Lecture 20 – Cytoskeleton and Actin
Intermediate filaments/lamins.

Made up of a number of building blocks such as rope like polymers.
Three phases of actin polymerization.

G-Actin -> Nucleation -> Elongation -> Steady State

Minimum of 3 Subunits to come together -> Slow nucleation.
Actin’s ATPase cycle. Actin tread milling.

Steady State: The point where growth of the + end is equal to the depolymerization of the - end, and
that’s because of the difference between the two critical concentrations (0.12 for + end, 0.6 for - end)

The + end continues to grow because it’s above its Cc but the - end will shrink because it’s below it’s
Cc.

Despite this, the filament will move even at steady state.
Calculations related to net growth rates, on-rate constants, off-rate constants, and critical
concentrations of cytoskeletal polymers.
(Note: the rate constants for actin will be provided if necessary. Exam questions may include hypothetical
polymers as well.)

Net Growth = Subunits Added - Subunits Lost (K[on] - K[Off])

Cc = Subunits when Net Growth is 0 = KOFF/KON
Location and architecture of different actin networks.

Cell Cortex: Right underneath the PM is a cage of wire like actin filaments that provide mechanical
strength to the PM.

During Migration: Cells from protrusions at the front end called fillopodia and lemellipodia, as well
as stress fibres, are all formed by actin.

Cytokinesis: Forms the contractile ring that closes and pinches

Adherend’s Belt: Where cell-cell adhesions occur.

Phagocytosis: Form cross linkage of moving endocytic vesicles
Lecture 21 – Actin Regulation
Mechanism of forming.

Grow the filament.

They polymerize linear actin filament arrays.

Dimer of forming FH2 domain is a small ring with a hinge in the middle. Ratchet motion.

Have FH1 domains that has bound profiling-actin. Feeds the profiling onto the end of the actin
polymer.

Formin can make a filament grow quicker than it would simply by collision. FH1 are essentially
giant pockets full of Actin.
Actin cross-linking (specific cross-linkers not tested)

Alpha-Actinin

Cross Linkers have 2 opposing filament domains. They take the two opposite filaments and stick
them together. Have two actin binding domains that bind two actin filaments.
Listeria comet tails and the leading edge.

Found in raw milk and cheeses. Causes no harm, but don’t want an infection if pregnant.

When listeria infects cells, it shoots around with comet tails. The tail is made up of actin filaments.
It infects cells, replicates, builds the tail that propels it through your cells such that it hits the PM
and bursts out into the extracellular space. The force of actin polymerization allows it to break
free of its host cell and go and infect neighbouring cells.
The logic of the dendritic actin network (Arp2/3, cofilin, profilin, capping protein). How does each
protein work? What does each protein contribute to the network? How would changes to the
proteins in the network affect cell migration or Listeria motility.

Listeria comet tails are nucleated via the Arp2/3 complex.

Arp2/3 mimics an actin nucleus and nucleates daughter filaments at 70 degree angles. It must be
activated.

Arp2/3 resembles the base of an actin filament, and then actin subunits add to it, giving rise to
daughter filaments. Binds to the sides. The 70 degree angle is optimal for pushing out of the plasma
membrane.

WASp activates Arp2/3.

o
WH2 domain (WCA) binds to actin monomer (on the W portion)
o
The A or Acidic domain will bind directly to the Arp2/3 complex, inducing a conformational
change that brings Arp2 and Arp3 together.
o
WH2 domain loads the first actin monomer onto the complex.
o
Thus Arp2/3 and the initial actin monomer form 3-actin like subunits which can easily
elongate.

Listeria are coated with ActA, a protein that mimics WASp. Also an activator of the Arp2/3
complex.

o
Will activate all inactive complexes, will nucleate a dendritic network of actin filaments to the
tail of the listeria which allows it to propel itself forward.

Profilin recycles actin, and blocks the - polymerization.

o
Will bind to ADP subunit.
o
Will accelerate the rate of which ADP comes out and ATP comes in, making way for a new
ATP Profilin Actin.

o
Will then disassociate after the Actin has bound to the end of the filament.
o
Prevents - end polymerization.
ADF/Cofilin Severs Actin Filaments

o
Depolymerization. Severs the actin filament in two, severing it.
o
Create more free ends so that depolymerization is quicker.
o
ADF/Cofilin binds to ADP Actin Subunits. Cofilin dissociates from the monomeric form as it
prefers to bind to the filament. Will be recycled.

Together, Cofilin and Profilin accelerate actin tread milling.

o

Thymosin-B4 binds to ATP Actin and inactivates it, keeps actin in reserve.
Capping Protein prevents futile polymerization.

o
CapZ caps Actin Filaments. Wants to cap those that are behind the leading edge.
o
Takes 1.5 seconds to find and cap a filament. If it were faster, no actin filaments would grow. If
too slow, would allow for futile polymerization in the opposite direction.
o

Tropomodulin caps the - end.
Listeria motility is driven by the coordinated action of 6 Proteins

o
ActA is at the surface of it, activates Arp2/3 Complex.
o
Arp2/3 nucleates branched filaments.
o
Profilin and Cofilin work together to accelerate tread milling.
o

Profilin recycles actin.

Too much ADF/Cofilin you’ll stop moving because you’re chopping at all the actin and
nothing has time to nucleate.
o
CapZ caps filaments that aren’t pushing
o

Absence of this they’ll move too slowly.
o
TAKING ANY OF THIS OUT WILL BREAK THE SYSTEM.
o
Listeria and Leading Edge are exactly the same.
Lecture 22 – Contraction and Chemotaxis
Components and mechanism of contractile bundles (non-muscle myosin-II).

Back of the cell, actin exists in contractile bundles that have the ability to be of one length and then
shorten. While shortening, they generate force, which causes the cells to pick up their feet.

o
Bundle is attached to the focal adhesion.
o

Integrins are components of the focal adhesions.



Integrins are attached to the cellular matrix as well as contractile bundles
Myosin produces force. They are motor proteins that convert chemical energy (ATP) into
mechanical work.

o
Binds to AF, undergoes conformational change, exerts force on AF.
o
Conformational change is coupled with the release of phosphate along with ATP
Hydrolysis.
o
MII forms contractile bundles.
o
Non-Muscle MII forms bundles that pull actin filaments inward.
o
Myosins interact with AFs in a polarized fashion, they bind and pull the - end towards the
middle of the bundle so the acting must be organized anti-parallely.
o
They disassociate the integrins.
Mechanism of small Rho-family GTPases (Cdc42, Rho, Rac, GEFs, GAPs, and downstream effector
proteins).

They trigger three types of actin networks.

Stress fibers: Formed by Dominant Active Rho

o
Rho has small motif that allows it to bind to the PM.
o
Is OFF in the GDP State.
o
Signal will bind to the receptor, allows the receptor to interact with a GEF: Exchanges the GDP
out of Rho and sticks a GTP Rho: Activating it. Protrusion generated from conformational
change of GTP in Binding Pocket.
o
Rho, now active, can bind to Effector proteins that are downstream. These proteins have
binding domains that only bind to Rho in its GTP form.
o
Effectors: Turn on the Actin Cytoskeleton.
o
How to turn it off? GAPs, activate the GTPase of Rho, forcing Rho to hydrolyze its GTP and
switch back to GDP mode.
o
GEF means Go and GAP means STOP.
o
Rho activates Formins by relieving auto-inhibition. Formins have a a Rho Binding Domain
(FH1 and FH2). Formin is normally folded on itself. Rho Binding Domain on Formins autoinhibit it.
o

In the presence of Rho, will be relieved, allowing the polymerization of actin filaments
which go on to form STRESS FIBRES.

