1

LECTURE 4: Cytoskeleton I - Filament Proteins September 22nd

1. Basic cytoskeleton
- Figure is a neuron
- Against actin (blue) and tubular (red)
- Actin filaments occupy the periphery of the cell; send out projections in a certain direction
- Tubulin — elongated

3 types: pg. 891

- Actin Filaments (AFs, or microfilaments)

- in the periphery (cortex) of the cell
- Microtubules (red)
- longer structures
- organize for mitosis
- Intermediate filaments
- intermediate in properties to previous two
- not as rigid
- tend to have more flexibility

FIlament Construction

- Each filament is made up of smaller parts i.e. subunits

- Subunits form the filaments — come together and form specific structures
- b/c of subunits, allows them to be compact and disassemble and fuse to different parts of the
cell
- In development, cells are not only dividing, they are also going to where they are supposed to
go (travel long distances)
- Actin filaments assemble in the direction of movement and more actin subunits (soluble in the
cell)
- Along comes a signal (nutrient source) which makes cell respond; the ones assembled then
disassemble and reassemble on the other side — advantage of having a globular
cytoskeleton; allows us to respond and be more flexible
- Green (MT) or red (MF)
- Fibroblasts moving - actin filaments involved; reorganization of MT and actin filaments
- Plasticity in the structure of the cytoskeleton is what allows us to respond to the environment
- Subunits are designed to maximize strength and dynamics (plasticity)
- MT — looks like a cylinder
- Sheet of protofilaments — to form MT
- Take one protofilament — snap very easy; easy to move one subunit than splitting in half
which helps in dynamic stability
- if there were strong ionic bonds — rigid skeleton but we need something dynamic
- 13 protofilaments makes MT
- More protofilaments together — harder to break the entire sheet than just one subunit —
thermally stable

Nucleation
 2

- Potential disadvantage: takes time and more energy to build structures up from scratch
- Certain amount of time is required to produce the MF or MT
- If they are already made, it is easier to add and remove
- But to build one back up that is completely broken down, takes a lot of energy

Lag phase — form an oligomer (starting to form a structure i.e. a nucleus of a structure)
Growth phase — once nucleation has been performed and we have a rudimentary filament, we
experience the elongation phase where there is addition of more
Equilibrium phase — (In the beginning, it grows on both ends) — now it reaches an equilibrium
of coming on and off

No lag phase if its already built up

- faster

Tubulin

- Heterodimer
- Subunit of tubulin (alpha and beta)
- GTP is the source of energy
- Both alpha and beta have an area for GTP
- Hydrolysis only on beta
- 13 protofilaments lined up
- they have a plus end a minus end
- + = beta — where growth occurs (when a lot of subunits are available)
- - = alpha

Actin

- Monomer
- Have a + and - end
- Region of high growth vs. region of limited growth

Treadmilling and Dynamic Instability

- involve both growth and shrinkage

- catalyze hydrolysis of ATP/ GTP
- “T” or “D” form — triphosphate or diphosphate
- ATP/GTP caps — hydrolysis is slow compared to the rate of growth
- Rate of addition exceeds the rate of hydrolysis so there is elongation?
- Critical concentration — subunit addition equals subunit loss; steady state

Treadmilling — Actin Filaments

- Filaments added to the ATP-actin

- + and - ends refer to the shape of the protein
- ATP-actin high, addition at both ends
- ATP-actin drop,s addition greater at the plus end
 3

- the availability of actin subunits has gone down — more growth at one end because its
thermodynamically favourable for that to occur
- At stage 4, tread milling or steady state. Rate of addition = rate of loss

Treadmilling — Microtubules

- High tubulin — growth at both ends

- As concentration becomes limiting, growth at the plus end — thermodynamically stable
- Putting subunits at one end and removing at the other end
- Polymerization is spontaneous

Treadmilling

- Rate of hydrolysis and Cc

- Treadmilling range is between the two critical concentrations
- Plus end is the T end and D end is the minus end
- T end — increase subunit concentration — tendency to take more of the elements and put
them in the growing filaments (strong, linear relationship)
- if we take away subunits, start to shrink
- shallower slope for the minus end; where thermodynamics is less favourable
- Fit together like lock and key
- Binding can still occur; for something to polymerize at the minus end, less probable
- Thermodynamic relationships; minus end has shallower, as there is more subunit
concentration, less growth at the minus end b/c shallower slope
- Totally different relationships between concentration and growth for both of them
- If reduced subunit concentration, enter a range where you’ll be losing subunits but there is
still growth; but as you move further back at a lower concentration, there is no growth
- Want to be in the treadmill range
- Can define a range
- Greater than T (so growth) and D will be shrinking
- Move around in the treadmilling area and have a balance of shrinkage and growth
- T < C < D, then we have treadmilling
- T < D — easier to get to that point
- Reaching the critical concentration of growth at the plus end is gonna take less time than
reaching the critical concentration of growth at the minus end

