1

Positive and negative feedback produce a cell cycle

Slow

Fast

Cell cycle oscillations

Inactive Cdk1-CyclinActive Cdk1-Cyclin

Active APC

2

Ubiquitin mediated proteolysis

Glycine

Lysine

Isopeptidebond

Similarities between phosphorylation and ubiquitination

>500≈500Number of human

modifyingenzymes

Protein activity,location, stability

Protein activity,location, stabilityRegulates

DeubiquitinasesProteinphosphatases

Demodifyingenzymes

NoNoReversiblereaction?

UbiquitinationProteinPhosphorylation

3

Different cyclins appear at differenttimes in the standard cell cycle

G1 cyclins overcome cell cycle roadblocks

Cyclin-dependentkinase inhibitors

(CKI)

4

Cancer cells lack G1 roadblocks

Cyclin-dependentkinase inhibitors

Standard Cycle

NORMAL

Cdk Inhibitors Lost

Cyclin-dependentkinase inhibitorsX

CANCER

Cyclin-dependentkinase inhibitors

Too much G1 cyclin

CANCER

OR

a. Cell cycle arrests

b. Damage repair

Cell cycle checkpoints

5

Partial DNA replication breaks chromosomes

Damaged DNA

DNA repair

DNA damage stops the cell cycle

Cell Death(Apoptosis)

DNA damagecheckpoint

6

Apoptosis kills damaged or unwanted cells

Life Sciences 1a: Section 3B. Mitosis Objectives Understand the steps of mitosis Understand the difference between equilibrium and non-equilibrium polymers Understand how GTP hydrolysis explains how microtubules grow, and shrink Understand how exploration with selection captures chromosomes Understand that exploration with selection is a general biological principle

Introduction Mitosis segregates chromosomes

Prophase -> metaphase -> anaphase -> cytokinesis -> interphase DNA replication is templated, spindle assembly is not Cdk1-cyclin complexes induce massive reorganization of the cell Nuclear lamina phosphorylation induces nuclear breakdown Phosphorylation induces chromosome condensation (compaction) Microtubules become more dynamic

The cytoskeleton The scaffolding and roadways of the cell Dynamic structures which are constantly assembled and disassembled Actin filaments (5 nm diameter), intermediate filaments (10 nm), microtubules (25 nm) Actin filaments and microtubules: polar, non-equilibrium, motors carry cargo in both directions

Microtubules Microtubules are composed of 13 protofilaments They are nucleated at microtubule organizing centers (centrosomes in animal cells) Equilibrium polymers must all shrink or all grow But with non-equilibrium polymers, some polymers can grow whilst others shrink Tubulin exchanges GTP for GDP as a free subunit and hydrolyzes the GTP when it polymerizes Growing microtubules are mostly GDP-tubulin, but have a cap of GTP-tubulin If the subunits of the cap are bound to GTP, the microtubule grows If the subunits of the cap are bound to GDP, the microtubule shrinks (protofilaments splay apart) Chance fluctuations can convert a GTP cap into a GDP cap initiating rapid shrinkage (catastrophe) Chance fluctuations can convert a GDP cap into a GDP cap initiating growth (rescue) At the level of single molecules, the same reaction doesn’t always happen at the same rate

Microtubules and chromosome capture In mitosis more microtubules are nucleated, but increased catastrophe rates make them shorter The centrosome nucleates microtubules equally in all directions Microtubules that interact with chromosome arms or centromeres are protected from catastrophe Their increased stability converts an initially uniform distribution of microtubules into a bipolar spindle

Exploration with selection (also known as search and capture) Stabilize a subset of random explorations from a starting point The next round of random exploration starts from these stabilized points Repeating this process is an efficient way to find very non-random solutions to biological problems This is a fundamental biological principle over a range of sizes and times; Protein folding: <10 nm, 1ms – 10 min Spindle assembly: 10 µm, 1 min – 1 hour Nervous system: 1 µm – 1 m, 1 day – 1 year Evolution: 0.1 nm – 100 m, 1 month – 109 years Reading: Alberts: pp. 579-591 (microtubules)638-654 (mitosis)

1

Different cyclins appear at differenttimes in the standard cell cycle

G1 cyclins overcome cell cycle roadblocks

Cyclin-dependentkinase inhibitors

(CKI)

2

Cancer cells lack G1 roadblocks

Cyclin-dependentkinase inhibitors

Standard Cycle

NORMAL

Cdk Inhibitors Lost

Cyclin-dependentkinase inhibitorsX

CANCER

Cyclin-dependentkinase inhibitors

Too much G1 cyclin

CANCER

OR

a. Cell cycle arrests

b. Damage repair

Cell cycle checkpoints

3

Partial DNA replication breaks chromosomes

Damaged DNA

DNA repair

DNA damage stops the cell cycle

Cell Death(Apoptosis)

DNA damagecheckpoint

4

Apoptosis kills damaged or unwanted cells

a. Phosphorylation controls cellular architecture

b. The cytoskeleton: roadways & scaffolding

c. Microtubules are dynamically unstable

d. Self assembly by exploration with selection:Chromosome capture by exploration with selection

Mitosis and the cytoskeleton

5

Mitosis segregates chromosomes

Mitosis: the movies

Aaron Straight

6

Templates versus self assembly

+

DNA Replication

Spindle Assembly

?

Events of mitosis

Cdk1-cyclin

Nuclear breakdown

Chromosome condensation

Microtubules more dynamic

7

Filament systems of the cytoskeleton

Actin filaments

Intermediatefilaments

Microtubules

Dynamics

Non-equilibrium(ATP hydrolysis)

Equilibrium

Non-equilibrium(GTP hydrolysis)

5 nm

10 nm

25 nm

Diameter Polarity

Monomers (entire proteins) are held together by non-covalent bonds

Microtubules are polymers

Prot

ofila

men

t

8

Microtubules are nucleated at organizing centers

Spontaneousnucleation

+

Templatednucleation

Polymerization

Equilibrium polymers can grow OR shrink

M

n+ M � M

n+1

Growing(high [monomer])

M

n+ M � M

n+1

Shrinking(low [monomer])

M

n+ M � M

n+1

9

Growth = kon[monomer][polymer _ ends]

Shrinkage = koff [polymer _ ends]

Finding polymer equilibrium

M

n+ M � M

n+1

Shrinkage = Growth

koff [polymer _ ends] = kon[polymer _ ends][monomer]eq

koff = kon[monomer]eq

koff

kon= [monomer]eq

At equilibrium

Non-equilibrium polymers can grow AND shrink

M

n+ M � M

n+1

M

n+ M � M

n+1

koff

kon= [monomer]eq

koff

kon= [monomer]eq≠

10

Microtubules can grow AND shrink

Tubulin hydrolyzes GTP

GTPGDP

GDPGTP

P

Free

On microtubules

11

Microtubules can grow AND shrink

time, ms

Microtubule growth, shrinkage, catastrophe, & rescue

time, ms

Add (0.2 ms-1)

Add (0.03 ms-1 )

(Rescue)

Lose (0.3 ms-1)

Hydrolyze (0.05 ms -1)

(Catastrophe)

GTP-tubulin

GDP-tubulin

12

Microtubules are more dynamic in mitosis

Mitosis

Increased nucleation

Increased catastrophe

Interphase

Spindle assembly: Exploration with selection

13

Exploration with selection is a general principle

1 month - 109 yr0.1 nm - 100 mEvolution

1 day - 1 year1 µm - 1 mNervous systemdevelopment

1 min - 1 hour10 µmSpindle Assembly

1 ms - 10 min0.1 - 10 nmProtein Folding

TimeDistanceProcess