Lamellopodia: Formed by Dominant Active Rac

Filopodia: Formed by Dominant Active CDC42.

o
CDC42 activates Wasp which also has an RBD. RBD binds to CDC42, relieving inhibition,
allowing the WCA domain of Wasp to bind actin monomers, inducing conformational changes
in Arp2/3 and giving rise to branched actin networks.
Logic of chemotaxis: how and why different actin networks appear. Crosstalk between Rho- family
GTPases.

We need them to interact with one another to establish the polarized actin structures we see.

Rho activates Myosin, makes it contractile.

CDC42: Has an effector protein that is a GEF for Rac. So activating CDC42 activates Rac. Rac
activates Rho because one of Rac’s effector proteins is a GEF for Rho.

Rho inhibits Rac because one of Rho’s effector proteins is a GAP for Rac.

o
This is done because you only want Rho in the back, obviously.

This is important for establishing a polarized array of actin.

WE WANT CELLS TO MOVE TOWARDS cAMP
Lecture 23 – Myosin and Muscle
Ultrastructure of muscle fibers, sarcomeres, etc.

Anti Parallel Actin Arrays - MII

Muscles are made of billions of these contractile bundles

Multi-Nucleated. Inside are Myofibrils, which have repeated units inside called Sarcomeres

Sarcomeres are made up of contractile arrays of actin filaments and MII filaments.

Sarcomeres are the basic unit of the “sliding filament theory”

o
Undergo rapid contraction upon muscle stimulation.
o
Type II Fast Twitch, 5-10ms between signal from brain and start of contraction.
o
40-45ms - Sarcomere contraction.
o
Type I, is slower but stronger.

Our muslces are made up of interdigitated networks of filaments that contract by sliding
relative to one another (interdigitating)

Relaxed: Long and slender cells. Contracted: Short and fat.

AFs within are being contracted by MII Bundles - Z Discs

Cell has to get fat because there’s fluid in-between the MII bundle and the Z Disk that
has to get displaced out.
The ATPase cycle of muscle myosin. Rigor mortis.

1 ATP Hydrolysis event by the M Motor Domain will lead to 1 Power stroke that the M gives to the
AF

o
1) Binds ATP, head is released from actin.
o
2) Hydrolysis of ATP to ADP + Pi causes the myosin head to rotate into a cocked, strained
state. M is now ABOVE the Actin subunit.
o
3) M Head will bind to the AF in the ADP+Pi state– Binding causes changes in the nucleotide
pocket that enables the release of the products of the ATP Cycle
o
4) Powerstroke, release of Phosphate pushes the Actin Filament to the left (like a rower). M
now remains in ADP state.
o

5) A new ATP binding releases M from the AF. ADP is released -> M is floating above AF
Failure of Myosin to detach (via ATP) causes Rigor Mortis.
Muscle accessory proteins (Tropomodulin, troponin, tropomyosin, CapZ). Mechanism by which
Ca2+ stimulates contraction.

The structure of Sarcomeres is set by capping proteins such as Tropomodulin (-) and CapZ (+)

Tropomyosin blocks the binding site for Myosin on the actin filament.

o
This is done to prevent continuous contraction.
o
In the presence of Ca2+, the TM will rotate, exposing the - end binding. Without it, it’s
blocking.

Motor Neurons causes a rapid spike in Ca2+ in muscle fibres, causing a rapid influx of calcium into
the space surrounding the myofibrils.

Troponin binds Ca2+ and pulls the tropomyosin out of the way.
Lecture 24 – Microtubules
Structure of microtubules. Basic cellular functions of microtubules.

MTs have 13 PFs. Found in all euks.

MTs provide structure to cells and enable long-range transport. They’re stiff.

MTs in Animal Cells are organized by the centrosome (which duplicates and goes to opposite poles
of the cell)

Fast Growing: +, Slow Growing: -

MTs form the mitotic spindle during cell division.

MTs are also the backbone of neurons.

o
Here, the MTs are densely packed.

Cilia of the Trachea are also specialized MT based structures. They beat (wave like)

Flagella (tails used for swimming) are ALSO Cilia. Movement driven by MT proteins.

MTs are built from Tubulin Heterodimers. (Alpha and Beta Tubulin Chain). When dimerized, it’s
irreversible. They’re held together via non-covalent bonds.

Head to Tail associations into Protofilaments (13).

Beta Tubulin likes to bind laterally.

o
As the helix wraps, there’s a place where B Tubulin cannot interact with another B Tubulin and
must interact with Alpha, that location is called the SEAM.
Dynamic instability and the GTP cap. Tubulin conformations during growth and shrinkage. Force
production by microtubule depolymerization.

Rapid growth and shrinkage: Dynamic instability.

MTs are the roadways for intracellular transport.

The MT end is made up of GTP-Tubulin (Not ATP as in Actin) and perhaps Sheet Structures.
Tubulin itself is a GTPase.

o
Will form a stable polymer structure but when GTP is hydrolyzed and the Pi is released you’re
left with GDP Tubulin Lattice which is unstable and will fall apart. It will collapse (not the case
in Actin, still stable)

GTP Stablizing Cap found at the growing end of MTs. Loss of the GTP-Cap leads to rapid
shrinkage. GTP Tubulin likes to be straight. GDP likes to be bent, thus GDP Tubulin at the back is
super strained, but it can’t part from the MT because of the CAP. As soon as that CAP is gone, the
MT will collapse, i.e.: Catastrophe.

However, Dynamic Instability easily allows cells to quickly restructure their microtubules.
Taxol.

Taxol: Drug that binds to and stabilizes MT, freezing them. Freeze cells in mitosis. The break down
and remodelling is important for the transition of a cell out of mitosis. When those cells die, using
taxol, they rupture in a way that creates a chain reaction of cell death in cancer cells. Especially
useful for breast cancer remission.
Lecture 25 – Microtubule Associated Proteins
Microtubule nucleation by g-TuRC at centrosomes. Microtubule severing enzymes (Katanin and
spastin). Logic of +TIP networks (EB1 vs. other +TIPs). Mechanism of MCAK.

The centrosome consists of 2 Centrioles, as well as Pericentriolar Material and Gamma-TURCs. One
centriole is always perpendicular to the other.

The Gamma-Tubulin Ring Complex (TURC) provides a template for the MT. They, like MTs,
contain 13 molecules of Gamma-Tubulin as well as associated proteins. Get arranged into an
oligomer that remains attached to the - end of the Tubulin as a baseplate, in order to be a
template for the new MT. However, it needs many dimers and in a stable structure in order to
elongate.

Severing Enzymes

o

They cut MTs in the middle. MTs thus become easily fragmented. Katanin and Spastin.
+TIP Proteins

o
Bind specifically to the GTP-Cap of MTs
o
+TIP Proteins track growing MT ends. Serve as markers.
o
EB Family Proteins recognize the GTP-Cap and the others hitch a ride! They
recognize EB1.
o
EB1 comes on and off really frequently. The comets you see are not the same EB1, they’re
a dynamic population of them that come on and off. Chill.

MCAK: Depolymerizes MTs

o
It is a type of Kinesin-13 located at the centromere. When we add MCAK, it chews the MTs
from both ends. They don’t undergo catastrophes however– So what happens is MCAK
BENDS the PFs back, tears the MT apart such that it falls off at about 50 Tubulin Dimers per
second.
Mechanism of XMAP215.

MT Accelerator (in terms of growth). It’s the opposite of MCAK.

It is an elongated molecule that has 5 Tubulin Binding Domains called TOG Domains.

What is does specifically: Increases the on rate constant of tubulin by about 5-10 fold.

What it does, is it only binds to 1 Tubulin: Wrps around, forming an XMAP+Tubulin complex.

However, under the right circumstances it can catalyze the reverse reaction– For example if you take
all of the Tubulin away. It functions like a catalyst. In the absence of Tubulin, it will depolyerize
MTs. If you add a little Tubulin, it won’t grow or shrink. Adding a large amount of tubulin XMAP
will react and quickly accelerate the reaction.
Analogies between MAPs and actin-binding proteins.

