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Lecture 10

Lecture 10 & 11 - Prof. Harris cell cycle and apoptosis.pdf

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Maurice Ringuette

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Lecture 10 & 11 - Prof. Harris November 8, 2013 5:39 PM Controls when cell divide. Cell diviosn is very imporant for organisms we start as a single cell and cell divide to form multicellular orgnaisms. Flipnside is cell diviosn should be controlled in adults as well. If not controlled, leads to cancer. Because it divides uncontrollably. BIO230 Page 1 When we think about eukaryotic cell cycle , we can define it as the repeated duplication of the contents of the cell and dividing into two. Picture: a daughter cell just bron from cell division. And before it divides again it has to grown. Ebcause of you divide without growing, its going to get smaller and smaller and smaller. That means it has to replicate its PM and internal mebrane systems. Has to replicate all the organelles before dividing. Proper replication of DNA is critical. If there are mistakes in the replication of DNA, and division of DNA, all of the cells in the body will be different from each other and this is the cause of cancer. When mistakes are made in DNA replication. The other aspect of this Duplication of DNA, is not only in the indivudal but also from generation to geeration to and segrgation to daughter cells. maintain the similarities between one generation to another, DNA has to be maintained in the basic form. There are some mutaitons leading to variation in species, but that species may has to be faithfully replicated. BIO230 Page 2 We can divide the cell cycle to 4 main phases. If we divide them again, we have the birth of a daughter cell. We see the duplication of the cells components are broken into 2 main sections. One is for the DNA, and the other is for the rest of the components. G - Gap pahse. Remember it as growth phase. In this all the other components of the cell are been replicated. The PM, the prganelles etc everything other than the DNA is duplicated in G1 and G2. In G1 there is a partial doubling, and there isa very specific phase called S pahse, S for synthesis. In that we are specifically talking about the synthesis of DNA. DNA content in the cell is duplicating. And that happens in isolation. And in the G2 the remaing things are doubled and then you have gotten two copies of everything in the cell and it is ready to enter M phase, which is mitosis phase. And now it is ready to divide, DNA is oulled into either end of the cell. And will be splitted equally. All of these steps are complex. So we need a temporal coordination of all of these processes. We need checkpoints to make sure everything is proper. So before the cell starts replicating the DNA, the cell monitors the resources and ask whether there are enough resources to duplicate. So cell is not going to run out of nucleotides half way through so it will make only a half copy of the genome when it wants a full copy. It will also make sure that all the DNA is replicated before mitosis starts. So it doesn't want to start dividing until. There are 2 full copies of the DNA. These are the questions a cell would ask. But what are the molecular mechanism underlying this? BIO230 Page 3 We refer to them as checkpoints to control the cell cycle. And there are specific check points when a daughter cell is born going through the gap phase when the synthesis of mateirals in the cell. The cell would check the environemtn before enerting DNA syntheis. Etc. Going from metaphase to anaphase: when chromsoems are been attached to spindle. The cell ensures that all chromsoems are attached to spindle. BIO230 Page 4 At the core of ther mechainsms controlling these checkpoints are Cdks. These are protein kianses and they have targets, they phosphorylate proteins that control the cell cycle. Cyclin dependent kinases When a Cdk is active, it gives a green light to pass through the cell cycle check point. We can think of this as the molecular machinery that is watching upstream the state of the environment etc, duplication of the DNA etc. if the signals are favorable it will activate Cdk, and the Cdk kinase activity will get turned on. And Cdk will phosphorylate the actual machinery that is responsible for each functions. Replicating machinery of DNA/ machinery making the spindle etc. One way these Cdk is tunred on is by binding to cyclin. These are 2 separate proteins. Cyclin binding to Cdk is one way to turn Cdk on, to turn the light green. So not only cycling binding to Cdk that turns that Cdk on. There are multiple steps. Focus on just cyclin Cdk relationship. What occurs during cell cycle is there are different cyclin Cdk checkpoints at different cell cycle stages and they provide the checkpoints for each stage. Start when a daughter cell has just been born. This daughter cell has would be growing for a bit in G1 and then it will have molecular machinery monitoring the environment, and if that machinery says the environment is good, then it will sends signals that will synthesize S cyclin. The protein S cyclin binds to S Cdk, activates the kinase. The kinase then phosphorylate proteins important for replication of DNA. We have an upstream signal that increases the expression of cyclin--> turns Cdk on --> Cdk is responsible for phosphorylate the machinery directly replicating the DNA. Once the DNA is replicated, a signal says it is replicated and in response to that cyclin is destroyed. So a specific cells, in