Treadmilling behaviour — MT

- white arrow head = growing end

- red — single subunit
- unlabelled tubulin is stationary
- subunits added to the plus end and lost at the minus end
- but appears to be walking like a worm

Dynamic Instability - MTs

- Minus end of tubules are anchored for the mitotic spindles

- Plus end — focus on this
 4

- Growth depends on concentration of tubulin and energy

- Go beyond tread milling range — addition exceeds hydrolysis
- Continuous transition
- Concentration is very high so produce GTP cap
- Addition of subunits eventually slows down
- hydrolysis eventually happens — continues and we see a CATASTROPHE
- all the GTP hydrolized to GDP — rapid shrinkage
- lose a lot of the length
- RESCUE
- now the concentration of subunits increases
- rate of hydrolysis is still slow but the rate of addition is high again with more GTP cap
- Dynamic instability is defined as the continuous transition between catastrophe and rescue

- When GTP is hydrolyzed, change in the structure of the MT

- Proteins undergo conformational changes which lead to functional changes
- Loss of phosphate changes the whole structure — subunits take a more bent shape (stable to
unstable conformation)
- (Catastrophe or depolymerization)
- fallen off subunit exchanges GDP for GTP which then joins back up and takes a part in
another protofilament
- curve shape — peel off
- energy is inside the protofilaments and hydrolyses releases that energy

Kinetics Dependent on [tubilin]

- More concentration of MT — more pointed tip

- Concentration of the subunits can dictate the shape of the growing MT or MF
- The tip would also affect the rate of elongation
- Different rates of elongation at higher concentration
- Rate of growth is concentration dependent

Dynamic Instability - MTs

- tracked overtime
- growing tip at the top
- long and shuts down at the end during catastrophe

Treadmilling and Dynamic Instability

- Act to maintain the length of the filament - depending on chemical cues

- Fastest way to grow filaments without nucleation is through tread milling and dynamic
instability — act to maintain length of filament
- CS is modular globular and the way its organized allows for very dynamic
- MT moves out searching for attachment sides
- Sometimes MT can come from two different sides and find each other and bind together

Intermediate Filaments

- Intermediate between MT and MF

 5

- Quantify force you apply — measure the angular deformation

- MT are not designed for that kind of a force — not meant to withstand sheering forces
- Actin filaments — survive longer but are also not meant to withstand the forces (forces in
muscle contraction but not deforming forces)
- Intermediate filaments — somewhat between the two properties; allows them to have a lot of
strength — can withstand greater forces/bending/twisting etc. — rope-like properties

- Soluble subunit of actin = monomonr, tubulin = heterodimer

- For IF - tetramer of four different proteins
- Less likely that the tetramer disassembles to single proteins
- Dimers are oriented in opposite orientation
- N terminus and C terminus
- Dimer — tetramer — two tetramers packed together
- tetramers are packed into an array of 16 dimers
 6

LECTURE 5: Cytoskeleton I continued September 25th

IF Filaments
- tetramers assemble and produce a rope-like

IF Types
- Epithelial (keratins)
- Most diverse family
- Type I and II keratin chains
- Strength in hair, nails
- examples of IF (Green are the IF — black circles are nuclei; purple shows the boundaries)
- Axonal (neurofilaments)
- Play a role in given strength and increasing the lengths of the axons
- Type L, M or H
- Growth, increased axon diameter

CYTOSKELETON II: Regulation

Accessory Proteins

- Filaments (AFs, MTs, IFs) are dynamic and under control of the cell
- Form higher-outer structures (e.g. mitotic spindle)
- Accessory proteins modify these cytoskeletal dynamics

Nucleation by gamma-tubulin Protein Complex

- allows for apparent movements of filaments throughout the cell

- takes part in nucleation
- alpha and beta tubulin — both bind GTP but only one of these is hydrolyzed (beta)
- gamma tubulin is an important accessory protein
- Spindle pole body in yeast and MTOC in animal cells
- GammaT is highly conserved in many different cell types
- Multiprotein complex — facilitates the process of MT formation
- MT are not formed in sheets; they are formed as rings from the very beginning
- Spiral forms as tubulin dimers are added
- Ring complex actively recruits heterodimer and tubular and form the cylindrical structure
- The minus end interacts with the ring complex (gammaT)
- + end grows actively

Nucleation by gammaT Complex

- Also in cytosol
- Time lapse shown in diagram
- The arrow shows an MT growing longer
- MT has daughters that go longer
- Tubulin complex allows for branching of MT
 7

The Centrioles and Centrosome

- 2 cylindrical centrioles
- Main task is to organize PCM — PCM contains gammaT
- Organize the PCM (containing gammaT) which organizes MT
- Centrosome divides and goes to opposite parts of the cell
- Still one cell but the centrosomes begin to organize the MT and come off from both the
centrosomes
- Form a star shape that goes off in all direction
- MT overlap and find each other with forms the mitotic spindle
- Triplets of structures organized; 9 triplets of MT