XMAPs function similarly to Formin in Actin.
Lecture 26 – Microtubule Motors
The ATPase cycle of kinesin. Role of the neck linker and intramolecular strain. Plus-end versus
minus-end directed transport (how do you choose?) Ultrastructure of axonemes (cilia and flagella)

Transport can be activated or inhibited by singling molecules. Cells will shift from high to low cAMP
environments.

Kinesin is a motor protein that walks to the MT plus end.

o
Converts chemical energy into chemical work.
o
TAIL DOMAIN: Where cargo will bind.
o
2 HEAD DOMAINS: Bind to MTs, walk one foot in front of the other to transport cargo.
o
Is two independent polypeptides that dimerize through coiled coil stalk that give rise to the
head domains.
o
Kinesin transports vesicular cargo.
o
Kinesin and Myosin (Actin) share a catalytic core that hydrolyzes ATP.
o
Kinesin has to take the hydrolysis of ATP in order to motivate large scale conformational
changes that drive its motion.
o
Thus it has two ATPase domains that have to talk to one another in a way that one detaches
while the other is bound.
o

1) Kinesin encounters the MT with ADP in both heads. ADP Nucleotide state has a
LOW affinity for MTs so when the Kinesin head has ADP it does NOT like to bind
to MTs and the reaction is weak. Kinesin will DIFFUSE, knocking ADP out of a
pocket when hitting th eMT.

2) The forward motor binds the MT, releasing ADP. NOW THE BP is EMPTY. The
nucleotide FREE state has a HIGH affinity for the MT, the foot is LOCKED.

3) Binding of new ATP into the FORWARD head induces a conformational
change. It causes the linker to fold or dock onto the head of the Kinesin. ATP
STATE IS STILL HIGH AFFINITY.

4) The NECK LINKER swings the rear, the trailing head, forward. (Like a power stroke)
The new TRAILING head (which was originally the forward) can’t hydrolyze ATP, to go
into ADP state, too quickly, otherwise it’ll fall off.

5) Now the new forward head has bound, it’s ADP has been knocked off, and is in an NT
FREE STATE and the back hydrolyzes ATP and releases Pi.

STRAIN: Accelerates the ATP hydrolysis of the rear end, such that when your foot is
unattached, it can hydrolyze ATP but it’s mad slow. So as soon as the other foot is down,
it’ll speed up the process. The strain is the signal.


Another thing: The strain prevents ATP binding to the FRONT pocket.
Mechanism of dynein

Dynein is a - end directed motor protein.

Has a Bipedal system (2) Hexomeric AAA ATPase ring domains with stocks that protrude.

The ATPase domain of Dynein rotates to move the motor.

o
The rotation is believed to drive the protein forward. The angle of the MT binding domain
moves, letting the protein rock forward.

Different cargoes by different kinesins.
Tug-of-war in flagellar beating, role of nexin.

Dynein however, powers the beating of flagella.

The tail is a bundle of 9 Doublets of MTs

The doublet MTs of Axnoemes are connected via Dyneins

o
Nexin: Creates cross links between the doublets.
o
Removing Nexin causes the MTs to slide past each other because there’s nothing holding them
together. Normally, with Nexin, they can’t slide because they’re physically connected via the
protein cross linker, i.e.: Nexin Links.
o

The frustration of sliding (the prevention of it) causes the Bending Motion.
Tug of War between opposing dyne ins gives rise to the flagellar beating.

o
If we could activate the Dyneins on one side, it would bend it that direction. Turning those off
and activating the other ones allows it to bend in the other direction.
o
So how does it coordinate? Tug of War between opposing teams of Dyneins on opposite sides.
o
So what happens when one team loses (in a tug of war), the winning team falls too
because suddenly the other team lets go. Same thing happens here after the
completion of the power stroke. Now there’s no force on the side that just won
because that team has now detached. During that time, the team that lost, get up,
grab on to their doublet and pull, and obviously they win.
o
Initial asymmetry gives rise to a stable oscillation. The coordination between
the dyne ins arises only from mechanical considerations. Specifics: The rate
of detachment of a motor from its filament of a Kinesin, Dynein, or whatever,
is FORCE DEPENDENT. Thus the greater the force its under the more likely
it is to fall off.
o
Once oscillation is established, it’s hard to break.
Lecture 27 – Mitosis
Kinesin-5 and monastrol.

Centrosomes separation via action by Kinesin-5 (Sliding in the middle)

Dimeric Kinesins tetramaraize through central region to form a tetrameric kinesin that has two heads
on either side.

Monastrol Targets Kinesin 5: Causes cells to have a mono polar spindle. The spindle poles don’t
separate and collapse on top of each other. Chromosomes remain attached to the outside but won’t
complete mitosis without two poles. Was ineffective in humans. Monastrol DOES kill cancer cells in
humans, but there is no death cascade as is the case with Taxol.

Antiparallel positioning of MTs allow Kinesin 5 to slide them apart. (Otherwise they’d both go in the
same direction and tehere would be no sliding.)
Spindle positioning by cortically-anchored dynein.

Astral MTs anchored out by Anchored Dynein

Comes from Cytoplasmic Dyneing which is anchored at the cell cortex.

MTs interact with the cortex and Dynein binds them and walk towards the - end, reeling in the MT
towards the cortex and pulling on the spindle pole to which that MT is attached.

Removing Dynein disrupts spindle positioning = Bad.
Poleward flux of microtubules from kinesin-13’s.

MTs flux toward the poles. They are continuously being depolymerized at their - ends
and depolymerized at their + ends. Similar to tread milling effect in actin. This process allows for the
movement of Spindle Poles in Anaphase.

Flux is driven by Kinesin-13s, the depolymerases located at spindle poles.

The depolymerization at the poles will suck the MTs INTO the spindle pole, creating a reeling effect
where the spindle MTs are fluxing towards the - end.
Kinetochores as microtubule sleeves (Ndc80). Force production by microtubule polymerization and
depolymerization.

Kinetochores are big sleeves for MTs. Chromosomes are attached to the spindle pole through a multi
proteins machine known as the Kinetochore.

They envelop the large segment of the MT.

They have to hang on to the MT that is shrinking because that will drag the chromosome to the
spindle pole of the new daughter cells.

NDC80: Arranged by the Kinetochore in such a way that it forms a ring that wraps around the MT.
As the MT begins to shrink, the ring will remain attached to it which will move with the shrinking
MT.

Protofilaments typically flare out– attaching a ring to the MT as these PFs flare out, they’ll push the
ring down. As the MT depolyerimzeis the PFs peel outward and this ring attachment gets pushed
down. Kinetochore is pulled along with it by energy released via MT depolymerization.

As soon as the GTP Cap is gone, the GDP state relaxes the MT into the fold it wants to have,
releasing energy. That energy, that release of strain in the MT is what is used to drive
chromosome motion.
Chromokinesins.

Kinesin-4. Those that bind to chromosome arms are called chromo-kinesins (with a DNA Binding
Site that is + end directed). If there are Kinesins on the MT arms that are interacting with that, you’d
push those arms to the metaphase plate. Arms always towards the metaphase plate because the K4s
are pushing the arms in that direction.

Chromosomes align on the plate because that’s where all the forces are balanced. Why do you want
these weak Kinesins bound here? Chromosomes are elastic, soft. If you put a strong and processive
Kinesin on them, they’ll pull the chromosome apart. Instead, the K4s provide a force that simply
pushes it towards the MP Plate but not so much as to destroy them.
Lecture 28 – Cytoskeletal Coordination
Control of Rho GTPases by microtubules

Without MTs, lamellopodia extend but don’t go anywhere.

Without MTs, you get no proper “zoning”, no inhibition, of the Rho GTPases to the front and
back.

