Lecture 11 – Actin I

- Cells need to move, what is the actin cytoskeleton, how is actin organized.
- Cells come in many shapes
o There’s stratified squamous tissue, cheek cells
o There’s a variety of shapes like in epithelial cells, shaped like flat pancakes due to thick
nucleus
o Squamous tissue is like cubed and stacked up. Not flat and cube in shape.
o Muscle cell, individual is a very long skinny cell that may span throughout a muscle fiber
and that has the ability to contract to become a shorter thicker cell in the process of
muscle contraction.
o The neuron has cell body which extends axon and dendrites.
o All the cells have the same genome, the same proteins, how can cells with the same set
of genes and protein building blocks create many different shapes?
- Cells are internally organized
o Have a sense of internal polarity, have the ability to position proteins in themselves in
response to geometric cues.
o With prokaryotes, not much of this organization because it’s just protein diffusing
around.
o In eukaryotes, there’s a complex 3D arrangement of internal components
- Cells import and export cargo
o The microtubules are the highway and there are various proteins attached to
microtubules and moving around.
o Cells must be able to receive signal and internalize it. Import using endocytosis. And
once the components are inside how do they move around?
o How do they establish these roads and who are the proteins responsible for the
movement itself
- Cells move and migrate into wounds
o Skin cells move around. If you cut your finger cells have to move into the wound to
repair it
o Secrete extracellular matrix, generate new skin cells.
o Immune cells migrate towards the wound

Cells construct an internal skeleton called the

cytoskeleton
- the skeleton of different types of cells has
different cytoskeleton
- cells accomplish the same thing by building
for themselves an internal skeleton
- made of 3 major elements, microfilaments
(actin filaments), microtubules, intermediate
filaments

The cytoskeleton plays key roles in cell physiology and metabolism

- responsible for shape, structure and stability. Whether its stiff or soft cell
- intracellular transport. Filaments create roadway network which cells move their internal
components and an architecture inside, so the cell knows whats top and bottom
- responsible for spatial organization
 - contraction and motility.
The cytoskeleton is made of 3 major
polymer systems
- Microfilaments: made of
actin. Long thin stress fibers
o must be polymers
They make proteins
that have the ability
to self-assemble into
structures.
o If they don’t need it,
they can break the
polymer system
down and rebuild it
in another way
- Microtubules: made of alpha beta tubulin heterodimer. Assemble into long hollow tube
- Intermediate filaments: made of various because there’s several different kinds of intermediate
filament building blocks that’s more diverse.
- Actin is big brother of the cytoskeleton. Microtubules are the kid brother. Discovered later in
1970s. Intermediate filaments are the forgotten stepchild hard to get good knockdown
phenotypes, not a lot of labs work on intermediate filaments.

The cytoskeleton is a type of ‘tensegrity structure’

- Microtubules = poles
o Very stiff
o Resistant to compression
o They’re big, thick and very stiff. If try to bend, thick Plexiglas like structure. Resistant to
compression. Overall, the highest mechanicals strength
- Actin filaments = wires
o High tensile strength
o If try to pull, does not stretch
o But flexible, if try to bend, it’s not that hard to bend.
- Intermediate filaments = ropes
o Elastic and flexible
o If you pull, it stretches.
- Cells throw together through polymerization process and this complex mixture of these like at a
construction site.
- Tensegrity structure: the Manhattan toy skwish color burst, tension held
o building held together by compressive forces.
o Foundation, rests on earth, then the of the house attaches and go up and whole thing
compressing down on foundation and presses down on earth
o Tensegrity structure held together by tension. Tension pulls it in multiple direction that
the combination of force is enough to suspend stick in air.
o Each are being pulled away from each other in a way it spreads the structure out

Tensegrity was coined by Buckminster Fuller and explained geodesic domes

 - Outside of structure is not held together by compression, only by tension. The biosphere in
Montreal is also a tensegrity structure.

Actin is an ATPase that drives cell motility

- In the actin there’s 4 lobes and an ATP binding cleft
- Discovered by Bruno Straub in 1942 a Hungarian biochemist
in the lab of Albert Szent-Gyorgyi
- At this time, already discovered myosin in muscle. At the
time, working on muscle tissue and knew that muscle tissue
had the ability to be viscous and fluid.
- If they added ATP to viscous myosin, it would become fluid
again, and then purified actin and found out actin facilitated
the fluidity of myosin.

Actin polymerizes into a two-stranded helical filament

- The actin polymer. The actin itself is asymmetric, ATP cleft on one side
- The filament formed is also polarized. There’s a + end that is the smooth
surface of the actin and the – end is the groove end
- Black dot to see helical of the path

Actin is major component of muscle

- If looking at myofibril, it’s mostly actin filaments. The interaction between
myosin and filaments during muscle contraction.

Actin filaments are arranged differently at distinct

intracellular locations
 Red is the actin that form differently in different
cells. In the microvilli it’s all on the top. Cell cortex is
around. Cells build a cage around the plasma membrane
 Cells that need to make tight connections: Adherens belt
 In migrating cells, there’s many different type of actin. Filopodia,
lamellipodium, stress fibers
 Roles for actin for endocytosis of structures. Phagocytosis, neutrophil
eats bacteria.
 Contractile ring, in mitosis or cytokinesis, in the end of mitosis there’s a
contractile ring that pinches the two cells in half.
Cells control: length, number, angle, bundling, orientation
- Cells must be able to control these aspects of the actin polymer

Microvilli and stress fibers contain bundles of actin filaments

- There’s parallel actin bundles in the stress fibers
- The actin filaments are relatively long in stress fibers
The leading edge contains a branched (dendritic) network of actin filaments
- A migrating cell, you can see the actin filaments are forming dendritic
network.
- Why do we want this type of network at the leading edge of a migrating cell
but stress fibers long parallel bundles?
- Must be some protein that make them branched.

Actin filaments drive cell movements

- Lamellipodium (leading edge) thick band of actin that moves out along with the cell.
- The brightness of actin is not homogenous, as leading edge moves forward, band stays relatively
constant.

Cells move
- focal adhesion is the cell’s feet, it’s where the cells touch the
surface
- extensions: extend out in the direction of movement. Through
extension of lamellipodium
- adhesion: make a new adhesion so the cell has to put its new feet
on the surface
- translocation: once it’s contact, it’ll have to bring it’s center of
mass in the direction of movement.
- De-adhesion and endocytotic recycling: pick up foot and then
recycle back to create a new foot
- Extend foot, extend skeleton, move center of mass, pick up back
foot.
- Cells do the same thing
- Actin filaments extend the lamellipodium and pull the cell
forward.

The leading edge pushes forward and stress fibers pull up the rear.

Lecture 12: Actin II

Actin polymerization occurs in 3 distinct phases

- Start out soluble in G-actin (globular) it’s the protein in its monomeric
form and has ATP in its nucleotide binding pocket
- Slow step which is the nucleation process. 3 of these subunits come
together to form a nucleus. Each one of the 3 will make 2 bonds with
actin. The trimer is stabilized by 2 bonds in each case
- Now, add actin to it called elongation and eventually hit steady state
where the amount actin to polymer is
the same as the amount coming off
The 3 phases of actin polymerization are observable in the laboratory
- getting 3 actin to come together at the same time is low
probability so it’s very slow step nucleation
- once the stable nucleus is made, it’s easy

Actin polymerization is described by rate constants

- actin is a polar polymer the structure itself is polar with a +
end and a – end
- can measure different rate constants at the 2 different ends
- measure association rate constant of actin subunits to growing + ends and you get
12/micromolar actin in the
solution per second it’s
concentration dependent
- Now the actin dissociation is
1.4s^-1 now
- Can get critical concentration
and that’s the concentration
where the on rate and the off
rate are balanced.
- 12*0.12 = 1.44 so then you’re at equilibrium from the + end
- But different rate constant at the – end.
o On rate 1.3 micromolar/second
o Off rate 0.8/second
o If you compare, you get critical concentration of 0.6 micromolar.
- You have a gap between the critical concentrations at the + end and the – end, we can describe
the net work to be subunits added – subunits lost
o Kon (subunits) – Koff
o Cc = [subunits] when network is 0
o If you know your two rate constants and you can solve for critical concentration
o Koff/Kon = Cc

Actin ‘treadmills’ at steady state

- There is a concentration at which the – end is
shrinking but the + end is still growing
- When you hit 0.6micro molar, it’s balanced but then
the concentration is still above so the + end will
continue to grow. Will continue to add subunits and
decrease concentration of actin further
o So then the constant is greater than the concentration in the negative end so it’ll start to
shrink
- Amount of polymer is constant but it’ll be translocating itself through space. The entire actin
filament would be moving through by polymerization.
- Cells have take this fundamental process of treadmilling and using that to drive forward motion.
 o Actin filaments rapidly treadmilling and the entire cytoskeleton treadmills the cell
forward.
- What is the regulatory apparatus that allows this?

Actin regulators allow cells to build diverse and dynamic actin networks
1.) Grow the filaments: Formins
2.) Cut the actin filament: Cofilin
3.) Cap the filament: capping protein (do not want actin to grow)
4.) Make branched networks: Arp2/3 (make dendritic branch actin networks)
5.) Cross-link the filaments: alpha actinin

Cross linkers have 2 opposing filament

binding domains
- Need 2 hands to grab 2 actin
- Cells divide whether the two hands
grab the same polypeptide or 2
polypeptides that dimerize
- Can we control the spacing between
the actin binding domains?
- Fimbrin and alpha actinin have
different spacing
- Alpha actinin is homodimer with antiparallel section
- Fimbrin has 2 hands close together and grab close together
- Spectrin: 4 polypeptides. 2 alpha 2 beta Can hold actin in diverse form
- Filamin: single polypeptide with hinge in the center that allows it to cross link actin filaments in
weird figures

The leading edge of a moving cell is a branched actin network

- Even though cell moves, thickness of band stays constant
- The forward motion is caused by the polymerization of new actin subunits pushing it out, there
must be other processes that those actin filaments depolymerizing the actin from the back end.

