1

Exam notes

Means are 76, 70 and ? Mean of means is

Approximate grades after 3 exams

• Screens for anti-cancer agents have identified a variety of naturaland synthetic products that disrupt MT assembly or function

• Many drugs/treatments disassemble MTs (block assembly)

– Examples with medical relevance:

• Vinblastine/vincristine… some leukemias… from lilly family

• Podophyllotoxin… warts

• Griseofulvin…anti-fungal

• Drugs/agents that stabilize MTs (promote assembly)

– Taxol… ovarian cancer

– from bark of Western Yew

Testing the role of MTs using pharmacological agents -“inhibitors”

Taxol stabilizes MTs and prevents cell division

2

MTs provide a scaffold for organizing the ER and Golgi

ECB 17-23

Green = MTsRed = GolgiYel = overlap

Green = MTsRed = ERYel = overlap

Centrosome

ER

MTs

Golgi

17.3-microtubule_ER.mov

Disassembly of MTs with drugs fragments ERand Golgi

MBoC (4) figure 16-62

Red = MTsGrn = Golgi

Nocodazole

Control

MTs are used for vesicle transport in some cells: Fast axonal transport

Cell body (“soma”) AxonNerve terminal

(“synapse”)

Outward (“anterograde”) transport

Inward (“retrograde”) transport

ECB 17-15

“+”“-”

Nucleus

MTs oriented with plus-ends “distal” (towards synapse)…

*

Microtubules

Kinesin motors power “anterograde” transport (to synapse)Use ATP hydrolysis to walk towards plus-endNumerous kinesin-related proteins

3

Kinesin uses ATP hydrolysis to “walk” towards the “plus-end” of MTs

Similar to myosin II, may have common evolutionary origin

But movement of two heads of kinesin are coordinated, unlike myosin II

The kinesin family: Motors for vesicle transport

ECB 17-17

2x Light chains

2 x Heavy chains

N-terminalmotor domains

Kinesin

bind cargo

Transport vesicle

(vesicles notto scale)

Minus-end

Plus-end

17.5-kinesin.mov

MTs are used for vesicle transport: Fast axonal transport

Cell body (“soma”) AxonNerve terminal

(“synapse”)

“Anterograde” transport

“Retrograde” transport

See ECB figure 16-14

“+”“-”

Nucleus*

***

*Microtubules

MTs oriented with plus-ends “distal” (towards synapse)

Kinesin powers “anterograde” transport (to synapse)

Cytoplasmic dynein powers “retrograde” transport (to cell body)Uses ATP hydrolysis to walk towards minus-end

Cytoplasmic dynein: a minus-end motor for vesicle transport

See ECB figure 16-16

(vesicles notto scale)

2x Light chains - bind cargo

2 x Heavy chains

N-terminalmotor

domains

Kinesin

Minus-end

Plus-end

Cytoplasmicdynein

2 x heavy chainsMultiple light andintermediate chains

Dynactincomplex

Transport vesicle Transport vesicle

“Cytoplasmic” dynein uses ATP hydrolyis to walk towards MT “minus-ends”

Cytoplasmic dynein, “dynactin complex” plus other proteins link MTs to transportvesicles (cargo)

Axonemal dynein and cytoplasmicdynein are different, but related,motors

4

Tail of motor protein determines cargo specificity

ECB 17-18

17.6-organelle_movement.mov

Three cytoskeletal arrays are linked to one another

MicrotubulesIntermediate filaments Microfilaments

25 nm 25 nm25 nm

Linkages are via an array of binding proteins and motors

END CYTOSKELETON

Lectures 21 and 22: The regulation and mechanicsof cell division

• Today - cell cycle (regulation of cell division)

– Cell proliferation

– The eukaryotic cell cycle

– Measuring the cell cycle

– Models of the cell cycle: from fungi to frogs

– The cell cycle is regulated by cyclin-dependent kinases

• Next time - mechanisms of cell division

5

A cell cycle is one round of growth and division

mitosis

cytokinesis

Growth and division must be carefully regulatedUnregulated cell growth = cancer

Cells only come from pre-existing cells

CLEAVA~1.AVI

CLEAVA~2.AVI

Most cell growth occurs during “G1”(6-20+ hrs; duplicate organelles,double in size)

DNA replication occurs during “S-phase” (4-10+ hrs)

“G2” prepares cells for division (1-6+ hrs)

G1+S+G2=“Interphase”

Division = “M-phase” = “mitosis”and “cytokinesis” (<1 hr)

A “typical” cell cycle for animalcells is 24-48 hrs long, but varies

The eukaryotic cell cycle is partitioned into four “phases”

ECB 18-2

2C(unreplicated DNA,diploid chr #)

4C(DNA replicated,diploid chr #)

4C 2C

2C 4C

C = amount of DNA in haploidbefore replication

Cell cycle times vary

(pH~1)

6

Adapted from MBoC figures17-5 and 17-6

DNA content (arbitrary units)1 2

Num

ber

of c

ells

Cells in G1

Cells inG2/M

Cells inS

Can determine phase of cell cycle from DNA content

Where are cells in G1, S, G2 andM on plot?