Nucleation of AFs

- Primarily in the periphery of the cell

- Actin is the most dense at the cortex
- ABPs or ARPs

Initiation of AFs (unbranched)

- Formin — ABP
- Two part protein (dimer)
- Large protein compared to the monomer of actin
- Goal is to orient actin monomers to form a growing MF
- GammaT complex begins to form a ring at the very beginning
- Formin does something similar so they can be pieced together to properly form a MF
- Formed in a complex manner
- Actin monomers — spiral-like
- Subunits fall in behind
- As the subunits behind, every actin monomers make sure the alignment is perfect
- For the thermodynamics to be favourable, need to have the correct orientation of the
monomers of actin (proper alignment)
- e.g. muscle spindle

Initiation of AF Branches

- In this case, different utilization of the same protein actin but more in the periphery of the cell
- ARP2/3 complex produces branching in the periphery of the cell
- The ARP2/3 complex binds the minus end of the actin filament
- Lamallapodea? — this is how its found
- Optimal angle of 70 degrees

Control of Subunit Pools

- Regulate/control concentration of subunits
- they can sequester (take up or tie up temporarily)
- all reversible reactions ^
- sequestered proteins are not hydrolyzed — not going to polymerize/or take part in elongation
 8

Thymosin Sequesters Actin

- Thymosin sequesters, but profilin recruits monomers

- Thymosin sequesters actin and make its addition to the + end less likely (inhibition)
- Profilin facitilates the process
- Competition between the two
- Only happens at the positive end (growing end)

Stathmin Sequesters Tubulin

- Stathmin binding prevents polymerization

- Sequester tubulin and take them out
- Decreases the effect of concentration of tubulin (the free tubulin)
- Promotes dynamic stability
- tendendy for losing the GTP cap — the protofilaments change shapes and bend; weak molec
bonds are not strong enough to keep the tubulin together and it falls apart

MT-Associated Proteins (MAPs)

- Bound to MT and there is an arm that projects out which can bind to an MT right beside it
- another MAP called tau — different length moving off
- Cells that have a higher expression of the protein tau have a more dense packing of MT
- MAPs can determine the packing of the MT
- These can help determine the structure of MT and MAP2 is in neuronal cell body; dendrites
are going off in different directions
- Axons — tau proteins
- tau are important in Alzheimer’s disease
- Not able to bind MT as well—spacing is not regulated as well as it should be
- Creates neurodegenerative process
- Tau protein changes its solubility

- Hyperexpression of MAP2 and tau — evenly spaced out uniformly

AF Binding Proteins

- Cofilin destabilizes AFs

- causes release of actin filaments back into the subunit pool
- forces some mechanical stress
- binding of the protein interacts with the actin filament and causes a stress
- important in treadmilling, turnover
- important in disassembly
- cell locomotion
- binds to sides

Tropomyosin — affects AFs and bind to sides and not the ends
- Binds to the side and tend to stabilize

Modifications at AF Ends
- “capping”
 9

- bind to ends of the growing filaments

- CapZ — elongation occurs at the (-) end; binds at the (+) end and makes it very unlikely that
it will interact and grow at that end so we’re left with growth at the (-) end
- Strong slope showing the trend; increase monomers get fast growth
- but if we cap off, we grow at the minus end
- dynamics are limited to the minus end
- slows down (regulate) growth
- T-form — unhydrolyzed form of the nucleotide
- Whether its hydrolyzed or unhydrolyzed has an effect on the structure of the actin protein —
determines how easily it will polymerize
- thermodynamics favourability
- Tropomodulin binds at the (-) end

Modifications at the MT Ends

- Can also stabilize MT

- Bind to the side of these MT
- Formation of MT in axons — the MT proteins aid in packing of the MT; not just
thermodynamics
- not cell undergoing mitosis

Cross-Linking Proteins and AFs

- Higher order structures

- Using actin to build higher order structures
- To build the cortex of the cell — not just actin; many other types of accessory proteins
associated with actin contribute to produce cross-linking proteins
- domains structures to bind to actin monomers; placement of these regions along the protein
that determine the structure being produced
- 2 major groups; bundling proteins and gel-forming
- 2 examples of bundling (fimbrin and a-actinin)
- gel-forming (spectrin and filamin)
- red domains are the actin binding domains
- fimbrin — 2 very close actin binding domains; put actin very close together
- a-actinin — dimer that has 2 actin binding domains
- gel-forming proteins — large molecules with actin binding domains in very spaced out regions

Actin Bundling Proteins

- a-actinin
- spacing out actin filaments
- fimbrin — very closely associated; more spaced out than the other in actinic
- myosin shortens muscle spindles

Gel-forming Proteins
- Actin in RBC — gel or network
- gives RBC their flexibility
- cell dimer greater than the dimer of capillary

Gel-Forming Proteins
 10

- Filamin
- how actin associates with itself and creates a higher-order structure

