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Lecture 17

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University of Toronto Scarborough
Biological Sciences
Dan Riggs

Last time we talked about the cell cycle and mitosis and the events that go on during mitosis. Today we will review that and discuss two main things. The first of those are some aspects of chromosome condensation and the second is chromosomal movements. When the nuclear membrane breaks down in prophase, the chromosomes might find themselves all over the interior of the cell. They have to move to the middle in conjunction with each other to lineup on the metaphase plate. Then, once they are in line, they have to move in opposite the direction. We will see how this can take place. This is a review of the last slide from last lecture. Figure 14 – 11- Keep in mind what is already taking place. The chromosomes have duplicated in S-phase, they have gone through G2 and they got a signal for anaphase to begin. Then what happens? During prophase, the chromosomes are very visible. During interphase, they were very decondensed and you would not be able to see a strand of chromatin through a microscope. Now as they enter mitosis, chromatin condensation takes place and the sister chromatids become visible using a microscope. Some dramatic changes take place in the cytoplasm as well. The normal cytoskeletal array of microtubules disassembles and is replaced by a different type of microtubule organizing center called the spindle apparatus. Microtubules from the spindle apparatus attach to the chromosomes and pull them to opposite poles. The nuclear membrane then breaks down. We will review cytoskeletal MTOCs or microtubule organizing centers. The chromosomes are captured by microtubules, the nuclear membrane is no longer in place, the microtubules invade the space of the nucleus and capture the chromosomes at the kinetochores and begin the movement towards the equator or the middle of the cell. This is Prometaphase. When they arrive at the middle of the cell, they are said to lie at the metaphase plate. You can see that the pairs of sister chromatids exist there and that they have connections emanating through both opposite spindle poles. The cell needs to conduct a check point, to make sure that both pairs of chromosomes are aligned at the metaphase plate and that they are attached to microtubules coming from opposite spindle poles. The cell needs to be able to ensure itself so it conducts a check point to make sure that those pairs of chromosomes are aligned at the metaphase plate and they are attached microtubules coming from opposite spindle poles. They do this by monitoring the tension from each pair of poles, that is, if they are being pulled to the left or to the right. When that tension is equivalent, it tells the cell that the chromosomes are aligned in the middle.* See blackboard animations on checkpoints.* Once they pass the checkpoint, the cell is told that all the chromosomes are at the middle of the cell attached to opposite poles and now the cell initiates anaphase. Anaphase is the poleward movement of the sister chromatids. Sister chromatids lose their affinity for one another and get pulled in opposite directions. The poles also move apart. So there are two different movements that act called anaphase A and B. When the chromosomes arrive at opposite poles, they begin to decondense. The activities that require them to be condensed that is the separation of chromosomes don't need that anymore. The chromosomes decondense so they are more accessible to enzymes like RNA polymerase and normal types of transcription and translation will begin again. Since you have separated the two chromosome bodies from each other, this is a eukaryotic cell; to begin the next step of the cell cycle compartmentalization is needed. You need to reform the nuclear membrane around each of the two sets of chromosomes. Later, the cytokinesis will divide the cytoplasm and the cell has duplicated the chromosomes, segregated them efficiently and correctly to opposite poles, built two new nuclear membranes, divided the cytoplasm, and turned one cell into two identical cells (at least usually). This slide was shown last time, but today we will talk about some of the events that cyclin dependent kinases mediate in cell cycle transitions. Figure 14 – 5 shows that there are two primary transition points for a cell. One of those is the decision to go from G1 to initiate a new cell cycle by approving new replication. That is, the transition from G1 to S-phase is mediated by a complex in yeast called START. Later, a different complex called MPF (maturation promoting factor) is going to mediate the transition from G2 to M phase. Remember that the complexes exist or consist of a couple of subunits. One of the subunits are a cyclin dependent kinase – kinase is an enzyme that phosphorylates other proteins – but not just any proteins, they are specific targets of the cyclin dependent kinases and now think about what those targets might be. What things have to happen in order to move from G1 to S-phase using the start transition or from G2 to M phase is in the MPF trigger. Let's talk about S-phase very briefly. DNA replication has to be initiated. The centrioles, and the cells that contain them, must be replicated because those are going to be the microtubule organizing centers (MTOC’s) or the spindle apparatus. When we talk about DNA replication and the licensing of DNA replication, part of the figure 13 – 20 says something to that effect of cyclin dependent kinase phosphorylates certain proteins involved in getting S-phase started. These are generically some of the events that have to take place in order for S-phase to begin. Now during mitosis what has to happen for anaphase to begin – what drives the cell from G2 to M phase? The nuclear membrane has to break down. You might remember that the interior of the nuclear membrane is lined by intermediate proteins called lamins. The chromosomes have to condense. Some other targets of MPF activity are two groups of molecules called condensin and cohesin. Lastly, the cytoskeleton has to undergo a dramatic reorganization to switch from the normal microtubule array which is present in most interphase cells, duplicate the centrosomes and they become part of the spindle pole bodies that segregate the chromosomes. Microtubule associated protein are some targets of MPF as well. Let's talk about these three things in a little bit of detail. First are the lamins. Remember that the nuclear lamina lines the interior of the nuclear envelope and is made up of primarily three proteins called lamins A, B, and C. What you can see from the cartoon here is that the they form interactions with each other and that makes sense in terms of making a lining in the nuclear membrane - look back to figure 12 – 3 you see a fairly interwoven mesh like network that exists and that made up of the lamins proteins interacting with each other. What is not immediately obvious is that chromatin is attached to the periphery of the nucleus by means of interacting