Molecular Bio L17+18

Treatment of cells with colchicine or vinblastine (microtubule destabilizing drugs) or with taxol (microtubule stabilizing drug) leads to a

•mitotic arrest. Cdk activity is high, cyclin B and securin are stable.

•Screen for mutants that overcome this arrest:

Spindle assembly checkpoint genes -

-Mad2 and others.

-Mad2 mutants enter anaphase prematurely, before all kinetochores have been attached to spindles.

Mad2/APC double mutants behave like

-APC mutants - arrest at metaphase. Therefore, Mad2 and the SAC function genetically upstream of the APC.

The APC is kept inactive until all kinetochores have made stable connections to

spindle microtubules from both poles

A complex of proteins, the Spindle Assembly Checkpoint (SAC) is active at unoccupied kinetochores (that are not attached to

•spindle microtubules)

The SAC component Mad2 is activated at these kinetochores and diffuses away and binds to

•Cdc20

Mad2 inhibits the activity of the APC/Cdc20 complex, preventing

•cyclin B and securin destruction.

Kinetochore / spindle attachment inactivates the SAC, leading to

•APC activation.

•Mad1 is recruited to unattached kinetochores.

Mad2 binds to Mad1

•Mad2 changes conformation (activated), released.

•Activated Mad2 binds and inhibits APC/Cdc20

MAD1 associates with unoccupied

kinetochores and brings MAD2

In prometaphase, some chromosomes are properly attached while others are still unattached or have monotelic attachments

Lack of microtubule attachment to these kinetochores -

•Mad1 binds, Mad2 binds, activated.

•Active Mad2 diffuses from these kinetochores to inactivate the APC/C-Cdc20.

In metaphase, all chromosomes are attached properly. This leads to inactivation of the SAC, leading to APC/C activation.

In anaphase the APC/C destroys cyclin B and securin.

securin destruction leads to loss of

•sister chromatid cohesion.

•Cyclin B destruction leads to inactivation of Cdk1, which leads changes in spindle behaviour that allow completion of anaphase.

Chromosome segregation in

anaphase, shortening of kinetochore microtubules and movement of daughter chromosomes to poles is an immediate effect of APC

the exposed plus end of a kinetochore wants to be polymerized but that can only happen in

anaphase

CDK1 prevents the transition into

Anaphase B

Anaphase B is when 1) a sliding force is generated between interpolar microtubules from opposite poles to push the poles apart; the interpolar microtubules also

elongate; 2) a pulling force acts directly on the poles to move them apart, causes further separation of chromosomes and further separation as it lengthens

Anaphase, •Initiated by APC/C mediated destruction of Securin and CycB

Anaphase A - Securin destruction

-> loss of

•cohesion - pulling forces of kinetochore microtubules partially separates sister chromatids

Anaphase B - CycB destruction -> lengthening of spindle to further

separate sister chromatids

Cytokinesis, the cell mb is being pulled into

a tighter ring

RhoA is essential for the assembly of the

actin/myosin contractile ring

RhoA - activated near central spindle (interpolar microtubules)

Active RhoA

associates with cell membrane near

•central spindle

•stimulates myosin II activation and actin filament formation at that site

RhoA recruits actin and myosin to the

contractile ring

Latrunculin A - depolymerization of actin. Rho A accumulation is

not affected, but MHC is.

RNAi knockdown of

MHC.

RhoA still accumulates, shows that myosin depends on RhoA, and that RhoA does not depend on it

ECT2,the RhoA

GEF

•ECT2 localizes to the central spindle (spindle midzone)

ECT2 is necessary for

contractile ring accumulation of actin and myosin

ECT2 RNAi cells fail to

undergo cytokinesis

ECT2 is necessary for RhoA accumulation at the

contractile ring.

ECT2 previously found to interact with a

spindle midzone protein, CYK-4.

CYK-4 is necessary for cytokinesis and for localization of

MHC, actin, ECT2 and RhoA.

Model for mitotic spindle dependent contractile ring assembly, CYK-4 associates with the

mitotic central spindle

•CYK-4 recruits ECT2

CYK-4 interaction activates

•RhoA-GEF activity of ECT2.