Changes in Cell Shape During Embryonic Development

- Neural tube forms on the dorsal region

- “Rolling” tube - AFs
- Cells begin to elongate — mediated by MT
- Rolling of the tube is by AF — pie shaped formation of the cells
- Example of how accessory proteins orchestrate changes in cells
- Accessory proteins change the conformation that change the length

Changes in Cell Shape During Embryonic Development

Induction by Extracellular Signals

- Controlled by ECS
- Received by transmembrane proteins
- WASP protein — when its activated, it leads to steps
- MF are long enough — plasma membrane pushing forward crawling toward
- Then destabilizing by cofilin
 11

LECTURE 6: Cytoskeleton III - Molecular Motors September 29th

IF don’t participate in vesicle transport

— EM
- The long bands are MT in the axon of a neuron
- Transported from the cell body to the terminal region
- The rest is what is a network of proteins — kinesins and dyenin (motors that use ATP and
transport the structures down the length of the cell)

Motor proteins
- Bind to cytoskeleton
- Dyenin and kinesins bind to MT — other end was a cargo that had a secretion of some kind
- Some sort of a net movement of protein or cargo
- Divide into 3 families
- 1) Myosins: AFs — muscle contractions
- 2) Kinesins
- 3) dyenins

Myosins
- II and V
- II = muscle myosin — muscle contraction
- V = unconventional
- V = like kinesins but doesn’t interact with MT
- Dark green = myosin head/catalytic motor region
- Catalytic heads are conserved; meaning that aa of these proteins are similar; 99% conserved
(not completely)
- b/c of the expression types of the motor proteins in different types

Myosin II
- Muscle type/conventional
- Heavy chains — greens
- Light chains — blues — assist in conformational change in the motor protein
- Heads are catalytic and provide force
- Tails mediate dimerization with other myosins
- Light chains amplify conformational chain
- Mediate conformational change
- Articulation of movement of the protein

Myosin Cycle

1) Attached: No ATP and the myosin heads are attached to the actin filament
2) Release: ATP bound, conformational change (away from AF) — releases myosin head from
the AF (thick filaments are the tails of myosin — lots of them)
3) Cocked: hydrolysis occurs, ADP and Pi. Hydrolyzed but the Pi is still there. Change toward
the (+) end of the AF
4) Force generation: weak binding, Pi release, power stroke, ADP lost, movement of the
myosin
Only one head can react; many many myosins in a thick bundle;
 12

Thick Filaments Formations

- Light green structures are the thick filaments

- Muscle spindles — important in these
- Bare zone — 2 myosins come together catalytic head at the ends and long tail line up — tails
dimerize and produce a bare zone

Sarcomere

- Bare zone = M line

- Actin filament in green
- The gray is the myosin
- Tropomodulin — caps the minus end of growing AF
- Cap Z — plus end; prevents elongation
- Z disc = composed of alpha actinin and CapZ;
- Induces shortening

Ca2+ dependence of Muscle Contraction

- Muscle contraction is dependent upon calcium

- Troponin complex interacts with Ca and moves the complex out of the way and allows myosin
head to come in
- Ca2+ released from SR (sarcoplasic reticulum) — can come from outside of the cell or the
organelles
- Direction is towards the cytosol for the electrochemical gradient
- Ca2+ intereacts with the troponin complex — Tropomyosin moves and exposes binding sites
- Myosin binds to AF — get muscle contraction

Myosin II S1 Fragment

- Isolating S1 fragment of myosin II (it includes the catalytic head and part of the neck)
- Isolated by the addition of a protease enzyme

Experimental evidence
- Glass slides — can see the filaments walking around
- Adhere to glass coverslips — there is ATP present and preceded and nucleated actin
filaments

Contractile Rings
- The contractile ring becomes smaller, falls apart (disassembling) so the actin subunits are
return to the pool
- Pointed end = minus end
- Barbed end = plus end
- Actin filaments on the contractile ring; two myosin heads/tails dimerize

Myosin V
- Unconventional
- longer tail region — larger steps
- processivity = there must always be one catalytic head in contact with the substrate
 13

- like walking; one foot on the ground

- these work my processive step; one head always in contact with the substrate
- not a problem with myosin II (single myosin head that interacts with actin filament)
- thick filament — long structure/series of them interact with the AF

Experimental Evidence
- lasers to manipulate polystyrene beads
- Change in pressure/energy near a bead
- Glass cover split put on the side; adhered to this are AF
- myosin V is added with polystyrene beads; attached with myosin V
- if there is ATP, the myosin fibers start walking around
- showed that they moved in a stepwise manner = b/c of these steps and tails regions that
move

Kinesins
- Radioactive formed of leucine was used and injected into the sciatic nerve
- traces of it were found further down the axon (further down the nerve)
- 410 mm/day was the rate at which the protein was making its way down
- important to carry things down to the terminal end of the neuron
- Pool of synaptic vesicles and transport them 410 mm/day
- Cell use a slow method to transport things; heading down and later on, more faster form like
filling and releasing vesicles take place
- make its way down
- at first, nothing down the other end; eventually, start getting kinesins down the other end