A pathogenic bacterium hijacks the actin machinery of human cells

- Listeria: found in raw milk cheeses
- It gets inside your cells and builds dendritic actin network out of your proteins and use your
proteins to rocket around your own cells.
- Does it to propelled itself out of host cell into extracellular space so it can go on to infect
another cell.
- It will shoot itself out of the cytoplasm into extracellular space to infect other.

Listeria “comet tails” are nucleated by the Arp2/3 complex

- Behind each bacterium, there’s a comet tail of actin and by understanding how this bacteria is
able to do that, we’re able to discover key players involved in this processes. Activators,
nucleators etc.
- Listeria comet tails are nucleated by Arp2/3 complex

Arp2/3 mimics an actin nucleus and nucleates daughter filaments at 70 angles

 - Actin related protein 2/3 complex.
- Eliminate the need to make actin nucleus. It builds a nucleus for the
cell such that the only thing that needs to occur is elongation.
- Conformational change occurs so arp 2 and 3 comes together loosely
together to match shape of actin filament to make template
- Then daughter template emerges at the 70 degrees angle
- This process preserves orientation. The + end of mother filament is
pointing the same way as + end of daughter filament
- The Arp2/3 complex must be activated!

The 70 degree angle creates optimal angles for pushing out the plasma
membrane
- There’s biophysics types that built a physical model of what’s
happening. Actin filament pushing into plasma membrane against
opposing force (load force)
- Actin network is assumed to be ridged network and the filament is
pushing it at an angle
- In order for polymer to push against anything, must have something
similar to a Brownian rachet
- Have something driven by thermal fluxuation that needs to be
rectified or set to motion similar to how Brownian C ring had to click
into place
o The vibration is the plasma membrane.
o Sometimes plasma membrane will push out far enough that new actin subunit can
sneak in and bind to the end of the filament.
o Polymer longer, membrane can’t bounce back anymore because it’s hitting the
elongated polymer
- Let membrane diffuse out, add subunit and can push membrane out in that way.
- They vary the angle to see the pushing reaction. That angle gives the new actin subunit a bit of
extra space to sneak in.

Wiskott-Aldrich Syndrome family proteins (WASp) activate Arp2/3

- A disease of immune system because neutrophiles are highly dependent on actin polymerization
for their migration behavior.
- Defects cause disorder.
- have 3 domains (W, C, A)
- The W domain (WH2) binds an actin
monomer.
- A (acid) domain binds to the Arp2/3
complex and then make complex go
through conformational change and then
add actin bound to the actin mimics on
Arp2/3 and then you have 3 actins!
Nucleated new daughter filament.
- The W domain (WH2) binds an actin
monomer.

Listeria are coated with ActA, a protein that mimics WASp

 - Adding Arp2/3 complex and WASp with 3 key domains, the nucleation lag phase is eliminated
and the amount of polymer spikes up because no longer need to wait to form nuclei.
- Already formed by putting Arp2/3 in correct complex and adding first subunit in the acidic
domain

The lamellipodium must overcome 2 major challenges

- #1: running out of actin
o Must make sure we have a constant supply of actin. Actin polymerization is
concentration dependent as the actin is consumed out of solution expect the net rate of
addition to decrease as a result, we result it to slow down but it does not.
o The rate of outward movement is pretty constant.
- #2: preventing futile polymerization
o How to prevent it from not pushing out of element
o A waste.

Profilin recycles actin and blocks minus-end polymerization

- Binds to actin subunits and it helps recycle their nucleotide in a way that has the added benefit
of blocking – end polymerization.
- We want actin to be adding to the ATP state, but when it comes off the polymer in
the – side, it’s in the ADP state
o Must recycle to keep energy source around
- Profilin cycle kicks out the ADP and puts an ATP in. profilin detaches and goes to
recharge a new actin filament
- It also blocks minus end addition because it’s bound to the smooth surface not the
grooved surface. Plus end growth is the grooved surface that binds.
o In order to bind to negative side, it’ll need to attach to smooth end but
profilin is there preventing it

ADP/Cofilin severs actin filaments

- Actin depolymerization factor or cofilin is the orange protein
binding to the ADP section of protein
- Actin binds to ATP state, so hydrolysis state that creates
actin-ADP-Pi and then phosphate release cause Actin-ADP
- Binds to ADP actin and cause fragment of sever into tiny
chunks and then because they have more ends and
depolymerization happens in the end
- Can rapidly shrink. Then Cofilin unbinds and then goes back
to bind and then Profilin binds to it.
- Actin is 2 stranded helical like a rope and Cofilin does,
untwists and unwinds the actin filaments so creates regions
of local mechanical weakness which are points of severing.
- Cofilin binds to ADP actin subunits, unwinds, creating regions
of mechanical weakness and then fragments of actin filaments that depolymerize.
Together, Cofilin and Profilin accelerates actin
treadmilling
- The Thymosin beta 4 keeps the subunit locked so
they don’t use it and when its needed release it
from the protein so polymerization can keep
going

Capping protein prevents futile polymization

- Not every actin filaments that’s growing is hitting
the membrane and to prevent futile, use Capz
- It takes the actin filaments and caps it. We have
ones at the + end and – end called tropomodulin
- Capping protein, is the rate it can find a new actin
filament end.

Listeria motility is driven by the coordinated action of

only 6 proteins
- New actin filaments that are pushing on the
bacteria and those filaments can grow for about a
second before capping protein caps them
- But during that one second, a new Arp2/3 complex is bound and
a new daughter filament is built and this cycle goes on.
- This works because capping proteins is slow enough to cap the
proteins but because they’re slow, it can still grow the branch
- The rate constant of capping that gets it just right

The movement of listeria can be reconstituted from purified

components
- Can have beaker of those purified proteins and can drop listeria
in the beaker and they will start swimming around.

Reconstitution enables measurement of each protein’s contribution

- Rate of listeria motion as a function of concentration of different
proteins
- Concentration of capping protein too high, all the filaments get capped and listeria doesn’t
move but if it’s too low, the lack of motion caused by different
problems the excess of futile polymerization.
- They all have an optimum concentration for listeria motion.
- No capping protein, fish bone structure.

The leading edge and listeria comet tails are functionally equivalent
 - The exact same network that’s a group of actin proteins accelerating the actin of proteins
pushing out of membrane
- This whole network is pushing the plasma membrane and migrating the cell

- They saw there must be some

activator for Arp2/3 complex
- And something that keeps them
sequestered and inactive
- There were unknown factors because
they were able to see that on the
basis of limited amount of
information the logic of this protein
network
- In order for Arp2/3 complex to work,
we’ll need other accessory proteins
to influence it

Lecture 13 Actin III

Contraction and chemotaxis

The leading edge of moving cell is a branched actin network

- Resembles comet tails built by listeria which hijacks the actin machine.
- Made of 6 key proteins, WASp, Arp2/3 make new daughter filament
- Cofilin and Profilin accelerates actin treadmilling
- Capping protein prevents futile polymerization
o It’s the kinetics of the capping that allows the whole system to work. 1 second to cap.
How do cells steer its self? chemotaxis

Stress fibers are actin filaments that can contract and

produce force
- How is it going to pull its backfoot up. It uses a
different type of actin to pull its foot up
- Stress fibers, they are actin bundles that can
contract and produce force.
- Blue protein called myosin in the middle
- Integrins are stuck to extracellular matrix and
they are central components of focal adhesion and attached to the feet in the back, is the stress
fiber. It’s able to contract in order to pull up the cell’s back foot
- These actin filaments are long linear actin filaments not the branched arrays in lamellipodium.

Formins polymerize linear actin filament

arrays
- The donut has ability to rock back and
forth. Rocks up to create binding spot
for actin subunit. (FH2 domains)
 - Once it rocks, it makes a new spot for another actin subunit to come in.
- There’s flexible arms.
- FH1 domain binds profilin-actin.
- the donut has long flexible arms that can grab profilin-actin and then feed it back on the chain

Formins add >10,000 monomers before detaching

- a lot of cycles, if the formin in open state and the if the other side accidently goes into
conformational state and the whole thing would fall off
- the entire machine can only rock up if the other side wasn’t rocked up.
- Highly progressive

Myosin is a protein that produces force

o Motor proteins, proteins that are able
to produce force. Can take ATP into
mechanical work
o Must be magnified in large distance to
make changes in the lever arm
o Same protein responsible for forces
produced by muscle contraction
- The nucleotide binding pocket produces big force.
- The ATP causes a swing in myosin cause actin filament to get displaced and pushed by the
conformation change in the myosin

Myosin translocates actin filaments across a glass slide

- Purify the head fragment and then add to microscope cover glass and myosin will all stick
- If you add actin filaments land on them and move around the surface of the myosin heads.

Myosins come in many flavors

- Myosin IIs involved in contraction

Non-muscle myosin-II forms bundles that pull actin

filaments inward
- antiparallel, myosin heads on both sides. And
all the ones on the left hand side pull towards
the right and vice versa
- Slide actin filaments in toward towards each
other.

Myosin-II bundles contract actin arrays

- Slide inward relative to each other in order to
contract

That’s how cells pull up their back feet. Slide actin

between myosin and detach backfoot

Different actin networks exist at the front and the back of the cell
- Cells must be able to put different actin networks in different locations in the same cytoplasm
- In the leading edge, there’s Y shaped. At the inside, there’s contractile bundles
 - How do they know where to put the stress fibers and where to put the leading edge?
- They can locally control what type of actin network they’re building. Must be able to build
branched network in the front stress fibers in the bac. Cells must know where their front is and
direction. Must be linked to sensing of outer environment (food etc) if food moves, reconstruct
Dictyosteliumis an amoeba that aggregates with other amoeba upon starvation
- as no more food, they crawl towards each other to aggregate into a large mass.
- Known as a slug. And once that occurs, they extend something that will come out of the screen
as a stock and at the end, there will be fruiting body
- If an entire colony of these free-swimming individuals, finds starvation they work together to
create a fruiting body and mass together. Send some of their stuff into fruiting body and it gets
carried away by things so to find new place for more food.
o What are the chemical signals that are drawing these amoebas together?
o Starts to secrete cyclic-AMP when its starving

Individual Amoebas move towards concentrated cyclic-AMP

- Scientist can cause them to move by exposing them to concentrated cyclic AMP
- When needle moved, the concentration of cyclic AMP gradient shifted and its able to reorient its
actin cytoskeleton.