MT Depolymerization in non-migrating cells induces stress fibers.

Removing MTs, Rho will be activated and you’ll form more stress fibers.

MTs alter the activation state of Rho Family GTPases.

o
Inactive GDP Form -> Interact with GEF -> Pushes them to GTP Form -> Interact with
Effector Proteins -> GTP state can interact with GAP -> Back to GDP (Inactive)

MTs must ACTIVATE Rac and INHIBIT Rho

“Pioneer” MTs penetrate into the lamellopodia and filopodia.

Growing MTs activate Rac. But we don’t know how.

MTs sequester, and inactivate a Rho-GEF (Which would normally put Rho in GTP form).
Myosin-V in vesicle transport. Multiple motors per vesicle. Centralspindlin and midzone
specification of contractile ring position.

MTs don’t always reach the PM. Cortex is in the way.

If you’re a Vesicle and you’re trying to fuse with the PM by hopping on a Kinesin, it carries you
there, but there’s a roadblock that Kinesin won’t allow you to get past.

Vesicles know that they’re going to hit that, and include Myosin-V on their surfaces. Essentially the
Myosin version of Kinesin. It’s two footed, and processive.

MV: Is the HAND OFF mechanism for Kinesins, which allow the vesicle to get into the PM
(Plasma Membrane)

All in all, Vesicles are frequently loaded with Kinesins, Dynein and Myosin V.

CONTRACTILE RING

o
The ring is a specialized contractile bundle made up of actin.
o
Process of Cytokinesis.
o
Essentially, it’s a stress fibre that wraps itself circumferntially around the cell.
o
The location of the ring is specified by MTs. MTs tell Actin where to be positioned. The
spindle “mid zone” is the region of antiparallel overlap of MTs emanating into the two spindle
poles.
o
Displacing that mid zone will move the position of the contractile ring.
o
THE SPINDLE MIDZONE IS ACTIVATING RHO! Rho, which activates Formins, which
relieves Auto-Inhibition.
o
Rho, in its active state, will bind to a Rho binding protein, relieving auto-inhbitions of Formins
which go on to assemble AFs.
o
We have to believe: Somehow, the position of the “mid zone” is serving to lead to localized
Rho activation and therefore localized Formin activity and construction of contractile bundles.
o
A Rho-GEF and Rac-GAP are transported to the mid zone via KINESIN.
o

The trimeric complex is known as the Centraspindilin Complex and moves the central
spindle and constructs the ring. Made up of 3 proteins (MgcRacGAP, ECT2 (Rho Gef)
and Mklp1 (+ Directed Kinesin)

Therefore you will transport your GEF and GAP to where you need them.

Need to transport both the GEF and the GAP because its not only the case that you need
to turn on Rho but it helps to inhibit Rac because you really don’t want any dendritic
networks formed at the location of the contractile ring, you want to keep it specific to
contractile bundles. You do that with this complex.
When the + ends are mislocalized by the spindle severing event (in the experiment), the
spindle midzone moves which leads to a new orientation of the GEFs and the GAPs onto
either the anterior or posterior side depending on where you sever the spindle
Lecture 29 – Cell Cycle 1
Cyclin synthesis as a “timer.” Cyclin-CDK as functional unit. Simplified one-cyclin one-CDK
network.

Cells can measure concentration, which comes from accumulation, which comes with time.

Concentration will increase as a function of time if the concentration of the kinase is determined via
protein synthesis. Cells know how old they are by knowing how much kinase they have
accumulated.

Cyclin concentration is the timer.

o
Cyclins are made continuously and destroyed periodically. When destroyed, the cells go into
mitosis. Leaving mitosis, the Cyclin count would generate again. It’s a simple timer.

Cyclin-dependent kinase (CDK) binds to Cyclin to define the “active” molecule. CDK
controls progress through the cycle. It binds to Cyclin to define an active Kinase module, used to
call the Maturation Promoting Factor, or MPK.

NEED TO KNOW: This guy is the active subunit. This is the substance whose concentration is
increasing with time. While Kinease is made constitutively, it is always present in high
concentrations, but it’s inactive in the ABSENCE of Cyclin (Obviously) so it needs that Cyclin
binding partner to become active.

o
Cyclin can bind to CDK and phosphorylate its substrate with the increasing accumulation of
Cyclin.

Free CDK2: Has a T-Loop: BLOCKS the active site. So, it’s normally inhibiting Kinase Activity.

o
But when Cyclin Binds, the T-Loop is pulled out of the way, revealing the active site of the
Kinase. Kinase can now go Phosphorylate things as you progress through the cell cycle.

This molecule is the fundamental timer of the cell cycle. Cells progress through the cell cycle by
monitoring the activity levels of CDK Kinases through the accumulation of Cyclins.
Control of cell-cycle phase by thresholds of cyclin activity. Logic of Cdc25 and Wee1.

Thresholds of CDK activity activate DNA Synthesis (S) and Mitosis (M).

M Phase: Level of CDK is high. Leaving Mitosis, Cyclins are degraded, clock is reset.

G1 Phase: Low CDK activity.

o
Make Cyclins, daughter cells begin to produce it, will bind to CDK and CDK becomes Active
o
CDK levels rise, pass a threshold -> That threshold is the time for the Cell to go into S
Phase, to synthesize DNA.

After DNA Synthesis: Cyclin continues to rise. Reaches a second threshold: Entrance into M Phase.
Ready to enter Mitosis! Cyclins degraded again.

But what if something happens, Cell Cycle must be stopped before S phase if there’s damage.

o
Wee1 Kinase keeps Cyclin CDK under control until cells have grown sufficiently.
o
Without it, CDK doesn’t wait. Wee1 phoshorylates CDK1 and keeps it under control.
o
Without it, Cyclins go out of control, cells become smaller and smaller with each division
because they’re not allowed enough time to grow

Now, CDC25 activates Cyclin-CDK to initiate Cell Division. It relieves inhibition of Wee1

o
Without CDC25, cells grow too large because Wee1 has shut off CDK all together. No
progression through Mytosis, CDC25 can’t turn CDK back on so the cell continues to grow.
o
CDC25 removes the inhibitory phosphate from Y15 (Which is initially phosphorylated by
Wee1) creating an active MPF.
o

Without CDC25, can’t turn MPF on. Cell can’t mature. Cell can’t divide.
So Wee1 is always competing with CDC25.

o
CDC25 wins when the cell enters mitosis.
o
Overexpress Wee1? CDK is always off. Cells get too large.
o
Overexpress CDC25? Mitosis is entered too quickly because CDK is never inhibited. Cells get
too small.

Cyclin CDK also phosphorylates DNA Replication Machinery. In S Phase, CDK phosphorylates
several subunits that causes the replication fork to fire.

o
It also breaks down the nuclear envelope via Phosphorylation, the phosphorylation of Lamin
filaments.
o
Chromosome condensing, centrosome duplication, spindle poles separation: These can all be
traced to some phosphorylation driven event of Cyclin CDK.
Lecture 30 – Cell Cycle 2
APC/C, Cdc20, p31, Mad2, Mad1, Securin, Separase, and Cohesin.

UB (Ubiquitin) is a small protein that gets attached to other proteins, making a large tag, recognized
by machinery that unfolds that tagged protein, pulls it into a cleavage chamber, cuts it and spits the
waste. (Get Rekt)

M Phase Cyclins are Poly-UBed by the Anaphase Promoting Complex or APC/C. It is a UB
Ligase, attaches UBs onto proteins. Those Cyclins will then be degraded by the Proteasome and
chopped.

o
If it becomes active too early, metaphase to anaphase transition will occur too quickly, leading
to incorrect segregation of chromosomes.
o
So how does it activate and find specific targets?

APC gets target specificity via Binding Partners. Is inactive by itself.