Kinesins (discovery)
- Axon can be mounted and played around with without microscope
- Can observe phenomena very easily
- The axoplasm was removed from the squid; and ATP was added; found that organelles
movement could be observed

Kinesin structure
- Head region conserved
- Tails are diverse
- Compared to myosin
- somewhat similar
- not the same part of the protein complex but the ATP are there
- Very different linker region
- Linker region interacts with catalytic core; not the same with myosin but for chines the liner
region is right in the core
- Kinesin arm swings around and brings the head form

Kinesin Cycle
3 phases
1. ATP binds to the leading head and induces conformational change in the linker region
2. ADP - bound to the trailing head (weak binding with the ADP head); bind is so weak that the
trailing head moves forward
3. Hydrolysis of the trailing head causes detachment; ADP dissociates from leading head
 14

Hydrolysis cycle is divided in attached and detached (half of its time in each of the two)
Longer hydrolysis than myosin II

Dyenins
- minus end directed
- MT motor proteins
- 2 major divisions (cytoplasmic — retrograde vesicular transport and axonemal — beating of
cilia and flagella)
- don’t interact directly with cargo

Cytoplasmic Dynein
- Does not interact directly
 15

LECTURE 7: Continued October 2nd

Dyenin interacts indirectly

Axonal Transport
- Kinesin goes to +
- Dyenin goes to -
- + — periphery (axon teminus)
- - — cell body
- Single vesicle may be associated with multiple molecular motors
- Carry them in one direction and then turn around and go in the other direction?
- Flow of cargo down an axon — kinesin where it was first discovered
- Leucine (radioactive) carried down axon

Vesicle being carried along Mts

Role of AF vs. MT-based motors

Myosin V — unconventional
- involved in organelle transport
- like kinesin
- pigment-cell is a cell that produces pigment
- pigment granules are carried throughout cells (not neurons but kinda have star-like shapes
that extend)
- pigment granules are sent around by kinesin and given off to myosin V to spread out
- ‘

Cell Cycle I: Intracellular Control

- Cells spend most of their time in G0 stage
- Some cells divide and stop differentiating
- Some cells can regenerate
- non-disjunction — does not align (no copy in the other daughter cell)

The Cell Cycle

- Checkpoints that prevent things from going wrong
- Most time in growth phase
- Mitosis is nuclear division

Why Control the Cell Cycle?

- Orchestra of many things happening

- Complex system of biochemical reactions that have to occur in a sequence
- If anything is performed incorrectly, there is a catastrophic outcome
- Molecular switches — turn things off and on; can cause the cell to re-enter the cell cycle
- If there is something wrong with DNA replication, the cell will not go into mitosis until the
repair takes place

Cdks Control of the Cell Cycle

- Cyclin-dependent kinase
 16

- kinase = protein that phosphorylates other proteins

Classification of Cyclins and Cdks

- G1/S cyclins —
- S-cyclins — bind during S phase and are required for DNA replication
- M-cyclins — goes up to allow cell to enter mitosis
- G1-cyclins —

Cdks are Protein Kinases

- depends on the type of protein that is activated
- site of phosphorylation is important
- you can phosphorylate protein and activate it or phosphorylate at a different site and
inactivate it
- NOT always gonna activate

Cdk Activity is Regulated

- M-cyclin is increased and leads to mitosis
- Triggers mitosis but in order to get out of mitosis, M-cdk needs to be deactivated
- S cyclin then comes in — Activates the S-cdk
- DNA goes around in that way

Activation of Cdk-cyclin
- Not as simple as just having the cyclin present
- cyclin comes in, conformational change
- activity of another protein kinase (CAK) activates Cdk
- Go from a partly active to fully active
- Need a CAK to phosphorylate

Inhibition of Cdk
- Wee1 = important in how cells get into mitosis
- Active Cdk (phosphate from CAK)
- Another kinase (Wee1) kinase comes and inhibits it by adding another phosphate
- Reversed by Cdc25 phosphatase
- Another way is p27 — inhibitor
- Bind with both the cyclin and Cdk to inactivate the protein

Ubiquitin and Protein Degradation

- Ubiquitin — marks cells for destruction
- Form chains — protein that marks for destruction have the ubiquitin chains
- Inside the proteasome — have a protease that breaks down the protein
- Can use the broken down subunits for other things
- Polyubiquitin chains binds to the proteasome — which chops them off into little groups of
amino acid
- Ubiquitin ligase — important

SCF and APC are Ubiquitin Ligases

- CKI — involved in inactivating Cdk cycle
- Destroy the inhibitor and destroy it to allow the Cdk to be active
- SCF leads to the destruction of CKI
 17

- F-box protein = adapter protein required in order to mark the protein for ubiquitation
- Before the F-box protein can bind the poly-ubiquitin chain to the inhibitor, the Cdk inhibitor
MUST first be phosphorylated — only this can be recognized
- The phosphorylated gets send to the proteasome — necessary to get to the proteasome
- Has a positive effect on the cell cycle