Rho family GTPases trigger the 3 types of actin network

- Dominant active Rho: Stress fibers
o If injected with that, it means the Rho is permanently turned on. The cell becomes all
stress fibers. All of the actin becomes stress fibers.
- dominant active Rac: lamellopodia
o If inject, nothing but huge lamellipodia
- dominant active Cdc42: Filopodia
o If inject, only get filopodia which only exists at the very edge of the lamellopodia. But
now it’s only filopodia.
- GTPases are in charge of actin organization
- How can a signal from extracellular space change activity level of Rho family GTPases?

Rho GTPases translate outside signals into changes in the

actin cytoskeleton
- GTPase exchange factor (GEF) activated after the
ligand reaches the receptor and then interact with
Rho and put a GTP into the Rho protein and take out
the GDP
- Now, you have an active Rho
- A conformational change in the Rho which will
enable it to interact with various effector proteins.
- To take off, use GTPase activating protein (GAP) take
off the phosphate from GTP to turn it into GDP again
- GEF = go, GAP = stop.
o Activity mediated by extracellular signal
- Have proteins that can sequesterion
- Dominant activate means mutation in GTP pocket and the Rho not able to be shut off and it gets
stuck
 - The effector proteins need to build stress fibers

Rho activates Formins by relieving auto-inhibition

- Rho needs to activate forming by relieving the
auto-inhibitory mechanism in the forming.
- FH2 arms, FH1 donut and then the RBD builds
into Rho and then so that the two domains can
not polymerize actin filament
- If it gets switched into GTP, will interact and the
FH1 and Fh2 domains are released and profilin-
ATP-actin will be taken by the FH1 domain and
then shuttled into the Fh2 domain

Cdc42 activates Arp2/3 by opening up WASp

- WASp also has Rho binding domains.
- When in active state, rho binding domain will bind
onto the Cdc42
- And then go through the Y actin nucleation

Cells need all 3 Rho GTPase to move

- they need stress fibers in the back and
lamellipodium in the front
- Scratch assay
- Confluent cells and completely cover the surface of
the petri dish. Then, we scratch across the petri
dish and get rid of a line of cells
- That creates a ‘wound’ in the cell as there’s a
stripe of cells missing.
- The cells adjacent to the scratch will sense
there’s nobody next to them anymore so they
start migrating into the gap. They’ll move into
the wound.
- If you plot % of wound that’s closed, and get rid
of any of the 3 rho family GTPases, so inject with
dominant negative forms, in all 3 cases, we lose
ability to close the case.
- Dominant negative means it cannot interact with
its GEF so it’ll be permanently turned off it’ll be
trapped in the GDP state because cannot interact
with GEF.
Rho family GTPases trigger the 3 types of actin network
- Active Rho interacts with kinase and phosphorylates light
chain myosin. Gives rise to myosin activity and gives rise to
contraction of stress fibers
- Rho both creates long stress fibers and triggers their
contraction
- Cdc42 triggers WASp and RAC triggers WAVE
- They also interact with each other.
- Cdc42 in its active state will trigger a Rac GEF, it causes Rac
to be turned on and the active Rac will dot he same to Rho
- It’ll trigger activity of Rho GTPase which will interact with
Rho GEF
- But once Rho is turned on it’ll try to shut off Rac.
- It’s the cross talk between the Rho family GTPases that
allow cells to create stable zones of rho family activity which is required to make stable zones

Extra cellular signals create zones of different Rho activity

- That signals spreads backwards into the plasma
membrane
- Eventually causing Rho activation in the back
- From these locations of high cyclic AMP you have a
biochemical wave of Rho activities.
o Cdc42, then Rac and the Rho activated
- At the front, high activity of GTPases especially Cdc42
and Rac
- As the Rho signal gets going and reaches the back, the
Rho will push back on the wave of active signal fighting
the spread of Rac activity to the back of the cell and the
competition between extracellular signal driving Cdc42 and Rac and reversal from Rho will
create stable zones of actin activity.
- When scientists move the needle, now the location of the high concentration of cyclic AMP
changes and that changes the properties of the membrane and get a new local region of high
Cdc42 and then spread back and then trigger Rho and then eventually pushes back.

The logic of chemotaxis is conserved from dictyostelium to human neutrophils

- From free living amoeba to our neutrophils right now.
- There’s also Rho Rac Cdc42 reaction and all those proteins and same set of 6 proteins in our cells
that drives this forward
- This circuit was evolved very early in eukaryotic life.
- It’s been conserved over a very long time
Lecture 14 muscle contraction

We’ve learned that cells have ability to put different actin net works in the front and the back of the cell.
- Back of cell stress fibers with non muscle myosin muscle fiber
- Extracellular signals create zones of different Rho activity.
o Cdc42 -> Rac -> Rho then inhibits Rac then goes back
- Myosin’s is a protein that produces form and converts chemical energy into mechanical work
(motor proteins). Drives muscle contraction and cell migration contraction
o It binds to actin filaments (head domain) ATP hydrolysis and phosphate release which
accompanies large conformational change.
- Myosin translocate actin filaments across a glass slide.
- We’ll see bundles of myosin that pulls actin filaments inward.

Muscles are made of billions of contractile bundles

- Made of many parallel myofibril and they extend and
they’re further subdivided into sarcomere.
- Sarcomere is the basic unit of muscle contraction
- Contains many actin filaments, myosin and bounded on
either side by the Z disk.
- Dontraction of them is the condimental event that leads
to contract
- If you have many sarcomeres, many myofibrils meaning
many muscle multinucleated muscle cell etc.

Sarcomeres are the basic unit of the ‘sliding filament theory’

- This idea was discovered in the early 1950s.
- Andrew Huxley also figured out the action potential.

Huxley and Hanson were able to

photograph a contracting muscle
fiber
 You can see thick bands
(filaments) and as the muscle
fiber contracted, size of thick
band was constant but the size
in between them was shrinking
 Able to see that the thin filaments (actin) were going in
to the thick filaments.

Sarcomeres are tightly packed arrays of actin filaments and myosin filaments
- The thick is the myosin band and then the thin is the actin filaments and can identify the Z disk
- If look at the Sarcomeres end on, you can see crystal impacting of thick filaments s and thin
filaments
- Nearly crystal in density of thick filaments and thin filaments. Around every thick, you can count
lots of thin.
- Thin filaments are surrounding so the myosin heads always have thin filament to grab on to
Sarcomeres undergo rapid contraction upon muscle stimulation
- Go from actin filament spread out to a contracted statement where actin filaments go inward
Contraction is FAST!
- Only 50ms for a Type-II muscle fiber (fast twitch)
o 5-10ms latent period between signal from brain and start of contraction
o 40-45ms contraction phase from fully elongated to fully contracted
- Very fast process. One of the challenges for scientists is that you must have a fast camera
- How does myosin produce this force and convert ATP hydrolysis and energy into
contraction/force

The crossbridge cycle couples 1 ATP hydrolysis to 1 “power

stroke”
- The myosin exerts force Is the power stroke
- Start at the state where myosin is dethatched to the
actin filaments and has recently bound to ATP
- It’s the binding of ATP that releases myosin head from actin filament.
- Once its in the ATP state it’ll be able to reversibly hydrolyse that ATP molecule

ATP hydrolysis puts myosin into a strained conformation

- Hydrolysis of ATP to ADP + Pi myosin head rotates
into “co cked” state
- Myosin is going between the co cked state and the
relaxed state.
- ATP Hydrolysis and recondenses while it’s waiting
for your brain giving the signal

Myosin binds the actin filaments in the ADP-Pi state

- Brain sends signal and the myosin binds to the actin
filament in the ADP-Pi state

Phosphate release relaxes the strain the myosin head

- Phosphate released triggers power stroke, large
conformational change push actin filament to the right

New ATP binding releases myosin form actin filament

- ADP falls out and then ATP binds and that releases actin
from myosin

We store strained energy in the ADP Pi state and release in upon

the Phosphate release and then we harness the release of
strained energy to move.
Failure of myosin to detach in the absence of ATP causes Rigor Mortis
- When animal dies and muscle becomes very stiff and hard to move the body of dead animal
because frozen muscles.
- This is caused because after death ATP in muscle is going down and without new ATP to bind
into the myosin head, the myosin heads become permanently attached to actin filament
- Frozen in the nucleotide free state. ADP has come out but because no new ATP to come in, can’t
detach
- freezing the thin and thick filaments to each other so they can no longer slide.
- When muscle tissue enters this state, meat industry considers muscle tissue into meat.

The “power stroke” is understood at atomic resolution

- Have crystal structures of myosin at different states
and can piece together at different conformations.
- Field gets reputation of eating its young because
you’d have to compete with all the old ones that
are in the field.

The neck domain of myosin acts as a lever arm

- have actin binding domain and nucleotide binding site and that’s
where the conformational changes happen that translates into
movement of green and blue domain here thought as lever arm.
- Motion of lever arm is what controls how far the actin filament moves
- The longer the lever arm, each conformation change leads to a bigger
change of the displacement
- As they lengthen that segment, velocity of actin filament movement in
glass assays started to move faster
- Each throw of actin is attached to longer lever arm.

What are the magnitudes of cellular forces?

- 1pN. = 10^-12 newtons
- KbT = 4.1 pN nm
- Myosin produces 5pN of forces over a step of 10nm
- Myosin produces 50pN nm of work with each ATP hydrolysis cycle
- Myosin more than 50% efficient in terms of how much energy its able to harvest and directly
convert into work.

