Lec 2 - Prof. Harris
October 24, 2011:13 PM
Trafficking eminating out from the nucleus and extenfding to the
BIO230 Page 1 Two linear polypeptides are coming together and binds non covalently
and forms a dimer. These dimers are alpha and beta tubulin. These
proteins bind to the GTP shown in red. Binds non covalently. They bind
to GTP and hydrolyzes. GTP is like ATP can be converted to GDP.
Alpha tubulin and beta tubulin
One end of the dimer is different than the other. Because they are
asymetric. What hapoens when These subunits are placed together to
form the protofilament. They are placed head to tail to make a
polarized filament. The polarity n the dimer is made into head to tail
Polarity: just that one end of the structure is different from the other
Polaried filaments end of the structure. The GDP exposed end we find an alpha helix. They
have a polarity These polymers and cells can make use of these polarity
to organize the cytoskeleton or to use the cytoskeltone (like running
13 of the protofilaments are arranged side by side. These are called
microtubnules ebcause they are tubes. Its hard to build them together
because they are ndividual dimers and they tend to ineract with each
other and falls apart. They are large tubes. The cell uses a template
complex to nucleate microtubules. This is called the Gamma tubulin
BIO230 Page 2 Gamma tubulin binds the tubulin heterodimers and assembles the
protofilament into tubes. Theres a minus end and plus end which
reates to the polarity of the polymer.
Blue: is the gamma tubulin ring complex. What the gamma tubulin
individual protein does is (shown in (light blue) forms a circle.
Gmamtbulin forms a circle which is a perfect template fot the
tube that extends from it. 13 gamma tubulins in a circle and 13
protofilaments growing out from that template. The other is
Gamma tubulin and plus end relates to its polarity.
Each red circle you see in the spehere corresponds to the blue gamma
tubulin complex you see.
Protofilaments into tubes
At the growing end there is a process called dynamic instabilty: they
Minus end grow and they shrink.
Grows away from nucleation site
The gamma tubulin forms a circle, then 13 protofilaments are growing
out of the template, the other proteins are supportin protiens whch
help the gamma tubulin make the template.
Centrosome - the orgnaizing centers. In the centere the two cylindircal
structures are called centrioles. The hole is called pericentriole
material. Right on the surface of the sphere is all the circles. The
surfaces are the gamma tubulin complexes. Each cmoeplx corresponds
to the blue circle in the gamma tubulin. Now the gamma tubulin can
emintate from all sides of these nucleation sites from the sphere. What
the cell is creating is a 3D spehere of microtubules. This helps it to allow
it to explore the 3d space in the cell. They have the potential to reach
all throughout the cell.
These growing ends are not static they undergo a process called
dynamic instabilty. They grow and shirnk.
There is a whole bunch of microtubules at the plus end. We can
watch these live as they are floresced. 1 and 2 They grow out and
comes back, recoils and comes back again. These ends are dynamic
and allows the microtubule to search the full 3d space in the cell.
Tubule 3 is growing out
Tubule 1 and 2 grown and comes back again.
This process allows the microtubules to search the whole 3D space in
Microtubules plays a key role in searching and capturing sites in the
cell. This is one way by exploring the 3d space that they can find
This behavior is excellent to explore the 3d space because the ends
are dynamic. Microtubules plays a key role in searching and
capturing sites. They grab onto chromosomes and pulls them. Its
through exploring the 3d space they find these chromosomes.
BIO230 Page 3 Structue is different when GTP bound its stabilizing, GDP bound
microtubulin is muc weaker, so then catastrophe occurs, and and then
you gain the cap again and grown
Growing and shrinking
Dynamic instabilty takes advantage of the fact that Tubulin subunits
Cap of GTP bound tubulin
hydrolyze their gtp. So there is a polarity in the ends of these filaments.
When subunits are added to the growing end,they are gtp bound. After
they have been incorportated into the tube and they hydrolyze the gtp
to gdp (green circles). If they lose this cap of gtp, if additoin to the end
is not fast enough of new gdp bound subunits, then catastrophe occurs.
The structure at one end its different from the other. When gtp bound
Catastrophe occurs its stronger. Weaker when gdp bound. The 13 protofilamnets will spray
apart and catastrophate. The gain and loss of gtp cap is the dynamic
instabilty. One way of doing this is by exploring the 3d space.
Microtubule polarity is going from the minus end of the cell to the plus
In vitro: This experiment: we have purified centrosomes mixed with
purified tubulin subunits surrounded by an artificial membrane making
a square. When centrosomes nucleates the microtubuesl, the
Microtubules we have a line that is coming out, and we have a line that
is coming out. They are the microtubules which are Shooting out
centrosomes. They start to poush on it so they move onto the center,
and then they reach a part when there is an equal push from both
sides. So the centrosome ends up at the center. This is what it means
by a coordinate system. So now The minus ends of the microtubule is at
the center, the plus end is at the outside.