cyclin Cdk complex that forms specifically before the stage and then cyclin is destroyed and turnd Cdk activity off. So now as we pass through, ew can imagine that the DNA replicating machinery has worked. It has replciated the DNA. The cell can monitor and if it has then M cyclin is synthesized. M cyclin binds to Cdk --> activates Cdk --> phosphpryalte machinery promting mitosis. When mitosis is over cell recognizes it -->trigger destruction of M cyclin so that Cdk is turned off.we have this machinery turned on in response to specific checkpoints. Important is : these machienries are also tunred off. Imagine that if Cdk was not turned off and cyclin was not destroyed, there will be a continuationactivation og the DNA replcaiton. And will cosntantly trying to either replicate DNA or form mitotic spindle. BIO230 Page 5 Major cyclin from vertebrates and yeast where most of the researches were done. Can see that as we go through the cell cycle. G1, G1/S, S, M there are different combination of Cdks in each stage. When we look here, the Cdk partner here, it is the same Cdk 2 used. The differnce is it is binding to different cyclin proteins. This will give a different conformation to the complex so it activates differt machinery downstream. In yeast: the Cdk is the same in every one of these checkpoints. And it is the difference expression and Destruction of different cyclins that gives the Cdk different activity towards different downstream targts. This is a good combiantorial control of Cyclin to Cdk. Cyclin binding to Cdk is key for activating Cdk, but there is this activating phosphate. We are looking at the Cdk. This region here is ATP and red ribbon structure is the kinase pocket/ the active site of the kinase. Its targets will eventually land on these pockets and the kianse will transfer the phosphate from the target protein. When it is inactive this pocket is folded up and is inaccessible for downstream targets. These two steps opens up the pockets so the target falls into the pocket and can be phsophoryalted. First step: cyclin binding leads to partial activation so it slightly opens. But it is not fully active what the opening does is, this is a distinct kinase which allows to phosphorylate the Cdk. So it is Cdk activating kinase. This phosphorylate a site on the ribbon and that phosphorylation opens up a binding pocket. So it is now fully open and the phsophate can be transferred to the target going into the kianse domain. BIO230 Page 6 Onece the kinase is active, some of the machinery performing the fuc tions in each stage of the cell cycle . If we look at the S stage. S Cdk activtiy inhibts an inhibitor origin recognizing complex. And by inhibiting this inhibitor turns this complex on, and it promotes DNA replication. So one of the target of Cdk is machinery which drives DNA replication. There is M Cdk in the M pahse. It phsohprylate multiple targets needed for mitosis . Promotes DNA replication One thing that is needed for mitosis is accesibilty of the chromosomes to the mT that are emanating out so the nuclear envelope has to break down. So one of the targets os the laminar in the nuclear enverlope which phsophrylate Cdk so the nuclear envelope has to break down. For mitosis Whole number of targets are needed yo activate Cdk. Oackages the chromoems up What tunrs them off? They can be tunred on by cylin and phoshprylation. What turns them off during the cycle? Destruction of cyclin. Synthesis and binding will turn it on. Destruction will turn it off. BIO230 Page 7 One example that affects M Cdk. This is called APC. This promotes the transition out of mitosis. This complex allows the completion of mitosis. So mitosis starts with an active M Cdk. So here is the active M cdk. When it is activating it is phosphorylating. Eg: the nuclear envelop breaking it down, MT to make the mitotic spindle, and chromosome to condense Here it is phosphorylating the mitotic spindle. As soon as it’s the spindle has seeperated the chromomes to either end of the cell, the two ends of the cells now wants to divide from one another and the two daughter cells goes back to the normal interface cell, ebcause they no longer have the mitotic spindle. So you want to shut the machinery down. When the signals are presents, eg: spindle has worked and the chromosomesare at either end of the cell, this activating protein subunit called Cdc20 I scitivated and it binds to APC and acitvates the comlpex. This complex acts with ubiquitination enzymes and adds ubiquitin groups to cyclin of the cyclin M Cdk complex. Ubiquitin is the question mark here. These enzymes can be added on to proteins, and that is a signal for the proteasomes to degrade. And APC is directing this ubiquitination machinery to M cycling once the spindle is properly formed. This gets ubiquitinated --> cyclin is destroyed and Cdk is shut off. And cell can leave mitosis. Regulation of cell cycle is important so there are more layers of regulation BIO230 Page 8 Cdk inhibitor proteins in another layer of regulation. Cyclin dependent kinase inhibitors. (CKIs) We have an active Cyclin Cdk complex. It has an activating phospahte, cylcin is bound. And this complex has already phosphorylated its downstream targets. But it cant be regualted. Cant be turned off by the action of one of the CKIs. Eg: P27. what P27 does is wrap around the entire complex and stops it from interacting with downstream targets. Just by binding to cyclin Cdk complex, Cdk complex is allr eady to go. But it is non covalently bouned to CKIs. This is another way to turn cyclin