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Lecture 18

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Biological Sciences
Dan Riggs

Lecture 18- Cell Cycle III: Biochemical Regulation of Mitosis and Mechanisms of Cytokinesis First we will talk about the mitotic process and then we'll talk about cytokinesis or how the cell divides the cytoplasm. There are two different ways that this can happen. First the animal cells where cytokinesis operates from an outside to inside mechanism. Second in plant cells which have a rigid cell wall and they build new plasma membrane from the inside to the outside. Review of molecular motors. They come in two varieties one called the dyneins - a class of motor proteins that track along the microtubules towards the minus end. What you can see here is that the molecule is taking its cargo and moving it towards the minus end of the microtubules. In contrast to that the kinesins are different family of motor proteins and they take their cargo and move towards the plus end. Both of these types of activities are involved in chromosome movement. To set the stage for the molecular motors do their job, they have to have a track to run on. The tracks are the mitotic spindles. The chromosomes are at the middle of the cell at metaphase and the sister chromatids have to segregate and have to be pulled to the opposite poles. There are several steps involved to get the chromosomes in the proper place so that they can go in the opposite directions. This sets the stage to talk about the different types of movements that occur during mitosis. Here is prometaphase. The nuclear membrane has just broken down, the microtubules have invaded the space of the nucleus and they may or may not capture the chromosomes. On the left-hand side of figure 14 – 33 you see the spindle fibers attached to the middle of the chromosomes. When the nuclear membrane breaks down some of the chromosomes may be at random positions but they all have to get to the middle. So pushing and pulling has to go on in order to get them to the middle. Movement 1 is where the two sets of centrioles which will become spindle pole bodies were duplicated and then move to opposite sides of the cell and they initiate microtubules as seen here with the plus ends sticking towards the middle and what happens is in movement 1, the kinesin motors hold on to one of them and they're trying to walk along the other. So they hold one of them stationary and exerting force on the other. The same thing is happening to the partner holding on to the opposite strand of the microtubule and pushing on the first strand. These two things force the two spindle bodies apart. Movement 2: to move towards a pole you have to employ a dynein motor. Remember dyneins are minus end directed and the minus ends of microtubules exist at the poles so if you need to move towards a pole you're going to use a Dynein motor. Movement 3: If you need to move away from the pole you can use the opposite type of motor called Kinesin motor to move you towards the opposite way towards the plus end. This happens both at the kinetochore as well as at several points along the chromosome arms where microtubules are passing by and are not connected to the kinetochore but rather are just passing by. Motors associated with them and the chromosome help to move them. This has been termed as the polar wind -like there is a force blowing from one of the poles towards the middle. And this is movement 3. Lets assume that this happens according to plan. Chromosomes are on one side, they came to the middle and now what you see by movement number five is that there seems to be balanced forces that are tugging and pulling from both ends. This keeps the chromosomes in the middle of the cell at the metaphase plate. This is movement number five. Movement number four is like movement number one. This is just a way to keep the poles apart such that the chromosomes can align in the middle and can be pulled in opposite directions. The activity of both plus and minus end directed motors at the kinetochore then is going to generate mechanical tension along the chromosomes in the middle. When the tension is deemed to be equivalent, that allows the cell to pass the spindle checkpoint. Then the cell knows to enter anaphase, separate sister chromatids, and move apart for anaphase. The events of anaphase are first, the sister chromatids lose affinity for one another. We're going to talk about the biochemical regulation of this. Then there are two types of anaphase movements. First, is referred to as anaphase A and that is the poleward movement of the chromosomes. Second, there is actually the separation of the spindle poles themselves that is the spindle poles were fairly far apart to begin with at the previous stages but now they themselves also move apart from one another. What this requires is both motor types of activities, changes in microtubule dynamics and the destruction of some regulatory molecules. At number six, the chromosomes have lost affinity for one another, and begin to move towards the poles. This is mediated by both Dynein type activity that is pulling them towards the pole and more so by a Kinesin like protein that is not exerting the kinesin motor function which you think would be pulling towards the plus end but rather it's a kinesin family member that acts as a microtubule depolymerizing agent. This is what causes the rapid depolymerization of the microtubule. As the microtubule is peeled back the chromosome uses that forced to be pulled along to the opposite pole. Movement number seven is similar to number one and number four. There is a separation of the spindle poles mediated by kinesin like motor operating on the polar microtubules. Something not shown in the textbook is that there is evidence that along the cortex of the cell if you think of this as interlining of the plasma membrane at the periphery of the cell, what happens is that there are dynein motors that enter there on the green dot and the dynein motors capture the astral microtubule and try to walk towards the minus end. Since the dynein is anchored to the side, they can only pull as they try to walk to the minus end. So the two forces that you see operating at number seven and number eight there act to push the spindle poles apart in number seven and also to pull the spindle poles apart which is movement number eight. Knowing how movement takes place and what happened during the stages, what