with lamins B. Chromatin exist all over the interior of the nucleus. It is attached at multiple points to the periphery of the nucleus by associating with lamins B. When the time is right, when MPF becomes active, one of its targets is lamins A and lamins C, shown by the red phosphate group added to those two. What happens is that this disrupts the interaction that takes place between the lamins so they release each other but they also release the chromatin. Now if you're thinking about chromatin compaction and condensation, the chromatin would not be able to condense if the nuclear envelope is holding it at various places. When it releases from the nuclear envelope and falls into the middle of the cell, it begins its condensation so the chromatin is now released and some other factors act on it in order to phosphorylate the condensation. Figure 14 – 14: think of what chromatin looks like in an interphase cell. It is decondensed but when DNA replication takes place what you have is one chromosome that has been turned into 2 sister chromatids transiently. A complex called cohesin is made up of two large proteins called SMC proteins (structural maintenance of chromosomes proteins). These are two large proteins that interact at both ends and there are some other accessory proteins associated with them (not important). Together this complex encircles around the two sister chromatids like a ring around the two sister chromatids that physically hold them together. This is what goes on during interphase. When mitosis or prophase starts, the chromatin begins to be decompacted and MPF and other proteins kinases phosphorylate some of the subunits of condensin and that makes them let go in most places. But, as you can see in figure 14 – 15 A, there is a pair of sister chromatids that appeared to be held together alone but are linked loosely in most places such as at the bottom where you can see some distance or separation, but they appeared to be held very tightly at their centromere. For a long time people thought the centromere doesn't replicate until just before division takes place but that's not what happening here. Actually cohesion is holding these sister chromatids together primarily at the centromere region during most of mitosis. It pretty much lets go of the rest of the arms of the chromosomes but still hold on tightly at the centromere. Next time we will talk about some of the events that reciprocate anaphase, that is, the separation of those chromosomes and what you see is that cohesin is targeted for the destruction of that chromosome. Cohesin plays a role early on in interphase, the early part of prophase less so but then at the end phase of anaphase is completely destroyed and the chromosomes can separate. Condensin, as its name implies, is involved in compacting or condensing the chromosomes. How does it do it? Condensing interacts with chromatin loops and it is a member of the same family of SMC proteins and has the structure of multi subunit complex similar to that of cohesin. What it does is something that looks like on the right-hand side of the figure. That is, it is a flexible hinge that is ATP driven and so both ends of the complex interact with chromatin loops and when the hinge closes it brings the loops closer together. In association with topoisomerase, it supercoils the loops such that they occupy less space and it pulls the super coiled loops together. In the end you make something like what is seen in the middle through the electron micrograph. This condensed chromosome consists of a coil around which lots of these condensed loops are arranged very close to one another. The third target involves cytoplasmic proteins and reorganizing the cytoskeleton. Figure 9 – 18 shows the centrosome which is a microtubule organizing center. You probably know that it has an arrangement of perpendicular pairs of centrioles and we have nine fibers of microtubules that are located together. These collectively are a structure that initiates microtubule formation. Over the cell cycle, these centrosomes are duplicated just like chromosomes are duplicated through an unknown mechanism. In early G1 phase you have a regular centrosomal array (which is the regular MTOC) and then what happens is that both of the mother centrioles are used as a template to make some daughter centrioles seen during S-phase. One of the things not printed on the slide is that cyclin dependent kinase governs this replication in some way. It is not clear but it is known that cells the centrosomal proteins are phosphorylated by the cyclin dependent kinase and that seems to be associated with their replication as the signal for them to replicate. Later as the cell cycle proceeds, the new daughter centrioles grow and become indistinguishable from their mother centrioles. Later as mitosis gets started there are other cyclin depending kinases and other kinases that phosphorylate them, which makes these two lose their affinity for one another. The two pairs, instead of being closely associated with one another, there are phosphorylation events which causes them to separate and move to opposite sides of the cell which sets up the mitotic spindle. In association with that, MPF activity also is involved in phosphorylating proteins that enhance microtubule initiation. Lots of microtubules are being formed to create this spindle apparatus. Mother-daughter pairs tend to stay together (semi-conservative). The rest of today, we will talk about the spindle apparatus and the microtubules associated with it and how they capture the chromosomes and how they move chromosomes around during different parts of the process. Let's review the dynamics of microtubules. Here we see a regular cell with the nucleus and cytoplasm and the main microtubule organizing Center in the centrosome. At a higher magnification looks something like this. Microtubules are made out of tubulin. Tubulin dimers come in and are added to the plus ends of existing microtubules to increase their length. You might remember this as a very dynamic process. Adding tubulin dimers to the plus end, microtubules go along and all of a sudden there are is a catastrophic collapse and they immediately disassemble but that is also associated with rapid elongation of other microtubules So it's a very dynamic series of events where microtubules are constantly initiated, become unstable, and the collapse. But the key rule which we will break sometimes during mitosis is that growth of the tubulin dimers occurs at the plus end and usually disassembly occurs at the minus end. We will break this rule in just a minute. Let's talk about chromosome movement. Let's assume that chromosomes have attached to microtubules, how can they move around and use those microtubules. There are molecular motors and in some measure those molecular motors hold on to the chromosomes and drive the chromosomes along the microtubules tracks. This is mostly active during prometaphase for something called congression. Congress means “to meet” so the process of congression
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