RhoA-GDP exchange for GTP activates

•RhoA.

•RhoA recruits actin/myosin.

A) Cells synchronized in metaphase at time 0

0-40: Metaphase

60-120: Anaphase, G1, ECT2 immunoprecipitates CYK-4 in

anaphase.

Phosphorylation of ECT2 (pTP) correlates with inability to bind.

CYK 4 can interact in anaphase, lost of phosphorylation of Ect 2 and kinase going away allows

binding

ECT2-pTP is used as the antibody to detect as it as

ECT2 can coprecipitate CDK4

Metaphase arrest cells

Add Roscovitine - inhibits Cdk1 activity -> allows

CYK-4 to bind ECT2

Therefore Cdk1 phosphorylation of ECT2 prevents recruitment of ECT2 to the

spindle midzone. Anaphase cyclin destruction permits ECT2 recruitment.

ECT2 IP in metaphase, no CYK4,

does not recruit in metaphase,

inhibits CDK activity, which allows interactyion to occur, is

no longer phosphorylated and can bind, therefore CDK activity keeps interactions at bay

•CYK-4 associates with the mitotic central spindle

During mitosis, CYK-4 is unable to recruit ECT2 because of

•Cdk1 phosphorylation of a site on ECT2.

•APC inactivation of Cdk1 results in dephosphorylation of ECT2 allowing it to interact with CYK-4 at the central spindle.

RhoGEF activity of ECT2 on central spindle leads to

•RhoA activation on overlying cell membrane

-RhoAGDP exchange for GTP activates RhoA.

•RhoA recruits actin/myosin.

BrdU Incorporation

Bromo-deoxyUridine (BrdU) is an analogue of dTTP.

Incubation of cells/tissues in BrdU results in its incorporation into DNA during

S-phase.

Cells are then fixed and BrdU is detected with anti-BrdU antibodies followed by a fluorescent 2ndary antibody.

G1: Cdk4/cyclinD (inhibited by

INK4)

G1/S: Cdk2/cyclin E (inhibited by

p27,p21)

S: Cdk2/cyclin A (inhibited by

•p27,p21)

These Cdks are activated in sequence to allow S-phase to occur.

G2/M -

•Cdk1/cyclin B

-

-cyclin B and cyclin A are both targets of APC/C

G1 phase = low

Cdk activity

APC/C-Cdc20 inactivated after anaphase

APC/C-Cdh1 becomes

active

Also: cyclin transcription is low and Cdk inhibitors present

-> low Cdk activity è G1 (stable state)

Cdk 1 with cyclin B and Cdc20-APC activity is

regulated in opposite wats from phosphorylation of APC

In G2 the APC is no longer

Phosphorylated, meaning its no longer active

•APC/CCdc20

activated by

-Cdk1 (in mitosis)

-inactivated in late anaphase when Cdk activity drops

•APC/CCdh1

-inactivated by Cdk1

active from late anaphase to

-S-phase (low Cdk activity)

•keeps Cdk activity low in G1

-inactivated in S-phase, G2 and M-phase (due to Cdk activity)

Cdk inhibitors bind and inactivate

Cdks

p27 (binds and inhibits any CDk-cyclin complex) and p21 inhibit

Cdk2-cyclinE and Cdk2-cyclinA

INK4 inhibits

Cdk4-cyclinD

ORC, Cdc6, Cdt1 and Mcm helicase =

= prereplication complex

G1 - low Cdk activity

•Cdc6 and Cdt1 bind to ORC

Cdc6/Cdt1 recruit

•Mcm helicase

= PreReplication Complex (PreRC)

•PreRC remains "licensed" throughout G1

Activity of Cdk2-cyclin A triggers

replication at origins, triggers prereplication to replication complex

•After completion of cytokinesis the two daughter cells are in G1.

APC/C-Cdc20 is now inactive (activity depends on

•Cdk1 activity)., needs to be phosphorylated

•APC/C-Cdh1 is active (activity depends on absence of Cdk1 activity).

APC/C-Cdh1 and Cdk inhibitors keep

•Cdk activity low.