APC
- Can lead to the destruction of securin (stabilized separase which causes chromosome
separation)
- Leads to the destruction of M-cyclin
- M-cyclin can be destroyed to inactivate the Cdk and allow it go get out of mitosis
- Activating subunit (Cdc20) activates APC/C
- APC works with E1 and E2 which produce a polyubiquitin chain on the M-cyclin
- Then cell can leave M-phase b/c of the degradation of the M-cyclin in the proteasome
- Positive effect on the cell cycle

SCF and APC are active during different stages

- APC-Cdc20 — help to keep M-cyclin low (inhibits APC-Cdc20)

- This is important in separase activation and anaphase (slide 17)
- peaks in anaphase (anaphase promoting complex)
- causes sister chromatids to go apart
- causes the activation of an enzyme that causes the sister chromatids to fall apart

Activation of M-Cdk Triggers Mitosis

- General activation — Wee1 kinase imparts phosphate onto M-Cdk but puts it on a site that
produces inactivation
- Activating phosphating but inactivating phosphating
- No progression b/c M-Cdk is not active until there is a signal
- Cdk phosphorylates Cdc25
- Cbc25 is a protein phosphatase - removes phosphate (inhibitory phosphate)
- Feedback loops — furthers the reaction
- Leads to the activation of its partners
- One protein that is active is going to phosphorylate other things
- Feedback enhancement — remove inhibition; prevents Wee1 from inhibiting M-Cdk on the
verge of mitosis

Checkpoints

LEC 8: Cell Cycle II — online Tuesday October 6th

 18

LEC 9: Cell Cycle III: Apoptosis Friday October 9th

- Scanning electron microscope

- Cells grown on a substrate and went through apoptosis

Apoptosis
- “Programmed cell death”
- Number of cells is, in part, controlled by regulation of cell death
- Differs from necrosis: trauma or cytotoxicity leading to lower ATP and lower NA+/K+-ATPase
activity
- Why does apoptosis occur?
- Process of purposeful death of cell; undergoes specific cascade of events
- Undergoes some pathological stimulation (one of the reasons cell dies)
- pH, ionic disregulation, contamination with bacteria or fungi, infectious tissue, damage,
trauma — NECROSIS
- Apoptosis is different from necrosis
- Apoptosis — large scale cell death
- Apoptosis is cell death in a neat way
- Dumping out material to the outside of the cell can be damaging for other cells

Apoptosis
- Developing forelimb of the house
- Loss of cells between digits; forelimbs start off as little buds
- Markers are fluorescently labelled — undergoing apoptosis
- Helps to balance cell division and cell death

General Characteristics
- In order for cell to undergo apoptosis, there are two defined pathways (very specific events
- Can be initiated by intracellular or extracellular signals
- Series of proteins involved in promoting apoptosis (two families of proteins)
- Important intracellular proteins necessary for survival are cleaved during apoptosis
- Orderly disposal of a dead cell

Structural Changes During Apoptosis

- Loss of adhesion = contract
- Blebbing as the cells are passed away

1. Chromatin condenses; shrinkage of cytoplasm. Contract and condense. Nuclear lamen

destroyed.
2. Nucleus fragmented; DNA “laddering” — breaking apart of chromosomal DNA; blebbing, cell
fragmentation
3. Apoptotic body is engulfed by phagocytic cells

Phagocytosis
- Asymmetric distribution of plasma membrane is lost (in step 2 in previous slide)
- Negatively charged phosphatidylserine then becomes exposed on the outside of cell
- The cell is then marked for phagocytosis by a macrophage
- Specific phospholipids help to make up membranes
- Plasma membrane is not created equally all the way around
 19

- Phospholipids in the cells can help to target that cell

Cell-death Mutants
- Free living nematods
- Caenorhabditis elegans is a nematode
- 959 somatic cells
- 131 apoptose every time
- Discovery of ced-3 gene that encodes proteins similar to mammalian protease
- Gene call ced-3 = responsible for the tidy death of these cells
- “caspases”
- ced-4 encodes Apaf-1; ced-9 encodes Bcl-2
- When cells undergo apoptosis, they refract light differently and can be easily identified
- ced-3 mutant = organisms don’t undergo apoptosis
- helped describe caspases = when we get rid of a gene, we stop a protein from being
expressed which stops apoptosis
- Apaf-1 and Bcl-2 are the mammalian versions of these proteins

Caspases
- Enzyme; similar to the ced-3 enzyme found in nematodes
- They are proteases that cleave important proteins for cell survival
- Cysteine residue residue at the catalytic side and cleaves other proteins at an aspartate site
(enzyme)
- Caspases may also cleave each other — leads to a cascade of events

Caspases Cleave Essential Proteins

- 4 examples of what caspases do Protein kinase
- When caspase cleaves CAD — it activates it (DNA fragmentation)