Consider a powerlifter squat

- 100kg squat, 500nM of work (half a meter), 5 x 10^23 pN nm
- Myosin produces around 50 pN nm of work
- When you compare, you can see how parallelized this reaction is
- Billions of myosin simultaneously engaging in parallel arrays to generate enough energy to lift
this weight.
Muscle face 3 design challenges
- Prevent continuous contraction
- Activate contraction (again)
- Freeze the structure of the sarcomere
o How do they not keep building and stabilize this structure
o Stop building actin

The structure of sarcomere is set by nebulin, titin, and

capping proteins
- Actin is capped. At the – it’s capped by
tropomodulin and at the + capped by cap z
- Actin is coiled by nebulin. Have a protein held both
of the caps and that sets the length of the filament
- Titin is the largest protein in our genome and it
spans between the 2 Z disk to help create that
spacing and as a elastic dampening and store
elastic energy in muscle fiber
- Maybe that’s why when you stretch you store
elastic energy in titin folds
- They fold together to establish the structure.
- There’s significant turn over in all of these
components. They’re maintaining in this dynamic
state

Tropomyosin blocks the binding site for myosin on the actin

filament
- Take a protein and stick it on the actin, myosin is there but
can’t get to the actin
- In absence of calcium, tropomyosin binds directly over the
myosin binding site
- the myosin is in the co cked state but cannot bind because
its blocked by tropomyosin

- Motor neurones transmit signals from the brain that activate muscle contraction
- Stimulation by motor neurons cause a rapid spike in Calcium
concentration in muscle fibers

The sarcoplasmic reticulum is a calcium storing organelle

- it triggers it to release calcium into the muscle cell

Muscle contraction is stimulated by the presence of calcium

- once you past particular calcium threshold, the rate of ATP
hydrolysis increases
Troponin binds calcium and pulls tropomyosin out of the way
- once it’s bind to calcium, it’ll rip tropomyosin out of the
way to expose the myosin binding site so the myosin in
the co cked state can bind to the actin.

Muscle contraction can exert massive force and refined

movement
- we can get big powerful muscle groups and harness into
big forces
- but can also parallel that into subtle sophisticated motion
like music playing.
- The creation of speech, movement of lips and tongue that allows us to create complex speech

Chapter 15 microtubules

Cytoskeleton made of 3 major polymer systems

- actin, microtubules and intermediate filaments
- microtubules are thick hollow tubes made of alpha beta tubulin dimers

Early observations: Keith Porter

- in the early 1960s
- there is thin little tubes in the plant cell (microtubules up against plasma
membrane) in the cw is the plant cell wall.
- Porter named these microtubules
- Gave cross sectional images knew they were made from 13 proto-filaments and know it was
hollow with a lumen
- Observed these in animal cells and concluded microtubules must be a universal part of the
cytoplasm of all eukaryotes.
o Organelle of all eukaryotic substructure is conserved
- Made out of alpha beta tubulin heterodimers
o Associate head to tail to form proto filaments. A stack and form longitudinal bonds.
Approximately 13 will come together laterally to form this hollow tube
 The seam is the location where the tubulin doesn’t line up in preferred
orientation. Want beta tubulin to bind to each other at the seam you get a slight
disconnect.
Microtubules provide structure to cells and enable long-range transport
- They’re resistant to compressive forces
- Serve for roadway network for long range transport.
- The cell’s highway system the cell uses to move around

Microtubules are found in all eukaryotes

- 3 big areas where they matter.
o Government funding lol 3 major research themes
- Mitosis: when cells undergo cell division, break down cytoskeleton and reconstruct with mitotic
spindle which is entirely made of microtubules
o Essential component
 o Poison mitosis by poisoning microtubules can treat cancer.

- Neurons: looking closely inside a neuronal process.

o it’s full of microtubule. Microtubule associated proteins are implicated in a large range
of neurogenerative diseases.
o Novel point mutation in tubulin gene that causes brain developmental defects.
o Mis regulation of microtubule function can cause brain diseases
o Alzheimer’s disease (tau) lines microtubules
- Cilia: thin hair like structures that line the trachea
o Significant number of diseases associated with cilia genes. Working on basis of ciliopathy

Microtubules in animal cells are organized by the centrosome

- It comes out from the centrosome and radially arranged.
- The microtubules look very static but microtubules are
continuously built, broken, broken down, and rebuilt
- Microtubule is constantly changing
- it’s being reformed continuously by the form of nucleation,
shrinkage, outgrowth.

Microtubules are roadways for intracellular transport

- secretory vesicles are being carried along microtubules by a type of motor protein called kinesin.

Microtubules form the mitotic spindle during cell division.

- Recently acquired a complex 3 D tomogram of entire spindle.
- The microtubules fills up the entire space makes up this complex structure
o The mitotic spindle is huge

Microtubules are the backbone of neurons

- Because neurons are so long they need to pass by information very fast

Microtubules in neurons are densely packed

- The orientation of the microtubules is different in axons and dendrites
- They’re polar. The beta tubulin towards the +
- In the axon, the + end is going towards the synapse and in the dendrite, it’s
mixed orientation.

The cilia of the trachea are specialized microtubule based structures

- They can beat, there’s a continuous movement of cilia and
pushing things out of your lung
-
Flagella (tail used for swimming) are also cilia
- The sperm tail is also made of cilia which is microtubules based
- Links between microtubules and motion of sperm and
infertility.
- They’re called dublets how can they give rise to motion of sperm?
Microtubules are built from tubulin
heterodimers
- It is a heterodimer of alpha and beta
tubulin.
- Polypeptide that are folded together by
chaperon complex
- This heterodimer is an obligate. Tubulin
is the dimer
- The dimers can associate head to tail to form protofilaments
- They are GTPases. Both the beta and alpha tubulin have a GTP binding pocket
o The hydrolysis state for beta tubulin is GDP it can go from GTP to GDP
o The state for alpha tubulin is always GTP. It’s not exchangeable and never hydrolyzed
and exchanged
- Beta tubulin is the one where the dynamics is. How does this polymer grow and shrink?
- Actin is ATPase and has ATP binding pocket and the dynamics for tubulin is different

Tubulin was first identified as the target of drug colchicine

- Natural product came from a flower autumn crocus found in the root system of these flowers.
They’re poisonous.
- They’re listed as a medicine in the Ebers papyrus oldest existing records of human medicine.
o Isolated colchicine used to treat gout. Inflammatory disease.
o Colchicine is binding directly to building blocks of microtubules (tubulin)

The rate constants of microtubule polymerise are less clear

- The + end and the – end do not have explicit rate
constants
- There are many different places for tubulin to land
whereas there’s only one place for actin to land in
the end of an actin filament
o You know precisely where the actin
filament comes in
- 13 possible binding sites in the end of a polymer tubule
- The + end is the fast growing end and the – end is the slow going end

The microtubule end is made of GTP-tubulin and perhaps sheet-structures.

- some protofilament are extending beyond
- the ends have elongated protofilaments
- What does the end of the microtubule actually look like?
- We know the tubulin is adding to the microtubule in the GTP
state
- Tubulin dependent on GTP on forming polymer. GTP = stable
- After GDP tubulin, it’s in the center region of the microtubule and it’s capped by the GTP tubulin
o GTP tubulin cap = GTP cap
 - This is what enables microtubules to have fascinating polymer dynamic.
- While it’s growing, imagine GTP tubulin is happen, it likes to be in a GTP polymer form. But if its
in the GDP state, it does not want to be in the polymer and get out. Most of the GDP tubulin is in
the center and would love to escape but it cannot because of the GTP cap. If catch up, then
collapse
Loss of the GTP-cap results in a switch from growth to
rapid shrinkage
- If the GTP tubulin subunits are lost and GDP
lattice is exposed, then it spring outwards in an
event known as catastrophe
- Protofilaments peel outward and the microtubule
will very rapidly collapse.
- It would hit plasma membrane and rapidly
disappear because polymer was depolymerizing
very rapidly disappear.
- Can start to regrow again and that process is
called rescue
- GTP-cap is lost because of random probability. Tubulin is adding, GTP is being hydrolyzed. It’s a
race. Can I add more GTP before the GDP catches up to me?

Tubulin can be curve or straight

- Took existing crystal structure of tubulin and made little protofilaments that would highlight
curvature. What tubulin is doing, it’s transitioning as it grows to a straightening process that
straightens out a process
- As it shrinks, it collapses down rapidly by structural transition to go from straight to curved
- Regulate these curve to straight transition. Making it like to be straight or curved based on what
it wants to do.
o Bending happens at the junction between alpha beta subunits in the heterodimer
- what does the end actually look like?

In textbooks:
- if you bind on the bottom of the structure, different rate constant than the top

Dynamic instability allows cells to quickly structure their microtubules

- the process that they grow, lose their GTP caps and collapse is
known as dynamic instability
- looking at a plot of microtubule length as a function of time.
Sawtooth patterns
- it grows steadily, goes through catastrophe, gets rescued and
then gets continuous pattern
o not what you would see with actin filaments
o as long as there’s enough actin subunits around, the actin
keeps growing and never shrink unless the actin falls below critical concentration
- in microtubule, even if lots of tubulin around, if lose GTP cap, you’ll collapse.

Dynamic instability was first deduced and alter observed directly

- working on solution of purified tubulin isolated from source
tissue
 - tubulin has complicated chaperon apparatus hard to get out of cell. The only cell type is
neurons. The only tissue that has neurons is the brain. Thought cycles and polymerization and
depolymerization get purified tubulin
- followed polymerization of microtubules in a test tube. The signal is the absorbance at a
particular wavelength. Overtime, observation increase as polymers form.
- Took the solution and plunged it and get sheer forces as fluid is forced to flow through needle
and causes polymer to break in the middle
- The point with light arrow is the point when you break
o When you beak polymer in half, cause significant reduction in total amount of polymer
- Take all polymer and break in half it collapses.
- Now we can see the things happening
- Homies measuring absorbance of samples and absorbance is measure of # of polymer in cell.
Passed tubulin through pipette to break if you break in half, the number should go down,
absorbance decreased a lot just by cut in half.