-APC/C-Cdh1 ubiquitinates cyclins (A and B) à degraded

p21 and p27 bind and inhibit

-Cdk2/cyclin A and Cdk2/cyclin E

-INK4 binds and inhibits Cdk4/cyclin D

-Also, cyclin transcription is low

Low Cdk activity -> pre-RC forms on origins of

•replication

•Cells that are terminally differentiated (most cells in our body) arrest (sometimes) permanently in a G1-like state - referred to as G0

•All cells have some mechanism of coupling cell division and growth.

In unicellular organisms the main determinant of rate of division is

•nutrient availability for that cell.

•In multicellular organisms the rate of cell division is largely determined by extracellular signals, typically from neighboring cells. Extracellular signals also control the rate of growth.

In most cells, the decision of when and if to divide is made in

•G1 of the cell cycle

•Entry into S-phase involves sequential activation of G1-Cdks -> G1/S-Cdks -> S-Cdks

•In multicellular organisms both growth and the cell cycle are highly regulated.

•Extracellular signals promote growth and cell division.

Extracellular regulators of the cell cycle are called

mitogens

Extracellular regulators of growth are called

growth factors

Extracellular regulators of survival are called

survival factors

•The effect of a given extracellular signal depends on the cell receiving it.

Many mitogens are also

•growth factors.

Many mitogens such as PDGF and EGF activate the

Ras/MAPK pathway.

Receptor binding ->

transphosphorylation

Grb2 - SH2 domain protein recruited to

receptor

Grb2 binds

Sos (GEF for Ras), converts it to GTP bound state

Ras-GTP binds to Raf and

activates it

Raf phosphorylates

MEK

MEK phosphorylates

MAPK

MAPK enters

nucleus

receptor kinase activity only stimulated when

ligand binds

myc and fos promotes cyclin

D and inhibits INK 4

MAPK phosphorylates and activates

transcription factors

Transcription factors promote expression of

myc and fos

myc and fos are transcription factors

myc and fos promote cyclin D transcription

and repress INK4 transcription

Accumulation of cyclin D and reduction of INK4 leads to

activation of Cdk4-cyclin D

Ras/Map kinase pathway, •activated downstream of many RTKs

the same RTK may also stimulate

TOR pathway

•RTK activation leads to recruitment of SH2-domain protein Grb2.

Grb2 recruits

Sos (the Ras GEF)

•Ras-GTP activates the first kinase in the MAPK pathway

-Raf -> MEK -> MAPK

MAPK phosphorylates transcription factors to

•activate them

-myc and fos promote cell division (and growth)

•ras, raf, fos, myc all discovered as viral oncogenes

Myc and fos promote

G1-Cdk activity

•Mitogen signaling leads to transcription of myc and fos.

myc and fos induce transcription of

•cyclin D as well as genes required for growth.

•and repress INK4 transcription

Mitogens and Cdk4/cyclin D inhibit p27, helping to relieve inhibition of

Cdk2/cyclin E

In G0/G1 cyclin E levels are low and all Cdk2-cyclin E complexes are inhibited by

•p27 or p21 binding.

•Cyclin D levels increase in response to mitogens.

Cdk4-cyclin D binds

•p27

-p27 does not inhibit Cdk4/cyclin D

Cdk4-cyclin D sequesters p27, allowing low level activation of

-Cdk2-cyclin E

•Cdk2-cyclin E phosphorylation of p27.

Phosphorylated p27 is recognized by

-SCF ubiquitin ligase -> targeted for destruction.

G1-Cdk activity promotes

G1/S

Cdk4-cyclin D and Cdk2-cyclin E phosphorylate Rb to

•inactivate it, thus activating E2F1

E2F1 drives high level expression of

cyclins A and E (and other S-phase genes).

An active Rb protein is deactivated by

active G1-CDK and E2F phosphorylating it (requires 2 phosphates)

E2F forms a postive feedback loop, promoting

its own transcription

This inactivation of Rb and activations of G1/S cyclin (E) and S cyclin (A) causes

active S-CDK leading to DNA synthesis

•E2F and other activator E2Fs are responsible for a rapid increase in levels of G1/S and S-cyclins.

E2F1 also promotes transcription of other

•S-phase genes.

•In G0 and G1, E2F1 is kept inactive by binding to Rb.