Procaspase Activation
- Initiator or pro caspases are inactive
- Important in the overall activation of the enzyme
- An apoptotic signal triggers assembly of adaptor proteins that active caspases these initiator
caspases. Bind to caspases and cause conformational change
- These caspases go on to activate executioner caspases by proteolytic interaction (cleavage)
- 2 inactive monomers of caspases dimerize
- Sites are cleaved away
- Active caspases then come together to form complexes
- Executioner caspases — breakdown lamina and cytoskeleton
- B/c executioner caspase can be activated by inactive caspase
- Sets up a chain reaction of activation
- Once they are active, cleave multiple substrates

The Caspase Cascade

- One caspase activates many cascades
- Cleaves cytostolic proteins and goes on
- Once it starts, its a chain reaction that keeps going
- Amplifies the signal — this is why it can happen very quickly
 20

2 Major Pathways

- Adaptor proteins recruit complexes in different ways

Extrinsic pathway
- Procaspase activation triggered from outside the cell
- Cell death receptors
- immune system e.g.

Intrinsic pathway
- Procaspase activation trigggered from INSIDE the cell
- e.g. intracellular damage
- e.g. via Bcl-2 family of proteins (note; this may involve an extracellular survival)

EXTRINSIC PATHWAY
- The signal might be another cell
- Adaptor protein called FADD
- Binding recruits the adaptor protein to associate with the death receptor
- Bring together pro cascades
- Since they are close together, they can dimerize
- Get an activation and once its activated, we have an activation of caspases that go on to
executioner capsizes
- From pro caspase to active caspase to executioner caspases
- Binding of the ligand to the receptor recruits the adaptor protein
- Binding changes the conformation of the complex

INTRINSIC PATHWAY
- Cytochrome c is within the mitochondria
- Cell before and after apoptosis
- Fragmentation of the nucleus is seen here
- Release from mitochondria
- cytochrome c is involved in the intrinsic pathway
- number of signals can be initiated

Intrinsic Pathway
- Apoptotic signal activates Bcl2
- Mitochondrial membranes are fluid
- Bax and bad are found in the mitochondrial membrane so when there is an apoptotic
stimulus, it initiates the formed pores that allow for the release of many things from the inter
membrane space including cytochromsome c

Formation of apoptosome
- in the same way that signal that initiated the whole thing was Fas, the adaptor protein helps
create the complex, the same things that happens with Apaf-1
- Casepase cascade
- Apaf-1 is activated by cytochrome c; there is a conformational change; when the car
domain is properly exposed, the different Apaf-1 protein complexes come together and
form the apoptosome
- Apoptosome recruit the caspases (dimerize b/c they are so close to each other)
 21

PKB = AQT?

- Survival factor - phosphorylates protein kinase B

- Removes Bad protein
- Inhibits cytochrome c
- Active Bcl-2 inhibits cytochrome c release
- If you inhibit production of Bax protein, release from mitochondria
- Inhibit the synthesis of Bax protein
- Stabilize p53 — promotes synthesis of Bax proteins

Apoptosis in the Developing Nervous System

- Recall NGF
- Neurons release NGF
- Concentration is very high and as you go further away, it gets lower and lower
- When connection is made, NGF promotes growth of these cells to the target; once the
release of these factors is abolished; there is no survival factor
- Without survival factor, Bax expressed and cytochrome c is released
- Forebrain protusions in the absence of the enzymes
 22

LEC 10: MITOTIC SPINDLE October 13th

- Fluorescence image — MT in all different directions

- dynamic stability (+ end = growing end reaches out looking for sites of attachment)
- nucleus broken down — just before metaphase
- centrioles have already divided
- stretch out so they can cast a net over the nucleus

Major Events of Mitosis

- Centrosomes duplicate (in G2)
- Dividing and spreading out to caste a net over nucleus
- Mitotic spindle formation (prophase)
- Chromosome condensation (prophase) — condense b/c there’s a lot of information and need
it to be in a compact form
- Breakdown of nuclear envelope (prometaphas)
- Chr. attachment to Mts begins (prometaphase)
- The metaphase plate

Increase Dynamic Changes

- Very important up regulation of dynamic changes
- During mitosis, M-Cdk is activated. Decrease in the activity of MAP and increase in kinesins.
- Kinesins — induces catastrophe
- Increase in dynamic changes of Mts
- Increase in catastrophes and rescues between the two states
- Help to solidfy/stabilize mitotic spindle

Components of Mitotic Spindle

1. Microtubules — Astral (grow in all different directions — become kinetichore); once they
come in contact with kinetochore, they become kinetochore MT
Kinetochore — associated with the kinetochore of the chr. pair
Overlap — overlap and interact with the other MT

2. Motor proteins
- Kinesin-related (+)
- Dyneins (-)
- Many of the MT end up being ejected from centrosomes

3. Chromosomes (chromatids)

4. Centrosome
- Centrioles
- PCM
- help to organize minus ends

Motor Proteins Contribute to Spindle Assembly

- Tail regions are different
- Tails of the motor proteins crosslink
- Heads are associated with 2 different layers of MT
- Kinesin associates with itself and other partners
 23