Cdk off. Regulation through regulatory phosphorylation. This phosphate is the activating phosphate which will turn this off. If we start here, this will be the active Cdk complex and cyclin is bound and the activating phosphorylation site is phosphorylated. We are talking about here is a separate phosphorylation site. The activating phosphate is still present. The overall complex is identical to this except now a phosphate is added at a separate site. This added phosphate changes the conformation so it can now no longer act with the downstream targets . This is a conformationsal change --> changes structure slightly --> cannot bind to downstream targets. This phosphrylation is through Wee1 kinase. This adds a phosphate group and Cdc25 removes the phosphate group. Wee1 can turn it off, and Cdc 25 will turn it on back again. BIO230 Page 9 Organized the ideas into this schematic. So activation of Cdk gives the green light to pass through. So if Cdk is on then it is going to be phosphorylating all the macinery needed to pass the cell cycle. One main thing that can turn this on is the binding of cyclins. Cyclins will bind and turn it on. Cyclins are controlled by their synthesis and degrdation Cyclin is destroyed --> one way of turning the green light off. Synthesis will turn it on. Even if cyclines were bound to Cdk and activates the phosphorylation the cyclins can still beturned off by two other mechanisms 1. When an inhibitory protein is present it will turn it off 2. And inhibitory phoshprylation occurs These layers of regulation, inhibitory proteins are controlled by the synthesis or degradation of the protein. And inhibitory phosphrylation will be controlled : if the kinase active it will promote the inhibitory phosphorylation and if the phospahte is active it will turn it off. Some exmaples BIO230 Page 10 Transition into S phase. This is controlled by a protein called Rb. Rb is controlling the synthesis of cyclin that will promote S phase. This is how a cyclin is synthesized to oush a cell into DNA synthesis, This is through the activity of a TF E2F. E2F drives the transcription of S cylclins. So the cyclins that are needed to bind to the Cdks are pushed into the cell for DNA synthesis [email protected] is responsible for making the cyclins that drive DNA synthesis. Cell wants to control this and donest want to occur imporperoy. So during development it doesn't want it to occur in improper places. It needs a mechanism to control E2F. This is what the protein Rb does. So if a cell is not receiving a signal to divide then Rb is a protein that wraps arund E2F and keeps it inactive. So Rbs normal role is to keep E2F inactive so you don’t have the synthesis of cyclins which will drive DNA synthesis. But if a cell recieves a signal that says you are ready to divide so you have an active G1 Cdk, this is from an early step in the cycle, this Cdk will phophprylate Rb and this triggers the dissocation from E2F. When Rb is phophryalted it releases E2F which can now drive DNA synthesis. We have an upstream signal which says either everything is going ok a signal is sent to activate the CDk which expels Rb from E2F and cells start to synthesize DNA. BIO230 Page 11 Rb stands for retinoblastoma protein. Rb is important for normal development, also important for cancer. Loss of both copies of Rb gene leads to eye cancer in children due to excess cell proliferation. So we can consider Rb to be a tumor suppressor. A tumor suppressor because Rbs normal actions is to stop cell cycle. So normally stopping of DNA synthesis, it tries to suppress cell proliferation. This is the normal role of it in cancer. . When cancer does develop in response to Rb we can imagine that the if we don’t have Rb activity. The E2F will alwaus be active promting DNA synthesis and cell division leading to eye cancer. Tumor supressor BIO230 Page 12 One step up from these are the Signals that the cells receive to divide. These are called as mitogens. We have a mitogen secreted protein that binds to a receptor. And it induces the transcription of a protein called Myc (bottom of the slide) , everything at the top of the cycle we talked about. Mitogen binding to receptor TK, and this activates Ras, and Ras binds to MapK. And then Map K cascade finally activates Myc. We talked about RTK and etc We talked about Ras over activity is linked to about 30% of cancers. We are going to see now why Ras activity is linked to cancer. And one of the major ways of this is that Ras leads to the activation of Myc expression. Myc is another protein that is now been transcribed in response to Ras signaling, and Myc increases cyclin synthesis and CKI degradation by regulating transcription. Basically promoting cell cycle through these two arms. Promitng cyclin synthesis and the degradtion og cyclin inhibitory proteins. BIO230 Page 13 Myc itself is a TF. When Myc is expressed it binds to the regulatory regions upstream of a whole set of genes. One of these genes is a gene for cyclin that drives the cyclin production driving the cell cycle forward. So it is producing cyclin and the another target of Myc is the expression of the SCF subunit. SCF subunit is linked to proteasome regulation and targets the protein P27 to the proteasome. P27 is the protein which noramlly wraps around the Cyclin CDK complex and keeps it inmactive, now the protein is been degraded. So we are making more cyclin Cdk complexes and unwrapping more cyclin CDK complexes from the inhibitory proteins. Both things are going to start the drive of cell cycle forward. How do they