APC has an active site that binds to UB in order to attach UBs to Proteins (Cyclin) -> That
is inactive without a Binding Partner.

So what cells do is inhibit Binding Partners, in doing so, they hold back the MetaphaseAnaphase transition.

THIS IS THE KEY TO THE SPINDLE ASSEMBLY CHECKPOINT PROCESS

o
An APC BP (Binding Partner) known as CDC20 is extremely important. Without it, the Cell
Cycle is defective.
o
Once Cdc20 binds to APC, it’ll Poly-UB Securin. Now if you do that, you’ll go into Anaphase,
leading to the transition and separation of chromosomes.
o
When aligning, building spindle, etc, need to block Cdc20, prevent it from binding to APC,
otherwise everything goes forward.
o
Wait Signal: Inhibition of CDC20.
o
Second BP: CDH1, becomes dephosphorylated after the transition, which allows it to bind to
APC. That one Poly-UBs Cyclin, allows for Cyclin degradation, resting the timer for new G1.
o
When do you want to activate CDC20? Until the chromosomes become bi-oriented.
o

What you’ll se when cells are in Anaphase is the sister chromatids will separate and
move towards the daughter cells.

Now, those are originally held together by Cohesion (Cohesion, get it) that wraps a
ring around the two sister chromatids and keeps them together. They have to
release the chromatids in order for them to move the daughter cells.
o
Second Wait Signal: Prevention of Proteolysis of Cohesion Molecules.
o

Cohesion is a multi-subunit complex wrapped around the two sister chromatids.

If the two sister chromatids are NOT lined up to go to opposite poles, then when
Cohesion breaks they’ll go in the wrong direction.
o
Cohesion molecules are broken by Separase (Separating the Cohesion, right?) They cleave
Cohesion molecules and allow the Chromatids to separate.
o
Separase is kept in check by Securin (For security, hah!): It’s basically bracers that remain
stuck in Separases mouth, unable to cleave Cohesion.
o
When APC and CDC20 are active APC will Poly-UB Securin, which allows it to
be degrated liberating Separase, Separase will cleave Cohesin, Sister Chromatids will
separate.
o
So we need to prevent CDC20 from activating APC too early.
o

CDC20 is inhibited by binding to Mad2, which exists in 2 different conformations. It
stands for Mitotic Arrest Deficient.


OPEN FORM: Cannot inhibit Cdc20.

CLOSED FORM: CAN inhibit Cdc20.

Thus we want to have a ton of closed Mad2, if we’re not ready to go through Mitosis.

When chromosomes are not aligned properly, they generate a lot of closed Mad2.

At the Kinetochore is another protein, called Mad1. Mad2 in the Cytoplasm (Open),
binds to Mad1. Mad1 will convert Mad2 into the closed state.

Closed Mad2 can leave, dissociate Mad1, inhibit Cdc20 by shutting it down.

Slightly more complicated: The first Mad2s that bind get converted into a closed state
and they stay stuck on Mad1.

Other open Mad2s can bind to the closed Mad2 on the side of them.


More tricky: Floating OPEN Mad2 will bind to CLOSED Mad2 (the two that are
bound to Mad1) and in a catalytic reaction, Mad2 bound to Mad1 will somehow
CLOSE those Mad2s

The reason? The backside of a closed Mad2 can convert another open Mad2 into
closed state, means that the protein complex with the closed Mad2 and the Cdc20
can also catalyze the conversion.

Which is to say an OPEN Mad2 can bind to the back of the surface of a closed Mad2 that
is ALREADY ATTACHED to Cdc20 (inhibiting it) and it can also be converted into a
closed state. So this can catalyze the conversion of many open Mad2s into closed
Mad2s.