- Sliding movement is possible

- Cause change in the overall length of mitotic spindle
- The overlap is gonna be longer
- Movement of MT is opposite
- MT move outward — on a large scale, the entire mitotic spindle increases in length

Motor Proteins Contribute to Spindle Assembly

- kinesins work at the overlap sites
- dyenins are important at minus ends where mitotic spindle has formed

Spindle Pole Separation

- Prophase
- KRPs push overlap Mts
- Beginning to elongate MTs
- More dynamic stability; more attachment
- Elongation and point of focus where the dyenins are
- Catalytic heads attach and walk closer to the centrosome but can’t walk anywhere cus the tail
ends are associated with the cortex

Chromosome Condensation
- Mechanism by which chromatin condenses to chr. pairs
- Occurs at prophase
- Cohesin is replaced with condensin
- Cohesin is cleaved by separase
- Anaphase promoting complex leads to the breakdown of cohesin
- Eventually to create a condensed chromosome so its easier to separate
- Different chromosomes have different sizes
- Cohesin right between the kinetochore (some left)
- Condensin is dependent on ATP hydrolysis
- Hydrolysis of ATP and phosphorylation of condension by M-Cdk

MT — Kinetochore Attachment

- Nucleus is gone and chr. is released

- The chr. need to be caught by the MS
- Astral MTs reach out b/c of dynamic instability
- Come in contact with the kinetochore — which is the site of attachment
- Astrals come out and make contract laterally
- Attachment of the kinetochore to the MT increases stability and reduces catastrophe
- Upon attachment, the kinetochore migrates back and produces a perpendicular attachment to
the MT which provides maximum stability
- Bipolar attachment — catches chr. pairs

MT — Kinetochore Attachment
- The way the MT attach to the kinetochore is indirect
- Presence of depolymerase causes MT to fall apart
- CENP-E walks in the + direction
- eventually they produce forces strong enough to pull chr. apart
 24

MT-Kinetochore Attachment
- Motor proteins: balance by astral ejection force
- Attachment of motors
- Chr. is the cargo; walk to the + end
- The force that causes it to pull is the dyenin motors
- Dyenines associated with the MT and kinetochore — allow the chromosome to move towards
the minus end of the centrosome

Consequence of Missing a Motor Protein

Poleward Flux
- Important in lining chr. at the metaphase plate and maintaining the alignment to stabilize
- Eventually comes to a phase where there is a balance
- Spindle poles = many MT that can do the same things simultaneously
- No direct binding between kinetochore and MT is b/c the structure needs to be modulated
and modified depending on signals received from the cell b/c we need to add or remove
tubulin
- Rapid addition of tubulin subunits and rapid loss of tubulin subunits and eventually the chr.
gets closer to the middle as more tubulin are added
- Treadmilling = like poleward flux
- Constant renewal
- Depending on where chr. are, there is a signal that modulates these addition and subtraction
until it gets to the centre
- At some point at metaphase, all the subunits are aligned but the length doesn’t change as
much (treadmilling)
- Fine balance is created b/c flux is in the direction of the pole and subunits appear to be lost
on the other end
- Flux goes towards the pole
- Explains what happens when all of this breaks down when chr. begin to separate

Summary: balance at metaphase plate

1. Motor proteins
2. Poleward flux
These provide balance

Anaphase A Separation
- Anaphase A vs. Anaphase B happen simultaneously
- Anaphase A is the poleward movement of the chromatids by the shortening of the kinetochore
MT
- Constant addition at plus end and constant deletion at minus; but if there is only a constant
deletion, then the overall length decreases
- Cohesins are lost
1. MT depolymerization at + ends by KRPs
2. Continual loss of T at - ends ends
- GTP hydrolyzed and blows apart
- MT are depolymerized b/c of KRPs
- Continual loss of T at the - ends without addition at _ end
 25

- Pulls chromatids

2 Separation Forces
- Disc shape = kinetochore
- Ndc80 complex is one protein complex that helps to connect the chr. with the MT
- No addiitonal energy is required
- GTP hydrolysis gives it the energy; utilized now and made when the MT came together earlier

Combined Model for Anaphase A

- Micrograph showing MT
- Dam1 rin
- At anaphase, depolymerase causing the MT to fall apart (GTP hydrolyzed and protofilaments
are curved)
- The model predicts that as it falls apart, the ring forms a collar around the MT and allows
transfer of the energy to carry the ring along to the minus end and carries the chromatid along
with it

Experimental Evidence
- Depolymerization drives enough energy to drive the chr apart

Anaphase B Separation
- Occurs in parallel with A
- In A, the centrosomes were in the same position but here they are separated
- Length of MT attached to the kinetochore does not change
- Further elongates the spindle
- When we stretch it, the chromatids get further apart
- Dyenins interact with catalytic ends attract with the + and and tails with the cortex
- Also pushing of motor proteins at the overlap MT (kinesin motors)

Action of Motor Proteins in Anaphase B

-