So this is the microtubule polarity going from the center to the outside
of the cell.
BIO230 Page 4 Polarity going to the outside if the cell and coming inside.
What can the cell do with this coordination?
Ski lifts from the center itakes people to outside.
Middle is the nucleus and taking them to the plasma membrane.
The cables --> has traffic so its like that in cargo
You can have microtubules minus in the cneter and plus at the outside.
There are many regulatory events which happesns as well and its not
We now have a ski life whcihc goes to the outside from the center.
This is like the biosynthetic trafficking. Transprtation from the ley resort
happening in the cell
BIO230 Page 5 What performs the ski lift function?
There are specific motors that bind to surface of microtubules -->
recognize the polarity of the microtubules --> and literally walk from
one end to the other end, along the microtubules
Dynein binds to the microtubule and walks to the minus end. (the
Kinesis walks to the plus end. Walks to the periphery of the cell.
In addition to binding to MT walking in a polarized way they also bind
Vesicles throughout the cell are been moves in a polarized way that
walks that vesilces along MT.
BIO230 Page 6 Experiment: role of microtubules in organizing vesicles in the cell.
Microtubules are in red, and we are asking about the position of the
golgi. All the vesicles that make of the golgi. To test the role of MT in
postiiong the golgi, we want to distrupt the MT and see what happens
to the golgi. This is a perturbation experiment. There are specific
inhibitors which can be used to distrupt the MT. like cortisol.
They are specific inhibitors you cant reat the system with and inhibit
MT. cortisol can distrupt MT. here the MT are stained in red. With
Cortisol treatment all the MT disappear and we see the depolymerized
MT subunits left.
What happens to the golgi is that its right around the nucleus.right next
to the nucleus there is a strong dot.that is the centrosome. In the MT all
the are eminatin from the nucleus and we just heard about the strucure
and there is acentrosome. The centrosome is providing a coordinate
system. We see that the gogli s fcused aroounf the centrosome. The
golgi particles floats freely away from the site and fills up the whole site
and the sytoplasm.
What will be the motor responsible fot the postioin of golgi? The motor
is walking cargo to the minus ends.
Golgi is right around the cukeas and all the microtubules are
emninating from it. The centrosome is the red dot.
Exmaple of how dynein and kinesin can work together and also
Shows how trafficking can be regulated.
We have balanced signals that regulate different motors which carry
vesicles of the cell.
The vesicles in the cell contain pigments.
Tropical fish: both are males: the dark one is acting aggressive and
turns itseld black : the white one wants to shine so it turns into a
lightbulb: there is a signalling in the brain which sends out signals to
change the physiology of the cell. The signals from the brain goes to
the cell and It changes the distribution of the membrane cargo and
the association with the cell MT. The pgiment filled vesicles are filling
the entire cell.
When the fish is dark the pigment filled vesicles is filling the entire
cell, because the kinesin and dynanin compete for the pigments in
the cell. Melanosme. These are pulled one way by kiensins and the
other way my dynein etc. so they have an even distribution.
When the fish wants to camaflouge titself a signal is sent to inhibit
kinesis. So you don’t have kinesin only dunenin. So they zip to the
center like what you saw in golgi.
Dynein moves to the center of the cell. And pigment filled are in the
middle and the cell is clea.
Has the effect of sequestering
This is how MT motor can control membrane trafficking system
BIO230 Page 7 The entire network can be remodelled. This is when the ski analgy falls
apart. Where the ski carts don’t come apart and completely reorgnaize
This is the kind of cell
All of the interface MT are broken down, and completely different MT
netweok is created which is the spindle. Spindle does its job, divides the
cell. The the two daughter cells are going to enter the interface and
reassemble in different ways and then back into its original state.
Go through the first slides , There is a nice comparison in the MT
BIO230 Page 8 Polarity relating to ATP hydrolysis.
Subunits of the polymers: these are monomers. (before it was a
dimer) . Monomers are also is a polymer. Has a - and a + end. They also
bind to small molecule: this case they hydrolyze ATP shown in yellow.
Actin subunits assemble head to tail so that the polarity of the subunit
is transformed into polarity.
There are only 2 protofilaments and its forming a helix.
There is a polarity for these filaments, - and + end, also polarity
associated with ATP hydrolysis
Like previously there was a polarity related to GDP hydrolysis.
When a subunit of actin is binding to ATP it binds to the filamnents in
an ATP bound state, after waiting in the filament for a whihle
hydrolyzes ATP to adp.