we will do is begin to talk about how this is regulated at a biochemical level. What you see is controlled proteolysis that is the destruction of proteins and this is intimately involved in the cell cycle progression. Looking at this figure we know that cyclins are very important. Remember from this figure (figure 14 – 5) the G1 cyclin comes up and then rapidly goes down and begins to be destroyed as cells begin to enter S-phase and likewise for the mitotic cyclins, once they have done their job of making maturation promoting factors to give rise to the G2 to M phase transition they are no longer needed so they get targeted for destruction. How was it that these proteins are recognized to be specifically destroyed? One of the things that happens to them in part is that they are modified by cyclin dependent kinases. Putting a phosphate group on can activate or inactivate proteins but it can also act as a signal for a different class of molecules called a ubiquitin ligase. This should be familiar since we already saw it in the context of post-translational control of gene expression. In the cell cycle there are target molecules that are recognized by one of two multi subunit complexes called SCF or APC. These two are ubiquitin ligases. Ubiquitin is ubiquitous in nature and all eukaryotes have it and it is a very conserved small protein that becomes covalently linked to the target protein and this gives the protein a passport to go to the proteosome and be destroyed. Figure 12 – 58 shows the cells that mediate cell cycle progression would now get modified by phosphorylation then they can become recognized by ubiquitin ligase (as seen in step two which would attach to the yellow protein) and this is the passport to go to the proteosome. Ubiquitin is cleaved off, the protein is threaded through the middle where proteases exist and that breaks down the protein into its constituent amino acids and those things get recycled. The end result is that you have destroyed some type of regulatory molecule and that changes conditions in the cell. Figure 14 – 26 is a very important figure for this lecture. Along the bottom you can see time of the cell cycle from G1 to S-phase and G2 to M-phase – anaphase is broken down into its component parts at the bottom shows what is going on at the chromosome level not too important. During mitosis you get the congression at the middle at metaphase and then they separate at anaphase. On the other axis are the activities of these two types of ubiquitin ligases. What you can see is that for the most part you can trace the SCF (the red dotted line) it is mostly present during interphase but not mitosis. The green dots are one of the two APC forms and they seem to be present only at the back end of mitosis and at no other time in the cell cycle. So obviously it must do something important there. The other APCcdh (the blue line) is not present during interphase but it comes up during the middle of mitosis and remains high until the end of G1. The key thing to remember is that APC (anaphase promoting complex) is regulated by two substrate selectors and that is why you see that the name CDH1 and CDC20. So APC will have two different binding partners at different times of the cell cycle. What happens during anaphase? We will look at CDC20 first. During anaphase the sister chromatids lose their affinity for one another and are pulled in opposite directions towards the two spindle poles. How does this take place? One of APCs binding partners is CDC20. When they get together they form a structure that recognizes certain molecules to add ubiquitin to them and destroy them. One of those is a protein called Securin which is an anaphase inhibitor (represented by a blue triangle with C). Now what happens is that securin has a binding partner itself. That binding partner is a protease called Separase (represented by a purple triangle with the S). Securin and separase are together for a large part of the cell cycle. When securin is targeted for destruction separase is released. Separase is not active when it is bound to securin which is why the securin is called an anaphase inhibitor. When it separase is released, it targets the cohesins for cleavage. Once the cohesins are cleaved then sister chromatids segregate. On the figure from the right, you can see this happening. APC binds to CDC20. Securin is destroyed. Separase then cleaves the cohesin rings that are surrounding the sister chromatids. The microtubules are pulling from both directions, but when the linkage or cohesin in the middle is destroyed then anaphase can proceed and the chromosomes can be pulled in opposite directions. CDC20 also has another important role in the cell. It is responsible for the metaphase checkpoint or what is sometimes called the spindle assembly checkpoint. It is responsible for helping to ensure that there is equal tension on all of the chromosomes which tells the cell that all of the chromosomes are lined up in the middle of the cell which satisfies the metaphase checkpoint and now it can start anaphase. It is not appropriate to do so before then because then you can get a situation that might be like trisomy 21. If all the chromosomes were in the middle with the exception of chromosome 21 then if mitosis happened early that one cell would get two copies of chromosome 21 so that is one way to get a birth defect. Figure 14-23 shows a series of micrographs of cells that their chromosomes are trying to align on the metaphase plate. The metaphase plate is represented by the purple diagonal line. What you can see is that there is a long chromosome pointed by little arrow which is not yet at the metaphase plate and is lagging behind not yet attached to the other side of microtubules. The cell then sends a wait signal or a halts signal until this can happen. So over time you can see the lagger chromosome moving into the pile of chromosomes in the middle and only then will the metaphase checkpoint be satisfied and the cell will proceed to anaphase. Some clever investigators have discovered a protein that seems to play a vital role in this. That protein is called MAD2. By immunoflorescence you can see that it is in blue are the chromosome pairs and green are the spindle apparatus, in pink is the immunolocalization of the MAD2 and it localizes to kinetochores that are not attached. Someway this says to the cell that some of the chromosomes have not
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