SIGNAL AMPLIFICATION.
o
Bi-orientation releases CDC20. Mad1-Mad2 will fall off. Will also fall of Kinetochore.
o
Tension causes the Kinetochore to stretch, leading to physical separation of components.
o
Away from Aurora B (Not sure if we need to know this) – Dephosphorylation of Kinetochore
components. Release of Mad1/Mad2 tetramers. Tetramers go on to activate P31 which binds to
Closed Mad2/Cdc20 complexes, releasing Cdc20, generating more open Mad2. Cdc20 binds to
APC, M-A transition occurs.
o
Cdc20/Apc complex interacts with Securin (Poly-UBed), leading to release of Separase.
Separase cleaves Cohesion, Sister Chromatids separate. Anaphase occurs.
Lecture 31 – Adhesion
Cadherins, cis- and trans- binding
Cadherins are a type of Cell Adhesion Molecule (or CAMs) - Cell-Cell
Cell-Cell adhesion is strengthened by adding up many weak interactions, like velcro.
Cis Interactions are LATERAL: Horizontal.
Trans Insteractions are LONGITUDINAL: Vertical
Cadherins are calcium dependent cell-cell adhesion molecules.
Cadherins attach/connect to the cytoskeleton via adapter proteins. B-Catenin
and P120 Catenin. They form an initial layer recognizing the intracellular surface of the
patch. Layering outwards, they eventually connect to the cytoskeleton.
Cell-cell sorting (E-cadherin, P-cadherin)
Cadherins confer identity to cells and cadherins will stick to those of the same type.
Thus Cadherins allow for sorting into clusters.
P-Cadherins are found in the placenta.
E-Cadherins are found in Epithelial Cells.
Role of calcium in cadherins and integrins
They have calcium binding sites. With the presence of calcium they will stick to
cadherins of the same type. Recruiting more and more to reinforce contact. When cells
stick to one another, they have to sense and activate signal transduction pathways and
gene expression pathways.
INTEGRINS ARE CALCIUM DEPENDENT.
INTEGRINS CAN BIND TO MANY TYPES OF ECM BELOW
ECM components: laminin, fibronectin, type-IV collagen, type-I collagen Collagen
processing, scurvy
The ECM is a diverse, cross-linked meshwork of pollers.
Type-IV Collagen, Laminins.
Many polymeric proteins have adhesion capabilities to themselves and to one another,
forming an elastic polymer meshwork.
Laminin is a MULTI ADHESIVE ECM PROTEIN
Shaped like an anchor and has the ability to bind to itself. Has globular domains
that are able to bind to other laminins via self assembly or to collagen.
Hook domain can bind to cell receptors and other protein moieties found on the
PM.
It can also form trimeric structures which build into large oligomeric structures,
this would be only if were self-assembling, which it is.
Collagen IV
Are networks of heterogenous polymers. Starts with a monomer, has an NTerminal
Globular domain.
They also oligomerize. The C-Terminal Globular domains can form DIMERS
whereas the N-Terminal can form Tetramers. Massive networks. Massive
meshwork’s.
Makes up the bulk of ECM.
Collagen is thus an important protein.
Collagen
Has a triple helix structure similar to ropes. 25% of the protein mass of the
human body is Collagen. We are ropes.
Type-1 Collagen
Make up the bulk of our tendons. Makes sense that they’re rope shaped. When
we eat a lot of meat, we’re essentially eating a lot of collagen.
Collagen Sysnthesis
Forms thick fibrils. Ribosomes make a collagen chain of A1 and A2. They are
modified post translationally to form the procollagen helix.
The helix is capped by two globular domains. The procolagen helix is shipped to
Golgi which will associate laterally into procollagen bundles, which will be
secreted into the extra cellular space.
The propeptides are thus cleaved off the ends by a protease. Now the collagen is
free to assemble into fibrils and cross link.
Scurvy!
Caused by a breakdown of this whole collagen processing.
Without collagen, it can’t heal wounds. Gums degrade because they’re subject to
a baseline level of damage while eating. Small tears in the lining occur, small
injuries never heal due to lack of collagen and the gums break down.
Scurvy = Lack of Vitamin C in the diet. It is a co-factor of an enzyme involved in
the hydroxylation of collagen pro peptides, allowing for the formation of
procollagen.
Integrin structure (α- and β-) and integrin activation
Made up of Alpha and Beta chains. - Cell-Matrix
Integrins are cell matrix adhesion molecules that straighten upon activation.
Inactive is bent when inactive. Low affinity. High affinity when active, and extended. Has
2 polypeptides, Alpha and Beta, that extend OUT and have attachment domains called
the propeller and BetaA domain.
Integrins, when unattached and inactive, their intra cellular domains and Alpha and
Beta TMDs (Trans Membrane Domains) are close to one another. So close that the intra
cellular domain interacts.
After it becomes attached, straightened, and active, the forces in the integrins allows
them to separate (think 2 strands instead of 1). That spacing allows the C-Terminal
domains to also be separated, allowing proteins to BIND to the tails.
The way that the cell recognizes that it’s integrins are attached is caused by a
physical separation of integrins upon binding the ECM. (Extra Cellular Membrane)
Integrins cluster into focal adhesion complexes and are important for cell migration,
the very bottom of the foot, the part that connects DIRECTLY to the ECM or the surface.
Integrins attached recruit various adaptors and signal kinases.
Lecture 32 – Short Term Signals
Structure, mechanism, and logic of GPCRs.
G-Protein Coupled Receptors: They respond to many hormone signals.
They have Transmembrane Helicies H1-7. An N terminus in the intracellular space
and the C terminus in the cytosol. (Think of Braces on Teeth, whereas the N
Terminus rests on the Gum (Exterior and the C terminus rests in the Cytosol
(Teeth) – Terrible I know) The C terminal loops are where you get interactions with
proteins such as G-Proteins.
Structure, mechanism, and logic of G-proteins.
G-Proteins are trimeric GTPases that transduce hormone signals from GPCR.
Trimeric (Beta, Gamma, and Alpha) Subunits The former two form a dimer. G-Alpha is
separate.
Membrane insertions are amphipathic helices, another domain of the G Proteins that
allow it to insert into the membrane without having some trans-membrane helix.
It’s a GTPase, and such will tranasition from GDP Bound (OFF), to an active
state
of GTP (ON)
A conformational change occurs, when bound to GTP.
GTP locks the arms that were originally open, the two domains (arms) bend
inward and interact with the moiety.
In the GTP state there is a protrusion that sticks off of the G-Alpha.
CONFORMATIONAL CHANGES ALL HAPPEN ON THE G-ALPHA. Thus it will
DISSOCIATE from the G-Beta/Gamma subunit in response to GTP binding.
Activation and inactivation of G-proteins.
Hormone binding to the GPCR RECRUITS the G-Protein.
The effector protein will do all the work and produce the signal. Hormone binds,
inducing a conformational change in the receptor. There is a NOTCH on the RECEPTOR
that MATCHES to the NOTCH on the G-ALPHA SUBUNIT.
Diffusion probably allows them to bump heads normally, but with no hormone bound
nothing happens.
When the G-Portein binds to the GPCR, it becomes activated. It triggers GTP Exchange.
Activated Receptor functions like a GEF, it’ll bind to the G-Alpha subunit, causing GTP to
bind to the G-Alpha Subunit, triggering dissociation of G-Alpha from the receptor as
well as G-Beta/Gamma. KISS AND RUN INTERACTION.
Now the G-Protein is active in the GTP state. And now it can interact with the effector.
Effector will transduce the signal
INACTIVATION
G-Alpha has a high intrinsic rate of GTP Hydrolysis so once it has GTP in its
binding pocket it will quickly hydrolyze it.
Thus G-Alpha is self-inactivating. Causes the signal to TIME OUT.
After interaction with the effector, GTP will be hydrolyzed. Back to the
resting state, assuming that the hormone has also left the receptor.
Ping Pong Chaining with the Receptor. Once the hormone falls out of the
receptor, the game stops because the effector protein will release the G Protein
but there’s no receptor anymore to catch it.
The effector AMPLIFIES GTP Hydrolysis.
THIS EXPLAINS THE FACT THAT OUR RESPONSE TO ADRENALINE IS
BOTH HIGHLY ACTIVATED AND INACTIVATED. As soon as the
adrenaline (hormone) falls out, the effector rapidly shuts off. That is
the LOGIC of Gene Proteins. Gene proteins allow you to quickly
activate and inactivate the signal.
The response to epinephrine: effector proteins and second messengers,
specifically
cAMP. PKA and its effects on glycogen metabolism (overlaps with Brown lectures)
Adrenaline/epinephrine is a hormone that triggers short term responses. Increases
contraction of cardiac muscle, the conversion of glycogen to glucose, the inhibition of
glycogen synthesis and the conversion of glycogen to glucose in the skeletal muscle.
Adrenaline signal is converted into a cAMP signal via the effecter Adenylyl Cyclase.
cAMP in turn, activates PKA. Active PKA will have two subunits that are
dissociated, freeing the Kinase to now phosphorylate targets.
PKA directly controls the molecules of Glycogen Metabolism.
It will stimulate the breakdown by phosphoyrlating GLYCOGEN PHOSPHORYLASE
KINASE (GPK) which will then Phosphorylate Glycogen Phosphorylase (GP) which
will break down Glycogen.
The Multi-Step process results in a significant amplification of the signal.
1 Epinephrine activates 3 Adenyl Cylclases (Effectors) Because ONE GPCR can activate
MANY G Proteins.