Microtubules are the target of broad class of thermotherapy drugs.

- Taxol, for breast cancer : yew tree. 1.6 billion annual sales ovarian breasts, lung cancers. It
stabilizes microtubules and causes mitotic spindles to freeze
- Vinorelbine
- These drugs are derived from natural products like plants

DZ-2384 is a synthetic derive of a natural product from a marine sponge

- Diazona sponge. Show that it would kill tumors with less toxic effects.
- No money : (

Microtubule-associated proteins control the dynamic of microtubules

- MAPs
Microtubules are the roadway for intracellular transport

Lecture 16: microtubules II: controlling microtubules

- The microtubule associated proteins and other regulatory factors that exist to modulate the
microtubule cytoskeleton, to create more or fewer microtubules.
- Cells have a range of protein they can use to modulate the cytoskeleton.
- A lot of these proteins. Look at cytoskeleton against microtubule cytoskeleton.

Microtubules are found in all eukaryotes

- In this case, they’re adding structural stability, important for constructure of mitotic spindle. M
- Mitosis, large target for chemo drugs. Taxol. To stabilize the microtubule cytoskeleton.
- Microtubules important in brain development.
- Neurite, found it’s absolutely full of microtubules and neurons are the cell type that’s highly
enriched in tubulin and as a result, brain tissue is main source for purifying tubulin
- Cilia and flagella also a lot of microtubules

Microtubules are built from tubulin heterodimers

- Protofilaments and polar molecules
- Has a beta and alpha tubulin
o + end is beta tubulin and slow growing – end is the one with alpha tubulin on the end
o GDP hydrolysis of beta tubulin is what draws dynamic of microtubules
 o Be
- Beta tubulin GTP binding pocket.

The loss of GTP-cap results in a switch from growth to rapid shrinkage.

- GTP cap at the end and when its lost, protofilaments will peel outwards rapidly shrinking causing
by catastrophe.
Dynamic instability can be directly observed in vitro
- The microtubules that have been stabilized by slowly hydrolysable of GTP GMPCPP and those
microtubules are very stable. We add to the reaction mix some fluorescent tubulin (green) and
see an elongation of green microtubule from the stabilized polymer seed.
- Dynamic microtubules grow from the microtubule extensions, they undergo catastrophe and
shrinks all the way down to the seed.
- Each horizontal slice is a frame of the movie
- Stack pixels in the Y direction and length of microtubule is shown in the X axis.

More complicated than that

- With microtubules there’s a set of proteins to regulate control, regulate, accelerate, decelerate
dynamic stability
- Polymerases: make microtubules grow faster. Makes polymer form
- Depolymerases, take microtubules apart or
- Tip proteins
- Severing enzymes: cut in the middle
- Nucleation factors: to nucleate new microtubules.
- Structure of microtubule + end and polymerases and depolymerases

Microtubules are born and reborn continuously

- All the white comets shooting out of microtubule organizing center. There’s + tip binding protein
that recognizes the binding end
- Continuously giving rise to new microtubules
- Constant sense of nucleation
- Reincarnation process. See microtubules born, get older and stars to undergo catastrophe and
all tubulin dimer

Microtubules are predominantly nucleated by the centrosome

- Who is nucleating them, who’s giving birth to them.
- Predominant but nucleate by centrosome.
- In the center, there’s a dot that is the microtubule organizing
center that is the centrosome
- How do we know that’s the major center.
- All the microtubules depolymerize.
- Can see the tubulin signal is entirely diffused and there’s
centrosome with sticks attached to it, shift temperature back up
to allow polymers to reform and all of it sprout back from the
centrosome.
- The centrosome consists of 2 centrioles, pericentriolar material,
and gamma-TURCs
o Surrounded by diffused electron dense material known as
pericentriolar.
 - Gamma tubulin ring complex (red rings) gamma-TURC
- They provide a template for microtubule nucleation

Gamma tubulin ring complexes provide a template for microtubule nucleation

- Cone shaped protein structure made of related family member known as
gamma tubulin
- Relative alpha beta tubulin exists only as a monomer and associated.
- The gamma tubulin ring complex was discovered early and at the time now we
understand how microtubules are nucleated
- They have a template and that template can elongate.
o For the last 25 years, the gamma tubulin structure is focused on the
ring structure and everything we known is been framed in terms of
this protein complex
o Nucleated by the gamma tubulin ring complex
- Not the only nucleation factor, major theme
- Characterize gamma tubulin nucleation independent pathway to figure out how it works and try
to build more sophisticated understanding to microtubule nucleation

Axon outgrowth is driven by microtubule nucleation but how?

- Zoom in at growth cone, would see a large number of new microtubules being formed in that
region.
- No gamma TuRCs in growth cones! Meaning they’re not the only nucleation factor
o Gamma tubulin independent in neurons. Not the nucleation pathway.

Doublecortin mutations cause diseases

- Found in growth cones. Linked to disease known as double cortex syndrome
- Single mutation, you can get one or two phenotype
o Type 1 lissencephaly. SMOOTH BRAIN SYNDROME
o If you’re type 1
o Brain does not have folds suffer from mental.
- If you have double cortin mutation and you’re a girl you have double-cortex syndrome
- Result in epilepsy and some form of learning disability.
- Cause: failure in neuronal migration
o Neuron born in ventricular structure deep in the brain and they migrate out long
distances to pattern the cerebral cortex. For some reasons, a single amino acid
substitution, you lose neuronal migration
o Located on X chromosome
o Males suffer severely because no other copy for females, there’s random X inactivation
so after the neuron terminate differentiates, activates one of the other X
o Some neuron get the healthy copy some get the mutated version. So you get mosaic
phenotype due to different population of neurons.
- How would double cortin be a cause of nucleation factor

Cells nucleate polymers in 2 ways

- Template by base-plate
- similar to Arp2/3 complex. Where you have a complex that sit at the base of the complex and
elongate off of it.
 - Actin cytoskeleton have other nucleation factors other than Arp2/3
o Spire: arrange actin monomers. Bring into close physical proximity so they stick together
to create fragment of actin filament then elongate
- The doublecortin is used to bind to tubulin dimers and arranging them
and forming a nucleus that way which is then capable of elongation.

MAPs can regulate nucleation

- Gamma tubulin ring complex unlike Arp2/3 is really lousy at activating
microtubules by itself.
- Needed WASP and need a W domain that can bind to monomer and it
might not function in similar ways which it needs ways in accessory
proteins to add to tubulin dimers
- MAPs that inhibit microtubule nucleation.
- Could be nucleation promoting MAPs, make them all produce
microtubules
- Nucleation to be efficient needs a lot of MAPs

Severing enzymes are ATPases that cut microtubules in the middle (ketanin)
- cuts appear in the middle and they become fragmented along their length.
- Hexametric ATPases. Have a central pore
- What they do is they bind to the C terminus of tubulin (end of
tubulin polypeptide) available to bind to in solution
- Down the length of microtubule, they’re the tubulin dimers they’re
the C terminal ends of polypeptide that are projecting into solution
o Severing enzyme has landed and has grabbed C terminus
and out into the pore.
o Using energy of ATP hydrolysis to grab the end and yank it
until it pops the whole tubulin dimer out.
o An unfoldase (they have pores and can grab end of
polypeptide and unwind it through the pore and break the 3D dimension)

+TIP proteins track growing microtubule ends

- End binding protein 3, it’s a +Tip because it binds to growing + end of a microtubule

+ TIP proteins bind specifically to the GTP-cap of microtubules

- How does it know to bind to that specific tubulin?
- Nucleotide state of tubulin end of microtubule has GTP cap
o Rest of is made of GDP tubulin

EB family proteins recognize the GTP-cap and the other hitches a ride
- Over 6 0 +TIP proteins and almost none of them can directly recognize the growing microtubule
end.
- The EB proteins can bind to growing end and many proteins bind
onto the EB
- Allow cells to target proteins to tip of microtubule
- + end ends up in focal adhesions, (foot), kinetochore during
mitosis
 - Cells want to send other things to the same place the microtubule has ended up by EB proteins.
- EB1 make sure its always here at the end where ever It happens to go

+TIP proteins are markers for plus-end features

- As time passes, the microtubule elongates and the end binding proteins signal
shows that protein is staying attached to microtubule + end
- Worked on doublecortin, DC appears to be binding to the growing end of
microtubule along its length, ended up discovering this protein also recognizes
the microtubule end
o How does Double Cortin recognize the plus end?
o Does it recognize the GTP cap?
- Tested to see if it would seem doublecortin would like GTP tubulin if it was doing the same as
EB1. No evidence that it was recognizing the nucleotide state of tubulin.

DCS recognizes longitudinal curvature

- Binding to the curved end because its flared out. Get microtubules to curve
in experiments and when introduced, found that the double cortin seemed to
bind preference to curved regions of microtubules.
- Bigger the curvature, more DCX signal.
o Measured brightness of signal as a function of curvature of the
underlying lattice.

DCS recognizes curved microtubule ends

- Add Taxol to kill end tracking
o Takes curved structure at microtubule ends and cause them to straighten
- When added Taxol, the straightening caused double cortin to stop binding preferentially to
microtubule end
- Can take advantage of naturally occurring point mutations use these as tools in the lab.
- Can introduce to wild type double cortin and purify a mutant version and ask how it behaves
- Several mutations in double cortin that would no longer recognize curved microtubules
- Also no longer bind specifically to growing microtubule ends
- 6 mutants lose both, 10 mutants lose neither.
o Both ability to recognize curvature and end.

Depolymerases remove tubulin from ends to trigger

catastrophes
- Depolymerize the microtubule by removing tubulin
from the ends and trigger catastrophes.
- If the ends are protected by GTP cap if the cap is
chewed off, it would collapse.
- Kinesin-13
o Use energy of ATP hydrolysis to remove the tubulin dimers from microtubule ends
o Physically bend protofilaments out and yank out the tubulin

MCAK depolymerizes microtubules

- Some microtubules attach to surface when MCAK comes in, the microtubules shrink from both
ends.
 - In controlled case, the microtubules are blunt if MCAK, curled protofilaments and it’s bending
the proteins out. Fastest rate is 50 tubulin dimers per second.