Which phase has most cells in it?Lasts longest?

ECB 18-2

Transition from one phase to another is triggered

We will take a historical perspective to ‘triggers’

Regulating the eukaryotic cell cycle:studies in four model organisms

• Marine invertebrates:– Surf clam (Spisula)

– Sea urchins and starfish

• Frog eggs and embryos:– Rana pipiens (Northern leopard frog)

– Xenopus laevis (African clawed frog)

• Cultured cells– HeLa (Human cervical carcinoma)

• Yeast cell division cycle (“cdc”) mutants:– Saccharomyces cerevisiae “budding” yeast

– Schizosaccharamyces pombe “fission” yeast

See HWK 618-619

7

1. Fission yeast “cell division cycle (cdc)” mutants define amaster regulator (trigger) of the G2/M transition

cdc2- (loss of function)

WEE2 = cdc2D

(gain of function)

“cdc”

“wee”

cdc13- (loss of function) cdc

cdc25- (loss of function) cdc

wee1- (loss of function) wee

“Wild-type” fission yeast WT

Mutant Phenotype

cdc2

cdc25

cdc13

wee1

G2 M

Genetic pathwayCdc2 promotes entry into mitosis

Nucleus

Egg in“M-phase”

Oocyte in“interphase”

Transfer M-phase cytoplasmto interphase oocyte

Oocyte “matures”(enters M-phase)

ECB figure 18-5

2. Frogs: unfertilized eggs contain an M-phase Promoting Factor

Transfer of cytoplasm from egg to oocyte inducesM-phase: “M-phase promoting factor (MPF)”

Not restricted to egg cytoplasm - Any M-phasecytoplasm will trigger M-phase

ECB 18-9

MPF activity cycles during the cell division cycle

Time

MP

F a

ctiv

ity

MPF peaks inM-phase

Interphase M-phaseM-phase Interphase

Peak MPF inducesM-phase

ECB 18-10

8

Time

MP

F a

ctiv

ity

MPF peaks inM-phase

3. Surf clams and sea urchins: the abundance of “cyclin”proteins varies with the cell cycle

“Cyclin” abundance varieswith cell cycle:

continuously synthesized,

degraded at end of M-phase

Cyclin B mRNA inducesM-phase when injectedinto Xenopus oocytes

Continuously label fertilized eggs with 35S-methionine

Analyze incorporation into proteins by SDS-PAGE

ECB 18-6

Ribonucleotide reductase(control)

Cyclin A

Cyclin B

Interphase M-phaseM-phase Interphase

Peak MPF inducesM-phase

Cyclin synthesis Cyclin degraded

Cdc2 gene product is a masterregulator of the G2-M transition

cdc2

cdc25

cdc13

wee1

G2 M

Three models of the eukaryotic cell cycle

MPF regulates entry into M-phase

Abundance of “cyclins” in clameggs varies with the cell cycle

Bringing it all together

Cyclin B mRNA (clam) induces M-phase in frog oocytes

cdc13 encodes a yeast cyclin B

MPF consists of frog cdc2 homologand cdc13 (cyclin B) homolog

Cell cycle control: from models to molecules

Inactive(weakly active)

Active M-CDK

“MPF” contains two components:cdc2 gene product = catalytic subunit of protein kinaseM-cyclin = cyclin B (CLB = cdc13): regulatory subunit, cyclins have no enzymatic activity

M-CDK = MPF = CDK1

Removeinhibitoryphosphate

ECB 18-11 and 18-12

PhosphorylateM-phasesubstrates Histones Lamins MAPs

cdc2

CLB(cdc13)

cdc2

CLB(cdc13)P

P cdc2

CLB(cdc13)

Pcdc2

CLB(cdc13)

P cdc25

cdc25(inactive)

wee1P

Positivefeedback

M-CDK (MPF)

Inactive

Inhibitory kinase

Activating kinase

M-CDK activity is also regulated by phosphorylationwee 1 is inhibitory kinasecdc25 is activating phosphatase, triggers activation of CDK1

“Switching on” M-CDK drives cell into M-phase

M-cyclin

9

M-CDK triggers its own inactivation“anaphase promoting complex (APC)”; targets cyclin B for degradation

Polyubiquitin

InterphaseAPC is turned off

cdc2

CLB(cdc13)

P

cdc2

cdc2

CLB(cdc13)

P

APCInactive

APCActive

M-cyclin degraded byproteosome

Anaphase

Accumulationof M-cyclin

CLB(cdc13)

Metaphase (mid-M)High M-cyclin M-CDK active

Telophase (late-M)Low M-cyclinM-CDK inactive

Prophase (early-M)Activation of CDK1 bycyclin and cdc25

M-cyclin accumulationactivates M-CDK

M-CDK activates APC

APC inactivates M-CDK byubiquitinating cyclin B

A cytoplasmic oscillator

Ubiqutin ligase

Review:

Time

M-C

DK

act

ivity

M-CDK peaks inM-phase

Interphase M-phaseM-phase Interphase

Cyclin synthesis Cyclin degraded

Accumulation of M-cyclin above threshold activates M-CDK and promotes entryinto M-phase; cyclin has no enzymatic activity

Activation of APC by M-CDK promotes cyclin destruction, M-CDK inactivation,and exit from M-phase

ECB 18-6

Multiple CDKs regulate progression through the cell cycle

M

G2

S

G1

ECB 18-13

S-phase cyclins

At least 6 differentCDKs and multiplecyclins in mammals

S-phaseCDKs

P

Active S-phase CDKs

Trigger M-phase

S-phasecyclinsdegraded…

P

Active M-CDKM-phase cyclindegraded

Trigger S-phase

M-phaseCDK

M-phasecyclins (B)

S-phase cyclins andCDKs trigger DNAreplication

G1-CDKs; drive cellsthrough G1 (won’t discuss)

Degradation of S-phase cyclinspromotes exit fromS-phase into G2

10

S-Cdk regulates DNA replication

Origin recognition complex -protein scaffolding forassembly of other proteins

Cdc6 increases in G1; bindsORC and induces binding ofother proteins formingpre-replicative complex

Origin is ready to fire

Active S-Cdk 1- phosphorylates ORC causing origin to fire = replication 2-phosphorylates Cdc6 leading to ubiquitination and degradationCdc6 not made until next G1 - prevents origin from double firing

ECB 18-14

Completion of critical cellular processes is monitoredat cell cycle “check points”

Is the cell big enough?Is the environment favorable?Is DNA undamaged?Yes? Enter S phase

Is DNA undamaged?Is DNA replicated?Is cell big enough?Yes? Enter M phase

Have all chromosomesattached to spindle?Yes? Proceed to anaphase

Of these, the G1/S checkpoint for damaged DNA is best understood

ECB 18-17

Prevents cell from triggering next phase until previous one is finished

RNA pol

The DNA damage checkpoint:p53 induced expression of an S-phase CDK inhibitor

DNA damage activates p53

Active p53 acts as atranscription factor to turnon genes, including p21

p21 protein inhibits G1/Sphase CDKs, blocking entryinto S-phase

Cell arrests in G1 untildamage repaired, orundergoes apoptosis(programmed cell death)

ECB 18-15

PP

p53(inactive)

P21 binds andinactivates S-phase CDKActive S-phase CDK

p53 (active)

Translation

Transcription

p21

DNA

p21 gene

Mutations in p53 inhalf of human cancers!

11

If checkpoint is activated

Or undergo apoptosis (in a minute)

neuronsmost plant cells

Exit cell cycle (temporary or permanent)

Zones of division and growth in plant roots

Meristem - zone of active celldivision

Zone of cell elongation - growthbut not division; Cells in G0

Zone of differentiation - cells ceasegrowing and terminally differentiate

Regulation of each zone is not well understood in plants but involves hormonesIn animals:

mitogens stimulate cell proliferation (block checkpoints)growth factors stimulate cell growth (stimulate biosynthesis, inhibitdegradation)

Arabidopsis thaliana

Only a fraction of cells still actively dividing

Apoptosis: A tale of tadpole tails and mouse pawswhat do they have in common?

Both processes involve “programmed cell death (apoptosis)”

Tadpole tails are resorbed during metamorphosis

ECB figure 18-19

Paws, hands and feet develop from “paddles”ECB figure 18-18

ECB - “programmed cell death is a commonplace, normal, and benign event. It isthe inappropriate proliferation and survival of cells that presents real dangers”

12

Necrosis (cell death following injury) often results in lysis, spilling the contentsinto the surrounding space and causing inflammation

During apoptosis (“programmed cell death”), cells remain intact and condense

Corpses of apoptotic cells are often engulfed by their neighbors or specializedphagocytic cells

Apoptosis is visibly distinct from necrosis

ECB 18-20

18.3-apoptosis.mov

Apoptosis is mediated by a “caspase cascade”“Caspases” are proteases; inactive precursorsactivated by proteolysis

Presence of suicide signals and/or withdrawal ofneeded survival factor activates first caspase incascade

Deathprotein

Survivalfactor

Inactive

Activated caspases degrade nuclearand cytoplasmic proteins (lamins,cytoskeletal proteins, etc)

Activated endonucleases cutchromosomal DNA

Active

Caspase(inactive)

ECB 18-21

Initial caspase proteolytically activatesdownstream caspases

…which activate additional caspases, and so on

Caspase cascade must be carefully regulated

Bcl-2 family of proteins are death proteinsForm pores in outer mitochondrial membranereleasing cytochrome c (respiratory chain)

Cytochrome c binds adaptor and complex activates first procaspase