Adenyl Cyclase can crank out a lot of cAMP. 1 Epinephrine = 1000 cAMP, but only takes 2
cAMP to bind to one of the subunits of PKA. Thus PKA is able to phosphorylate a ton of
Glycogen Synthase or GPK.
Then enzymes will be able to produce product based on their constitutive activity.
Sensitive Responses are due to this Cascade, this Signal Activation.
cAMP is a second messenger and almost every signalling pathway is going to have
some type of secondary messenger.
Concepts of differences in G-protein mechanism (inhibition, effectors, second
messengers, and Gα vs. Gβγ as carriers of signal).
Different signalling pathways will reply on different second messengers but they’re
common of signal transduction pathways, especially for short term responses.
It is NOT always G Alpha Subunit that does the hand over. Sometimes, G
Beta/Gamma will do the same job with Potassium Channels, causing them to
open. That will rely only on the intrinsic GTP Hydrolysis rate of G Alpha to shut
everything down, and not the Effector as well.
So in fact, the response of your body to adrenaline will depend not only on the ability of
your body to sense that adrenaline but also whether or not you have other inhibitory
hormones circulating in the blood, and if you have too much inhibitory hormone you
will become insensitive to epinephrine and if you have low levels of inhibitory
hormones you will be hyper sensitive to adrenaline because there will be no inhibition.
If we know that we want to tune the response to adrenaline, we don’t always want it to
be the case that just because adrenal gland has secreted adrenaline we don’t
immediately go into the fight or flight response. We want to have a body that is more
sensitive, more subtle. We want the body to be able to suppress the adrenaline
response and so to do that you have to have pathways that converge on a common
target such that you can create this competitive balance, thus the reason why second
messengers and effector proteins are used, they provide a node in this network were
stimulatory and inhibitory signals come together, and you can achieve a subtle and
balanced response to your hormone environment.
Lecture 33 – Long Term Signals
Growth factors, NGF and EGF. Mechanism of RTK activation.
When cells receive these, they know to proliferate. Tell your body that it is time to
divide.
RTK receives signals FROM Growth Factors
They bind to RTKs. They are Kinases, and as such will phosphorylate tyrosine
residues.
They end up dimerizing upon receiving a signal to form dimeric complexes.
Aberrant RTK Signaling is found in nearly ALL human cancers. One of which,
HER2 is over expressed in 25% of breast cancers.
Ligand binding induces dimerization of RTKs.
When EGF binds, jaw like clamps slam into the EGF, causing a loop on the
backside to protrude.
In the dimerized state, they’re protruding. The loops bulging out ALLOWS
the receptors to dimerize, rather. Thus you need 2 EGF receptors, both
clamping onto the molecule, pushing out the dimerization domains and
bringing the RTKs together.
What happens after they’re together? It brings their intra cellular domains into
close proximity, these domains are where the kinases are. Together, they
phosphorylate each other.
Following, the intra cellular domains become highly phosphorylated.
The highly phosphorylated domain of the RTK is the signal that the cell needs,
how the cell knows that Growth Factor has SUCCESSFULLY bound to its RTK.
This is CHANGE BY PROXIMITY, rather than CHANGE BY CONFORMATION in Short
Term Signalling.
RTK, GRB2, Sos, Ras, Raf, Mek, MAPK. Logic of SH2 and SH3 domains.
Now, RTKs will recruit additional signalling proteins that propagate the signal in the cell.
The signalling protein has something called a PTB domain, which binds to
Phosphorylated Tyrosine Residues (which are now highly phosphorylated at this point,
and WON’T bind otherwise)
These PTB Domains are SH2 DOMAINS.
SH2 domains thus bind to phosphorylated tyrosine residues.
They recognize Amino Acid sequences, and that’s how you get specific SH2 domains to
recognize specific RTks.
Ras is a small GTPase and the most common mutation in human cancer. (Rat
Sarcoma– Constitutively active Ras is a cause of 20-25% of all cancer, 90% in
pancreatic cancer)
It will go from GDP to GTP State, and will bind to downstream targets.
Cells will proliferate WITHOUT END, giving rise to tumours.
How does it become activated by RTK? What is the connection between Ras
activation and recruitment of SH2 domains via RTK?
Well, GRB2 and Sos (Help, Cancer! (?))– link Ras to active RTKs.
SH2 domain CONTAINING GRB2 allows it to bind to RTK by recognition of
Phosphorylated Tyrosins. Will recruit yet ANOTHER domain called the SH3
domain, which goes on to recruit SOS, which will bind to Ras.
Therefore Sos activates Ras. Thus there is a connection between SH2 and SH3.
SH3 domains are proline-rich peptides.
Sos is a GTP Exchange Factor, or GEF for Ras.
Sos must have proline-rich residues.
When Sos binds to Ras it turns it on. Ras signalling will now trigger cell
proliferation (Bad).
Propagates the signal inward.
Active Ras triggers a Kinase, a KINASE CASCADE.
Ras will turn on a protein called Raf. Normally inactive. Has an inward notch to bind
to the protrusion on active Ras.
Raf will only interact with Ras when Ras is active. Ras releases the inhibition standard of
Raf. Raf will go and phosphorylate things.
Raf is a GAP for Ras. GTP Hydrolysis will lead to the dissociation which helps shut the
signal down in “normal” circumstances.
Finally, Raf will phosphorylate a Kinase called MEK, turning it into an active state,
which will phosphorylate yet another kinase called MAPK and MAPK will finally activate
Transcription Factors.
The Take Way: At the end of the chain, all you’ve accomplished is to activate
gene
transcription and that comes from the phopsphorylation of TFs by activated
Kinase Molecules.
NOTE: MAP Kinase Cascade is responsible for “schmoo’ing” of Yeast.
Lecture 34 – Resting & Action Potentials
Basis of the resting potential. How resting potential would change in different
ionic
environments. Non-gated K channels, Leakage of Na through gated Na channels.
Nernst equation.
Action Potentials are “Traveling Voltage Waves”.
Ions MEDIATE the change in voltage.
If the number of ions is equal on both sides, there will be no voltage difference.
The thing that gives rise to the voltage is the SELECTED PERMEABILITY OF THE
PLASMA MEMBRANE.
NERNST EQUATION: 59mV Log([K outside]/[K Inside])
Selective Permeability creates an electric potential defined by this equation.
If we take the system with different concentrations of ions, all we have to
imagine is we make the glass selectively permeable to K. Concentration WANTS
to be equal on both sides. If greater on one side, ions will want to flow in the
opposite direction due to their chemical potential.
As soon as those ions flow, there will be an electrical imbalance. More ions
on the RIGHT than on the LEFT.
Now, as the ions flow, they’ll create an electrical potential that
COUNTERACTS their continued flow.
Therefore, many ions will flow across quickly but be pulled back by the
electrical potential imbalance created by the imbalance in ions.
SO, the EQ you reach is one which the chemical gradient, chemical
potential energy that WANTS to push the ions from LEFT TO RIGHT, is
counter balanced by ELECTRICAL POTENTIAL energy that wants to pull the
ions from RIGHT TO LEFT. Hence, EQ.
The equation gives us the final electrical voltage that exists across the semipermeable
membrane. The voltage will be such that the chemical drive for K to
continue to cross is EQUAL to the electrical potential bringing it back across.
That’s when you reach EQ.
Non-Gated K Channels ESTABLISH the Resting Potential
We see Non0Gated K channels partly open, allowing K to flow out of the cell.
K will flow, causing an accumulation of + charged ions OUTSIDE the cell relative
to the inside (-).
Enough will flow down its chemical potential gradient to create the voltage, then
the flow of K will be counter acted by the significant - voltage you’ve created
(inside). K will feel the tug to move out, down its chemical potential, or back
down its electrical potential.
We can measure that concentration and use the equation to estimate when the
resting potential of cells could be
Leakage of Na through Gated Na Channels
It’s more - than -60, but when measure it’s right about -60. The reason for the
difference is the Na Leakage, so membrane is quite selectively permeable to K
because the non-gated K channels allow for rapid flow of K but also has a small
amount of permeability to Na.
Na/K ATPase pump.
The Na+/K+ ATP-Pump establishes concentration gradients of ions.
Need to know: Power of 1 ATP Hydrolysis Event: Uses it to export 3 Na+ out, and
bring 2 K+ in, creating a difference in concentration of Na and K
Imbalance of ions here is not sufficient for the voltage that you experience across the
membrane.
Logic of action potential propagation: what makes it unidirectional and fast.
Action potentials involve the controlled opening of Na+ channels.
Propagation of the voltage wave down an axon mediates the controlled opening of Na
channels.
With APs, the propagation of the voltage wave is the controlled opening of Na channels
down the length of the axon. Inflow of sodium causes the next batch of channels to
open, etc, it’s like a wave.
The influx of ions will cause a very small and very local change in the membrane
potential so we can think that there might be some small tick (on the right) to the