XMAP215 accelerates microtubule growth

 Microtubule polymerase. The growth rate of dimer per
second and subunit per second.
 With XMAP215, apparent constant goes way up, how
does it make tubulin bind faster?

XMAP215 binds to tubulin dimers

 Forms complex
 With tubulin, it’s the ball and without it’s a stick so made a
drawing.

XMAP215 catalyzes the reverse reaction

- To lower the delta G star of the reaction. Which is what catalysts do
- Catalysts always accelerate both the forward and reverse reaction.
- Product exceeds reactants. What happens to the end of microtubule I the absence of tubulin
- Without extra tubulin around, XMAP215 depolymerized the microtubule
- If you add a bit of tubulin, you can hit equilibrium where it would stay constant length
o Add more reactants, acceleration of growth
o Because if can accelerate both ways:

XMAP215 is a textbook catalysis

- XMAP215 stabilizes a weakly-attached tubulin dimer

Sometimes you’re wrong about things

- Thought XMAP215 would wrap around tubulin dimer and
would do that structure. The little blue squares are domains
known as TOG domains (tubulin bind G proteins)
- In 2012, solved the co crystal of TOG1 crystal bind to alpha
beta tubulin heterodimer
- The ratio and the dimer is a 1:1 ratio.
- the TOG domains act progressive add new tubulin to microtubule ends

Actin filaments vs microtubule

- polymerase
o Actin: formin
o Microtubule: XMAP215
- Depolymerase
o Actin: N/A
o Microtubule: MCAK
- +TIP proteins
o Actin: Capping protein
o Microtubule: EB1
- Severing enzyme
o Actin: Cofilin
 o Microtubule: Katanin
- Nucleation
o Actin: Arp2/3
o Microtubule: Gamma-TURK

Lecture 17: microtubule III microtubule motors

- Microtubule based transport (kinesins and dynein)
- Long range transportation
- Microtubules undergo ‘dynamic instability’
- Microtubule dynamics is regulated by microtubule associated proteins
- Microtubules are born and reborn continuously

Long range protein transport is driven by motor proteins

- Microtubule cytoskeleton, + side pointing out ward and organelles
attached to microtubule via motor proteins
- Cytoplasmic dynein or kinesin family memory
- Secretory vesicles are carried by kinesins

Transport can be activated or inhibited by signaling molecules

- Cells can choose when they want to change location of their content
- The African frog has ability to change color can manipulate cells on surface of body called
melanophores.
o Can cause pigment molecules to change position
inside cells and that change in pigmentation can
cause color change on the animal
o Male frogs change as a way to indicate mate
- Organelles called melanosomes (pigment granules) and
have ability to be dispersed or aggregated
o Aggregated, darker pigmentation
o cAMP signal changes the distribution of the
melanosomes
o not only cells, ability to move things around, can choose when and where to move

molecules are the ‘roadways’ for the transport of organelles.

- Thin black line is squid giant axon was motion of dark molecules back and forth kinesin and
dynein.
- Thought electron microscopy, can isolate out microtubules with organelles physically attached
and can see microtubule network is the road way network upon the organelles are being
transported.

Design problem for transport

- How to put one foot in front of the other
o How to move?
- Some cargo in, some cargo out
o 2 way traffic. Can send some protein in and some protein out.
o Can figure out the bidirectionality of transport
- Cargo selection
 o Don’t want to send everything out, maybe just want to move one thing, how to choose
the cargo?
- Which road to take?
o How to decide which microtubule to go on?

kinesin of is a motor protein that ‘walks’ to the microtubule plus-end

- Converts chemical energy to mechanical work
- There’s 2 head domains (but actually the feet), a long coiled coil
that emerges from head domain and a light chain and tail up at
the top
o Cargo binding interface
- Head domains are the feet
- Dimeric molecule, 2 polypeptides
- There are linkers that lead into the stalk
that takes you to the cargo

Kinesin transport vestibular cargo

- How does the walk happen on the microtubule?

Kinesin and myosin share a catalytic core that hydrolyzes ATP

- Beta sheet surrounded by alpha helix
- The evolved protein domain that can convert nucleotide
hydrolysis to large change
- Catalytic ATPase core that then evolve to serve different
functions in cases
- Actin skeleton of microtubule skeleton

Kinesin is the world’s tiniest biped

- Has 2 feet, tiny, and walks on microtubule.

The way Kinesin walks

1.) Microtubule binding releases ADP from the forward, leading
head
o The collision of kinesin and microtubule has effect of
popping nucleotide out of the head that binds (the
forward head) releasing ADP and putting in
nucleotide free state
2.) Binding of new ATP in the forward head induces a
conformational change
o Against the head domain.
3.) The “neck liner” swings the rear, trailing head forward
o Rear head is up and it’s the new forward head and the
head in the back is the ATP bound kinesin head
4.) New forward head releases ADP, binds tightly and trailing
head has to hydrolyze ATP and release phosphate.
o If the rear head hydrolyses too fast, the motor will fall
 o Something must prevent the back head from hydrolyzing its ATP before the front head
has a change to bind.

- Need to coordinate the two ATP cycles need the two heads to be out of sync so one of them is
always attached to microtubule
- When both of feet down, and new ATP pops in, it will try to swing the other feet forward, but it
won’t be able to swing the back leg forward but the back leg has not hydrolyzed ATP
- 2 things:
o the back feet to not hydrolyze ATP until the front feet has attached
o Once the front feet comes down, it can’t mind to new ATP molecule until back feet has
hydrolyzed.
- the way they communicate is physical strain caused by simultaneous
biding to microtubule
- when kinesin walking, foot step kind of far, and as a result, when both
feet are down, the front foot feels a force directed backward to – end of
microtubule and pulled upward
- due to that pull, the ATP binding pocket becomes slightly closed so new
ATP cannot bind
o the back foot feels the forward pulling force forward + end and
the upward pulling clips the ATP to ADP and Pi
o hydrolysis reaction is accelerated in presence of strain.

Dynein is a minus-end directed motor protein

- dimeric made of hexametric ATPase domains
- has a long stalk with a microtubule binding domain

The ATPase domain of dynein rotates to move the motor.

- The rotation and detachment of the stalk will make dynein move towards
the negative end.

Dynein’s power stroke has been observed by electron microscopy

- In pre power stroke state vs post power stroke goes from sticking from one
part of molecule to the other
- Have 2 different kinds of cards working on these roads

Motor protein cargo is selected by adapter proteins

- Dynactin: links the cargo to the dynein molecule.
- There’s a set of these transport molecule, a set of kinesin (13 types of
kinesin) and only one dynein.

Dynein motors power the beating of flagella

- The tail beat sperm is due to motion of whipping of flagella.
- How does it happen?
The doublet microtubule of axonemes are connected by dynein
- Between doublets, there are dynein arms.
- In the doublets

A tug of war between opposing dynein give rise to flagellar beat

- It causes sliding of doublet relative to one another
through action of dynein
- Nexin linker. Now they attempt to slide the doublet but
because of nexin cross link, there’s a bending with
mechanical strain and causes entire structure to bend.
- There are teams of dynein on either side pulling on their
respective doublets causing the tail to bend in one
direction and then bend the other way when the other way
pulls down
- How to get the coordination of two set of dynein that don’t
require signaling processes.
- At the start of the beat, there are 2 teams of dynein pulling
against each other
o If somebody slips, now it’s 5 v 4 and because of that, the 4 people left now carry an
increased load relative to previously so become more likely to slip
o When one team wins, they fall back, and teams of dynein of opposite sides are
competing against one another. One side gains a transient advantage due to failure on
one side
o So dynein on losing side have to carrying increasing load in result, collapse
o So winning team pulls sperm tail to one direction, and the winning team detaches as
ATP hydrolysis cycle complex’s and the losing team starts to pull

Post-translational modifications of tubulin marks different

microtubule ‘loads’
- how to pick different roads. Tubulin itself can become post
translational modified in a variety of ways
- the C terminus of tubulin becomes glycerates, and all the things
which creates additional segments of polypeptide that emerges
off of peptide.
- Act as roadway markers inside cells
- Looking at neurons, the post translational marks in the axons
are different in the dendrite. When a kinesin lands on
microtubule it can somehow know which direction to go. Will it
lead me to dendrite or axon.
- A way of chemically encoding it in post translational
modification
- Referred as tubulin code.

Motor proteins build the mitotic spindle

Lecture 18: membranes
- Membrane manipulation

The cytoskeleton: cell shape, cell mobility and intracellular transport

- Leaving behind, but the cytoskeleton does the role for
everything

Membrane-bound organelle are everywhere.

- A electron microscope of a cell and there’s extensive network
of intracellular membranes
- All the organelles have membranes
- ER membranes can be stuffed full in a cell. How can it exist at
the same time as all the cytoskeleton elements?
o The cytoplasm is very densely packed, and it’s significantly
packed with membrane bound organelles
- How to manipulate membrane bound systems and keep track of
where the organelles are

And there’s a constant flow and trafficking between the endomembrane

systems
- Can tell from outside of the membrane what type of membrane it is
by the membrane tagging. (red, blue purple)
- How to create membrane of distinct shapes

The fluid mosaic model

describes a complex but
basically flat membrane
 Plasma
membrane
consists of

phospholipid bilayer with hydrophillic head and

hydrophobic fatty acid tail on the inside.
 Inside membrane there’s embedded proteins and
there’s different types of membranes embedded in the
bilayer.
 Seen the schematic that the bilayer is flat, the outer
surface of cell its okay to say flat, but looking into
intracellular membranes:

Intracellular membranes are often highly curved

 - All tiny spheres, and they vary in size and they are vesicle of different size
- Membrane bound organelle can have flat surfaces and then curve around like the ER
- The spherical sizes isn’t like minimizing surface area to volume ratio, cells are inducing specific
curvatures in specific membranes in specific place or time
- The geometry of membrane bound organelles is controlled by proteins.

