Lec 2 transcribed cytoskeleton- Prof. Harris.pdf

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Maurice Ringuette

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Lec 2 - Prof. Harris October 24, 2011:13 PM Trafficking eminating out from the nucleus and extenfding to the PM> 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 orientation. asymetric 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 motors across) 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 comples. 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 supporting proteins. 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. Dynamic instability 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 the cell. 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 these palces. 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 instability 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. Rescues growth Microtubule polarity is going from the minus end of the cell to the plus side. 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? Analogy: Ski lifts. 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 that simple. 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 center) 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 to cargo. Vesicles throughout the cell are been moves in a polarized way that walks that vesilces along MT. Minus end Plus end 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. centrosome a. 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 themselves. 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 cytoskeleton network. t 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.
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