membrane potential. The amount of Na that flows in will be small and the change in
the voltage will be restricted to a local area but if that flood of ions crosses some sort of
threshold, if you get enough Na in that small area, that causes a flood of Na (local) to
defeat the THRESHOLD for Signal Propagation. If enough Na comes in that you get
past it, you’ll send your AP.
Spike causes local flood of Na+ into the neuron.
Voltage-gated Na channels, voltage-sensing helix, channel-inactivating segment.
Voltage Gated Na channels lead to a flood of Na+ into the neuron.
Channel has a voltage sensing alpha helix (+ charges on it), because + charges like
to
be attracted to – charges, they’re mostly in the down state, close to the intracellular
side
Now if you have Na that flows in, due to deflection of the whisker that voltage sensing
helix will move because the membrane has become depolarized because the flow IN to
the cell has changed your electrical potential.
That changes the position of this voltage sensing helix from bottom to top.
Now the outside of the cell is more – and inside is more +. Causes the channel to
open up and these voltage gated Na channels can pass a lot of Na quickly so as soon as
they open, Na FLOODS into the neuron.
Reach EQ where inward flow (chemical potential energy) is balanced by the electrical
potential energy, that’s when the flow will stop. So the peak (+50) is equal to the EQ
potential for sodium.
NEXT STEP
When Na floods, it diffuses out. The diffusion of Na to the LEFT and RIGHT of the
AXON triggers the opening of the gates. RIPPLE/WAVE EFFECT.
However, we need the Action Potential to go towards the synapse, to the RIGHT
(theoretically)
That’s when the inactivation of the V-Gated Na+ Channel stops the flow.
The channel inactivating segment (ball) will throw itself into the Channel,
eventually leading to the inactivation of the channel. This makes the
pathway unidirectional towards the synapse.
Voltage Gated K+ channels (not Na!) will now reset the membrane
potential.
SHITTY SUMMARY:
So Na flows in, it diffuses left and right, when it diffuses left it encoutners Na channels
previously closed due to the activity of the segment, all it can do is have an action by flowing
right, the Na flows to the right, CHANGING THE MEMBRANE POTENTIAL, opening the Na
channels on that side. Back where you had recently changed the membrane potential. So…
You open your V-gated K channels, K flows out of the cell, leading to hyperpolarization and
then the
repolarization happens after your Na channel inactivation is released/relaxed. This is the
refractory period. Neurons can only pass so many APs per second, a maximum freq to which
APs can fire, that’s the time it takes for the segment to release its inhibition of a Na channel
such that it can become sensitive again
Peak voltage in action potential, Relationship to action potential voltage to
ionic
environment. Thresholds for action potential propagation.
Peak voltage in action potential: +40 mV
Threshhold for Action Potential Propagation: -40mV — -55mV, but can flux due to
sodium and potassium ions.
The potential required to move the voltage sensing helix of the voltage gated Na
channels. Once reached, the Na channels open and now your AP fires.
Lecture 35 – Synapses
Neurotransmitters and their receptors, especially acetylcholine. Ligand-gating
vs. GPCR
coupled receptors.
When the axon terminal or synapse becomes depolarized, when Na+ ions flow into the
terminal, when happens is the release of Neurotransmitters towards the direction of
Receptors for them.
Synaptic Vesicles hold neurotransmitter molecules, released into the extracellular
space between the avon and the cell receiving the signal. The cell will have receptors,
once bound, the receiving cell responds.
NTs diffuse across the synaptic cleft.
NTs are small chemical compounds, acetylcholine is used in muscle contraction.
They are small because they accelerate the speed of neuronal transmission.
Dopamine: Reward
Serotonin: Happiness.
Two types of Neuroreceptors: Ligand-Gated, and GPCR.
Role of calcium.
Axon terminals contain voltage gated Ca2+ channels. Ca2+ influx TRIGGERS exocytosis
of neurotransmitters. Flows into the extracellular space into the axon terminal.
Acetylcholine (AC) receptor, is an ion channel. Binding of AC triggers influx of Na into
the muscle. Ca essentially triggers muscle contraction. Na releases Calcium. Occurs
through depolarization of the muscle cell membrane. Triggers Ca release channels of
the sarcoplasmic reticulum to open.
The NT Receptor: Is a LIGAND gated ion channel. This is the AC Receptor. It has two
binding sites for AC, they cooperate with one another. Facilitates binding of a second
AC. When both are bound, the Gate opens and Na flows into the muscle cell.
GPCR: The binding of the NT is going to recruit G Protein which will be pushed into the
active state and the active G Protein is then going to open an ion channel if it needs to
depolyerize the synaptic cell.
The synaptic vesicle cycle: V-class ATP pumps, Neurotransmitter-H+ antiporter, SNAREs,
synaptotagmin, complexin, Neurotransmitter-Na+ symporter, dynamin.
It’s driven by a proton gradient within the synaptic vesicle itself.
There’s a V-Class H+ pump that uses the energy of ATP hydrolysis to shuttle protons
into the synaptic vesicle.
Operate by hydrolyzing, instead of synthesizing, to push protons up a proton gradient.
The pump pushes protons in, acidyfing the environment inside the synaptic vesicle but
that acidification is used to import NTs
While the proton goes inside to outside, the NT comes from the outside to the inside.
Packaged by proton neurotransmitter antiporters that take protons out and bring
NTs in.
Eventually, you reach an eq where the concentration of the NTs inside the vesicle is
high that there isn’t sufficient free energy from the release of the proton down its
chemical gradient to bring a NT up its chemical gradient.
Logic of the synaptic vesicle cycle: docking, recycling, kiss-and-run, and excess
capacity.
Synaptic vesicles are docked at the plasma membrane by SNARE complexes.
They have to be docked at the PM: Why? You want exocytosis to be fast. As soon as the
axon terminal is depolarized you want exocytosis to happen instantly. Makes sense that
if you want that, you don’t want to wait for the vesicle to diffuse to the PM, you want it
docked there. Machinery that executes exocytosis is the same as the machinery that
docks it; SNARE Complexes
SNARES: Have a transmembrane domain; rods! They have helices that protrude from
the rods, and the helices will wrap around one another to form an alpha helix bundle;
the bundle holds the synaptic vesicle membrane in close contact with the PM at the
axon terminal. It is the SNARE complexes that dock the vesicles.
NOTE: BOTOX UNDOCK SYNAPTIC VESICLES, PREVENTING AC RELEASE
To trigger excytosis:
Ca2+ influx.
Causes membrane fusion and NT release.
Ca channel opens, Ca flows in, vesicle fuses and NTs flow out in response to the Ca
signal.
That signal is sensed by a sensor protein called Synaptotagmin. Synaptotagmin has
Ca binding sites and the binding of Ca trigger exocytosis.
The SNAREs are spring loaded and ready to fuse the membranes. If released, they
would! But complexin binds to the v-SNARE/t-SNARE complex and inhibits its ability to
cause exocytosis.
As long as complexin has bound to the v-SNARE/t-SNARE complex it is inhibited from
doing what it wants to do; smashing membranes together.
Synaptotagmin: Has two diviots in the small purple domains, those are the Ca binding
sites and when Ca binds to it, synaptotagmin kicks complexin off of SNAREs.
Allows the SNAREs to merge. The Synaptic vesicle is bound, NT is released.
NOTE: The kiss and run mechanism makes some sense because neurons spend a lot of time
building synaptic vesicles, putting these proteins on their surfaces, and well “if they
completely flattened and all those proteins diffused away in the PM, all the work that you’ve
done to create the vesicle is lost” and you can’t then re-endocytosis, you’d have to do that all
independently, put them back in the right ratios and so forth. So maybe instead, you
preserve
the structural integrity of the vesicle by only releasing the NT but keeping the rest of the
vesicle intact.
Reuptake of NTs occurs by Na+ Symporters.
Recycling of the Synaptic Vesicle requires the GTPase.
Clathrin mediated process. Vesicle coats, endocytoses and the coat is lost.
Vesicle is now uncoated.
Protein that pinches the vesicle back off into the cytoplasm and completes the
last step of endocytosis is Dynamin.
Need to “re-protonize” your vesicle afterwards. Takes time.
Lecture 37 – Guidance and Migration
Structure of growth cones and leading processes.
Neurites -> Axon -> Growth Cone -> Elongation of Growth Cone -> Others specified as
Dendrites (Receiving), which will also elongate -> Dendritic Spines (Points where contact
is made between Dendrites and Axons of adjacent Neurons)
Re-arrangements of the Cytoskeleton will be done via Actin Filaments and
Microtubules.
The direction in which neurone migrate and axons progress is ultimately governed via
Guidance Queues.
Role of microtubules in cell migration and growth cone motility.
Netrins, semaphorins. Attractive vs. repulsive guidance cues.
Lecture 38 – Cell Death
Intrinsic vs. extrinsic cell death pathways.
Mechanics of self-destruction.
CED-9, CED-4, CED-3, Egl1.
Bax, Bad, Cytochrome C, Apaf1, Bcl2, PI-3 kinase, PKB, 14-3-3. Murder: Fas ligands and
Fas
receptors. Granzyme and Perforin.