So where does membrane curvature come from?

- The inverse of radius of curvature. Has units of per micron
- How to specify curvature?

Lipid themselves have an intrinsic curvature

- Some of them are happy forming flat membranes (phosphyl choline PC) the
acetyl side chains are parallel to one another from the phospholipid head
group
- As a result, when the lipid stacks, they form a plane.
- PE, it’s acetyl chains emerge with an arc, so when they interact side by side,
you get curved structures.
o If cells modulate the concentration of different lipids in their
membranes, the lipids have different intrinsic curvatures.
o Specific species of lipids will have a tendency to sort themselves based on curvature of
the region.
o The PE is preference concentrated in highly curved region because acetyl side chains are
able to engage in thermodynamic favorable formation
o The PE can tolerate the curvature, sorts on the side.
o Cell’s don’t use this as primary method of sorting out membrane curvature
o Cells aren’t doing that much at this level, there are active methods.

Consider the uptake of lipid from the bloodstream

- How can cells internalize a Low density lipoprotein particle (LDL) bad cholesterol.
- Can get total cholesterol count and get HDL and LDL ratios, too much LDLs or VLDLs, those are
the ones that are predictors of cardio vascular disease.
- Cells need to be able to get this particle out of extracellular space and internalize it.
o LDL particle tagged with iron made it easy to see and when they did it, create dark black
puntae and it forms a hole in the plasma membrane and in the hole there are LDL
particles
o Further along, can see that the region where the invagination of membrane occurs has
gone into the plasma membrane
o A thin neck connecting to the sphere and all the
LDL-ferritin goes inside the cell.

LDL particles are endocytosed into clathrin-coated vesicle.

- On surface of plasma membrane, there’s LDL receptor.
- The apoB protein on the LDL binds to the red string of
beads
 - The binding triggers the recruitment of many inquire protein to the inner surface of plasma
membrane
- The AP2 complex (blue) the notch binds to the intracellular tail of the LDL receptor.
- AP2 recruits clathrin.
- Clathrin increases curvature of the plasma membrane
- As the clathrin is recruited, continuous movement inside and then you make a clathrin coated
vesicle.

Clathrin is a trimeric protein that assembles into triskelion cage

- They polymerize into cage like structure (teal is one clathrin) the rest of clathrin
coat forms a cage like organized structure
- The trimeric proteins are binding to Ap2 complex and these portions pile onto
the piece of membrane until its fully engaged by clathrin molecules
- Then cargo comes into the cell for downstream processing.
- You’re bending a plasma membrane that doesn’t want to be straight, where is
the energy coming from? Don’t get directed movement in cells for nothing

Endocytic vesicles are driven inward by actin polymerization.

- endocytosis assembly factors are any proteins recruited
to the site of endocytosis by triggered receptor
- They recruit Arp2/3 activators like WASP
o Green is what we’re trying to internalize
- The WASP triggers Arp2/3 activation and actin network
will push against plasma membrane
 It pushes the
endocytic cargo
into the cell and
that provides
energy/force to
bend the
membrane down
so the clathrin can
continue to bind and shape into endocytic sphere
 Energy comes from actin polymerizing.

Dynamin is a GTPase that pinches off membrane

- Go from endocytic vesicle that’s mostly covered in clathrin to a fully
engulfed vesicle. There’s still a neck
- Dynamin pinches vesicle off to allow it to enter the cell
o Also does in mitochondria.

Dynamin creates thin membrane tubules in absence of GTP

- A lipid bilayer that has been put over glass surface.
- Take this lipid bilayer and dynamin to it.
- Get formation of very thin membrane tubules
o There’s no GTP in solution no nucleotide to drive this GTPase
 o Gets trapped in step in the cycle that is in the nucleotide free state
- The formation of thin membrane tubules. Formed region of tightest membrane constriction

In the presence of GTP, those thin tubules are severed

- Fragmentation and severing of membrane tubules into tine vesicular bodies
- Can see when the GTP hits because of tension in the system

Be able to oligomerize around tube in nucleotide state and through hydrolysis of GTP shops the
membrane in pieces.

Dynamin forms tetramers that assemble into a membrane collar

- Dealing with tetrameric protein. They’re tetramer that forms in cis
- 4 polypeptides are cis tetramer
- Green balls are PH domains, and they interact with lipids. They interact
with membrane and red is GTPase domain
- All the PH domain warps around and turns into a large helix.
o GTPase domain interacts in trans
o 4 GTPase domains from 4 cis tetramers are going up and down
relative to thin membrane tubule
o the precise mechanism where oligomer constricts to get
membrane scission

Membrane scission by dynamin may

occur by a constriction mechanism

- dynamin warps around

membrane tubule and
squeezes the membrane
tube
- it’s like a boa constrictor. They twist and switch
- increase helical pitch of oligomer and strangle membrane that when membranes get so close to
one another they pop and fuse into separate lipid bilayers.

BAR domain proteins are membrane-bending bananas

- They are shaped like bananas
- If there’s gaps between protein domain and lipid bilayer, it’s a
weak binding
o If the lipid bilayer adopts a more curved, it’ll increase
the amount of binding interface between binding
domain to the phospholipid head groups and now it’s
trapped in head state and its strong binding
o The curve becomes more curved at first due to
Brownian motion
- Bar domain comes in range of shapes
 o Highly curved to a flatter membrane and to an inverted curvature.
o To tune membrane curvature
o By knowing the curvature you’re after, you can infer the identity of bar domain protein
you have

How to go from inside to go outside and blow a bubble? Exocytosis.

ESCRT proteins enable ‘reversable-topology’ membrane scission

- Budding yeast, mother cell produces bud but needs to snip off
- ESCT oligomerizes the intramembrane surface to bring the
membranes closer together

Reverse topology membrane scission occurs in budding and cytokinesis

- Separate 2 cells in half.

ESCRT proteins assemble into conical spirals known as bells

- Can achieve reverse membrane fusion

Lecture 19 Protein sorting

Membrane bound organelles are everywhere
- Intracellular membranes even though lipid bilayers prefer to be flat and talk about protein that
shape the protein

Consider uptake of lipids from the blood stream

- When LDL binds to lipid bilayers it gets engulfed and LDL particles are endocytosed into clathrin
coated vesicle
- Recruit proteins to bottom surface (cytoplasmic face) and recruit clathrin and make endocytic
vesicle to round up.
o Dynamin pinches off the neck of vesicle.
- How do cells know where to put their proteins?

And there’s constant flow and trafficking between the endomembrane

systems
- Specific locations, number of different processes in the
endomembrane system
- Vesicles emerge out from golgi body.
o Some are moving out to plasma membrane for secretion
- Cells have 2 big things to figure out
o Figure out how to put proteins into particular compartments
 How did it know to go there?
  Secretion of digestive enzymes. Proteases that need to be secreted how does it
know?
o If have vesicles that contain particular proteins, that’s on the inside. The knowledge
about what’s inside the vesicle has to be available outside. The colored coats can show.
 Identifying compartments with maybe a tag?
- Essential task for cells to do.

How are proteins sorted into compartments?

- The mRNA gets translated by ribosome. The first chunk of the protein
produced is identified as a signal sequence
- The information about where the protein is supposed to go is encoded in the
amino acid sequence itself.
- Protein zip codes
o Gunter Blobel discovered this. The information in amino acid sequence told cell where
to place the protein
ER proteins are often translated by ER-bound ribosomes
- Intended for secretion.
- The transmembrane receptors are also included as they go through
secretion pathway as well.
- The rough ER contains ribosomes that translates proteins that go into the
lumen.
- It passes through a channel to get into the ER lumen.

Cotranslational translocation is initiated by signal

recognition particle
 Being translocated across the ER membrane while its being translated
 Initiated by signal recognition particles.
 The ribosome sees the mRNA, sees it, and start making the polypeptide chains.
Out the bottom emerges the signal sequence. It’s hydrophobic and doesn’t like
getting exposed. The SRP has a hydrophobic binding groove so the recognitive
sequence specific hydrophobic sequences and bind to them.

SRPs dock the ribosome on the ER membrane

- The SRP dock it. The SRP receptor and it sits close to the channel the protein
has to go through. Dock the ribosome on the signal recognition particle
receptor
- And see both are in the GTP state (both GTPases)
- When the two GTPases touch and interact, they trigger hydrolysis of GTP in
both independent protein domains
o Get hydrolysis of GTP in the SRP receptor and the SRP.
o This hydrolysis dislodges the SRP molecule and receptor and as a
result, releases signal sequence and detaches the receptor from this
channel which is now a translocon and allow emerging polypeptide to enter the
channel.
- Now the ribosome can continue to synthesize and move into ER lumen

The signal sequence is cleaved by a signal peptidase

- Now can translocate into the ER lumen
 - Ribosome leaves and its made now a folded protein in the ER lumen which is what the signal
sequence wanted it to do.

How does a protein-conducting channel

prevent leaks?
- it allows a protein to go across, how does it
not leak?
- Must push polypeptide chain through the
hole. How to not let it leak?
- If you open, it has to pass through amino
acids of very different sizes
o Tryptophan, and Arginine, glycine

 Somehow, this hole has to be able to dilate and

let in large hydrophobic side chain and allow
only glycine through. (CALCIUM LEAK not good
Hydrophobic signal sequence displaces a ‘plug’ in Sec61
- The red alpha helix in the translocon.
- The membrane is in the plane and looking at the side of the translocon. The
pore is at the top where the translating polypeptide will go in and in the
absence of a protein going in the channel
- The signal sequence unplugs the plug as its hydrophobic.
- It’s plugged until you unplug it and the only way to do it is with a signals
sequence
- How does it allow sidechains of different sizes to go through?

A ring of isoleucine’s acts as a gasket during translocation.

