CED-9
5. CED-4 forms octamers in the cytoplasm
6. CED-4 octamers convert inactive pre-cursor CED-3 to active CED-3 (caspase holoenzyme)
7. Destruction of proteins and cell death
e.g. lamins, cytoskeleton adhesion proteins, etc.

Goal of cancer

Avoid apoptosis (cell death)

Cancer

Disease of cell proliferation; induces uncontrollable division due to removal / modification of cell cycle regulatory mechanisms

Cellular changes contributing to cancer development

1. Sustaining proliferative signalling \-- activate mitogen pathways in the absence of mitogens

2. Evading growth suppressors \-- e.g. Sic1 inhibitor in G1/S boundary; CDC25 in ATR pathway in G2/M boundary

3. Activating invasion / metastasis \-- translocation from primary growth location and onset of secondary growth

4. Increase nutrient supply via angiogenesis / other mutations

5. Resisting cell death \-- disabling death pathways e.g. apoptosis

Mitogens

Growth factors from external environment that promote cell proliferation; if absent, entry into G0

Metastasis

Translocation from primary growth location and onset of secondary growth

Angiogenesis

Formation of new blood vessels to increase nutrient supply

Proto-onco Genes

Normally promote cell growth
When mutated, converted to "oncogene"
Usually gain of function
E.g. ras, myc, src

Tumour-suppressor genes

Normally inhibit cell growth
Can be spontaneous or environmentally induced
Usually loss of function
E.g. p27, p53, Rb, ATR

Compare / contrast proto-onco and tumour-suppressor genes

1. Proto-onco normally PROMOTE cell growth, tumour-suppressor normally INHIBIT cell growth

2. Proto-onco normally GAIN of function, tumour-suppressor normally LOSS of function

3. Examples of pro-onco are ras, myc, src and examples of tumour-suppressor are p27, p53, Rb and ATR

4. Proto-onco genes need to be converted to oncogene upon mutation; tumour-suppressor genes mutated more traditionally either by environmental or spontaneous causes

Multi-Hit Hypothesis

Need combination of multiple mutations to progress toward a malignant tumour; need to be UNIQUE, interfering with DIFFERENT PATHWAYS, and confer a UNIQUE GROWTH ADVANTAGE

Adenoma Polyposis Coli (APC) Protein

Inhibits wnt signalling to create cells with more myc, causes loss of regulatory control

RAS / MAPK

Normally promote growth; with a gain of function mutation, becomes independent of GF's

p53

Prevents over-proliferation by arresting the cell cycle, senescence, apoptosis

Activated by stress e.g. DNA damage, shortened telomeres, hypoxia, etc.

Active form is a tetramer of p53 sub-unit, therefore highly susceptible to cancer \-- knockout 1 sub-unit to lose function completely

Major target for cancer: delete or inactivate ATR, overexpress or amplify Mdm2, delete or inactivate p53

ATR

Inhibits Mdm2; deleted or inactivated in cancer

Mdm2

Inhibits p53; over-expressed or amplified in cancer

Restriction Point

Once reached, cell is committed to another round of cell division and becomes mitogen-insensitive

G1 Restriction Point

Major target for cancer: delete / inactivate p16, overexpress / amplify G1-CDK, delete / inactivate Rb

Rb

Transcriptional repressor of E2F

E2F

Transcription factor that induces gene expression for cell cycle progression e.g. S-Phase cyclin