- Acts like a rubber ring which prevents water from escaping from the
side.
- Looking inside structure of the translocon, will see the very center of the
pore (pore ring) the plug has been removed and can see green isoleucine
side chains and they are ringing around the pore.
- Because the isoleucine themselves are hydrophobic, repel ions that
otherwise might escape
o Escape the structure has enough flexibility in its 3D fold that as
the protein polypeptide chain is being pushed, the ring of
isoleucine’s can expand out to accommodate a large tryptophan state and relax to
original state when glycine
- The energy source: ribosome burning energy as it translates.
- Can have a case where a protein translocon has a small pore but can push something big
through it because we do have a source of energy.
- This might be a protein you want to excrete to the extracellular space.
o One of the other class of proteins are like transmembrane receptors.
o They have transmembrane helices
How does the translocon extrude transmembrane helices
- the luminal side, N terminus and C terminus. This could be the LDL
receptor. The N terminal binds to LDL (ends up on the exterior of
the cell) can see the tiny transmembrane domain and can find the C
terminus domain in the cytoplasm.
- Many times of transmembrane domains, ones
where the order is reversed. The N terminus is
in the cytosol.
- how do we make these helices? And allow
them to live inside membrane if they’re going
through the Cotranslational process?

The translocon extrudes STA sequences into the ER membrane

- stop transfer anchor sequences
- when translocon encounters the sequence, it extrudes the sequence in the ER membrane
- the ribosome and the mRNA is bind to an open translocon
- pushes polypeptide into lumen and cleave the signal.
- What happens if the ribosome starts producing hydrophobic amino acids? Those won’t want to
go into the ER lumen, as the ribosome is making polypeptides, the hydrophobic ones won’t want
to go into the lumen
o Get the formation of a
hydrophobic helix in the
translocon and that sequence is
called a stop transfer anchor
sequence.
o It tells you to anchor that section
into the plasma membrane
- Ribosome is still working, but the
polypeptide isn’t going through into the
lumen but just making a tail into the
cytosol
- Now you have type I tarnsmembrane protein.

Sec61 opens like a clam shell to allow hydrophobic a-helices to exit

- A way out of the channel through the lateral exit to lipid bilayer
- Can see that the basket of alpha helices is dividable into 2 sections.
Transmembrane helices 1-5 and transmembrane helices 6-10
o Following the structure and a hinge, the two sections act like
independent protein folds connected by a hinge
- Some how, that hinge can bend and the translocon can open up like
a clam and allow the stop transfer anchor sequence to exit into the plasma membrane.

Translocon and associated machinery are studied using microsomes

- Purification of the ER
o The ER fragments into tiny structures called microsomes.
o Breaks ER up to fragments into smooth and rough ER
 o Then centrifuge into sucrose density gradient and the rough ER will be further down the
gradient and separate out the structures
- Tiny vesicles of ER with associated ER ribosomes caught in the middle of a translation
- Repeat this by giving cells specific mRNA, and then see if it ends up inside the microsome
o if it does, it has a signal sequence

Microsome experiments can determine whether a protein was translocated.

- Treat microsome with detergent, and then add protease no more intact protein left in the
sample. Anything on the outside of protein will get degraded.
- Or, add just protease. Can see that anything on the outside of protein will get degraded, but
anything inside will be protected by the lipid bilayer.
o Take protein, take mRNA and send the cells only the C terminal half, and see if it goes
into the microsome.
o Now, we know the C terminus is in the first half.
- Can identify the ER targeting sequences.

Different sequences target proteins to diverse organelles.

- In many cases, protein is fully translated in the cytoplasm
- Then if there’s targeting sequence, they it’ll be carried by
proteins to their destination.

For any location the cell wants to put a protein, need a defined sequence of amino acids that
can be defined as a zip code, then need a protein that can read the zip code and then carry the amino
acid to the proper receptor such that the protein gets put into the compartment
- For ER it’s during translation but for other proteins, it’s usually after translation

Transmembrane helices are formed during translation: you can see why they’re always perpendicular to
the plasma membrane. That’ why the transverse helix in complex I is weird because it runs parallel.

But how are secreted proteins delivered to the plasma membrane etc?
- We figured out how the proteins are brought to the ER, but how does once the secretory leaves
the Golgi towards the plasma membrane, how does it know the protein is destined for the
plasma membrane?

Lecture 20: secretion and vesicle transport

 - From Golgi to plasma membrane for secretion
- How vesicles maintain identity
- The endomembrane system is established
- Cotranslational translocation produces protein in the ER lumen, N terminal sequence
o Protein itself contains information of where to go and allow understand where to go
o Now vesicles, how do they know what’s inside?

It’s important to secrete things!

- Fundamental process in cell biology
- Cell growth
o Need larger plasma membrane, it’ll have to come from somewhere
o One process is the process of cell growth
- Insulin secretion
o Hormone signaling.
- Synaptic transmission
o Neurotransmission secretion

Camillo Golgi
- Developed silver staining that allowed us to see structures under microscope
- Now we know the study.
- Ramon Y Cajal used silver stain to discover connection of neurons.
- Can see specialized organelle that secretes vesicle.
-
Randy Schekman initially discovered 23 mutants with defective secretion
- Budding yeast with electron microscope.
- Look for strains that couldn’t secrete protein
- Mutants then get filled with secretory vesicles that can’t get secreted.

Sec mutants fall into distinct categories

- Translated from ER, move to Golgi, Golgi produce vesicles, fuse with plasma membrane and
secreted.
- Yeast can fail to import protein in the ER, then can’t get into Golgi and cannot be secreted
- Failure to produce ER vesicles: protein can’t leave the ER
- Failture of ER proteins to fuse with golgi
- Failture to produce golgi vesicles (enlargement of golgi and red protein in golgi and not in the
secretory vesicles)
- Failure of golgi vesicles to release (cannot fuse and release to plasma membrane)

Vesicle budding by coat proteins can be reconstituted in vitro

- Purify components and put back together to see if he can rebuild
- By taking particular cocktail protein and put in bilayer, they could cause vesicle binding.

Design problem for vesicle transport

- Coat vesicles to make and identify them
- If you cover a vesicle, will prevent it from sticking to plasma membrane and fuse
 - Remove the coat for fusion, but meaning when that happens, we don’t lose track of what’s
inside. Keep a tag associated with vesicle to maintain its identity.

Secretory vesicles are coated in proteins that specify their identity and shape
- The green is the coat protein. And its attached to the vesicle

Different coats for outward (anterograde) and inward (retrograde) transport

- Ourward COPII and inward COPI transport
- In clathrin coat, it did that so we know there’s several species of protein
coat.

signal sequences determine which kind of coat proteins

- Blue ball, identity of protein on the outside shows which amino acid
function to recruit which type of coat
- The membrane cargo receptor protein
- Assembly of these coats need to be reversable

ARF family proteins are GTPases that control vesicle coat assembly
- Sar1 binds to membrane in ER called Sec12.
- Sar1 start as GDP and when it binds, it turns into GTP state
- In GTP state, inserts a hydrophobic N-terminus to separate cytoplasm
from ER lumen.
- Arf family will then recruit coat proteins. they play a role
in shaping the vesicle as they have intrinsic curvature
- Recruits Sec23/Sec24

GTP hydrolysis of ARFs trigger coat disassembly

- GTP hydrolysis event lowers affinity for ARF causing coat
to disassemble
- A GTP timer/switch
- Switch ARF to GTP to pull up vesicle and then to GDP to dissociate the coat proteins.

Rab protein maintain the identify of uncoated vesicles

- They maintain identity of uncoated vesicle. GTPase and it has anticap helix in the
membrane
- As you move out from nucleus to plasma membrane, there’s a sequence of Rab
numbers
- As you move in, you also have different types of rab
o Once it attaches, they’ll see oh I’m a Rab __ vesicle, put me in ___
o Rabs tell cells what type of vesicle it is
o As it moves through, the rab identity changes over time.
o How does that identity change?
 You can see initial vesicle with one Rab and then change to have another color
Rab on it
 Sorting tag system
 o Once you’ve made this far, change tag.
- As secretory vesicle moves, rab identity changes

Cognate pairs of SNARE proteins mediate fusion

- Hit its target! In terms of secretion want it to hit the plasma membrane and fuse to it.
- V-snare (vesicle snare) is the protein in the membrane fo the vesicle of cell.
- Encounters T-snare (target snare) they oligomerize and clamp vesicle on its final target

Oligomerization of SNARE complex proteins initiates vesicle fusion

- The proteins wrap and make a SNARE complex to smash the
vesicle across plasma membrane which fuses
- Then left with cis snare complex and T snare and NSF comes in
and unwinds them.
- How does vesicle get moved through cytoplasm?
Vesicles are transported by kinesins and dynein
- They need to know which vesicles to interact with
- Adaptor proteins interact with Rab proteins on vesicles.
o This recruits motor protein to vesicles in a way to send to the right direction

Microtubules do not always reach the plasma membrane.

- Directly under is cell cortex which is cross link of actin filaments.
- Attaching myosin to the kinesin (dimeric) called Myosin-V
o It’s a two footed processive myosin
o Undergoes similar walking as kinesin
- Discovered not by muscle or myosin person but by geneticist by animal coloration

Mice and gerbils without Myosin-V mutations have dilute coloration

- Dilute coloration is passed through genetics.
- Dilute locus actually encodes for a myosin.
- The unlocking that actin and myosin play a role in vesicle secretion
o Pigment molecule secretion that bind to hair or fur that causes pigment

Vesicles are frequently laded with kinesins, dynein, and

myosin-V
- when it hits actin cortex, the myosin grab onto actin and
carry it through the actin cortex to the plasma
membrane

How much information is encoded by the endomembrane

system?
- the chain for the protein has a code in it which when
translated by ribosome produces amino acid stretch to
tell where the protein goes
- a huge host of proteins that exist to recognize signal
o has evolve to know
- cognate pairs of SNARE proteins, Rabs, rab effectors
that interact