do it ? Note that they both are G1 stage cyclins. So theya re feeding into the Rb E2Fpathway just described. So G1cyclin and Cdk complexes will phohprylate Rb which will remove E2F, and this is a TF which drives the expression of cyclin to S phase. E2F activity goes up because it is inhibitors are been phosphorylated. Also the cells the E2F gene is a target of Myc. So Myc drives the expression for the E2F gene. Now not only there is a less inhibtor for E2F but tE2F is also building up at higer and higher levels. We can see that Myc is extremyl potent promoter in the cell cycle. You can think of upstream, RTk, ras upstream of it and they all are potened the activation of the cell cyle because they feed into the cyclin production and activation of the S phase. Myc has normal roles during cell division and . And physiology and Myc abnormal over activity will lead into cancer because of excess cell proliferation. Drives into the S phase. Because Myc is forcing cell division it is called oncogene. oncogene BIO230 Page 14 We are looking at the kinases that will be inhibiting Cdk and phosphatases that relieves that inhibition. This is the transition from gap pahse into Mitosis. The cell is making sure that all the DNA is replicated, the environment is favorable to start to build the spindle and separate the DNA into two poles. So one of the signals that tell the cell its ready, is the synthesis of M cyclin. Cell starts making M cyclin --> it binds to Cdk -->Cdk activating kinase will phosphorylate Cdk at the activating phosphate This complex would be ready to go, but it is held back from Wee1 which is the inhibitory kinase which phsohpryaltes at this site. So cell is produced one signal to say it is ready to go into mitosis, but it has a back up system just to make sure. It is not relying. It needs 2 signals to say that it is ready to go into M phase. Because it is critically important to get that right or there will be massive damage to the DNA. This inhibtory kinase is keeping the complex n check waiting for the 2nd signal just to be sure that the cell is ready BIO230 Page 15 So the second signal comes through this phosphotase, which will remove the inhibitory phosphate. It will become activated ,by removing the inhibitory phosphate and creates the M Cdk. Now this has to be activated somehow. And that is through this kinase called a polo kinase upstream. The main idea is that M cyclin and polo kinase are the two of the machenry the cell is using to recognize that it has gone to mitosis. Cell needs both of those to be active to creatrte the active M Cdk whichh will then promote the formation of the spindle. Another intriguing aspect of this in addition to having the backup system in place, is that when M cyclin if formed and you start creating those molecules you are basically building uo a supply of Cdk that is ready to go (it is phosphoryalted), except it has an inhibitory phsphate. This is where you have to thinkabout how many molecules are invovled here. It is not just only one molecule, but there will be 1000s of M Cdk. There will be 1000s of M Cdk phoshprylating 1000s and thousands of MT assoicated proteins, chromatin , and nuclear envelope associated proteins and so on. One elemennt of this is you can start buiilding all those 1000s of them in this inhibitory state. BIO230 Page 16 Now you only need one switch to turn them all on at one time. So one thing that do happen is when you do have the switch Cdc25 removes the inhibitory phosphate one thing M Cdk also does is, it promtoes the formation of itself. It does by activating Cdc25 moreby inhibitng the Wee1 inhibotrry kinase. You very quickly a little bit of it made and then it promtoes the formation of more and more. If you start with a 1000 moelcules, and if 50 or 100 is initially activated, then very quickly the 50-100 willa ctivate all the 1000 of them. When the formation of the spindle and the condensation of the chromosome, that will have to be a fairly synchronized process. You can imaginew that all the chromosomes condensing and the MT formation must be happening at the same time. This is then a good strategy to turn all those 1000 molecules on in a short time span. So they all can be turned on once to wor on the downstream amchnery in a synchronous way. We talked about ther egulation, entry into S phase with E2F. We have Ras Myc E2F promoting DNA synthesis. The other key element of dividing the DNA and entry into M phase we just discussed. With the inhibitory phsphate. One question is: What if there is a problem arising at some stage of the cycle? It takes time for the whole cell division to occur. What if it expresinces DNA damage in part of the process? Then cell wouldn’t want to comtinue with damage on and pass to the enviroienmt So cell would want to shut down the cycle, either less nutrients, unfavarable enviroenmt etc, it would want to shut down its cycle and wait until it gets better ir try to fix the DNA damage that is been done. BIO230 Page 17 Major player that can perform this is called p53. so we can stop the cell cycle in reponse to DNA damage. In here we are looking at the role of an inhibitory protein. If everything is ok in the cell without any DNA damage and is receiving all the signals to divide then p53 is always produced but it is always degraded. This might seem like a waste of resources. Why would cell make a protein and detroy it right away? This means that there will always be a ready supply of p53 to be used by the cell. All the cell has to do is to stop the degradation of p53 and its levels will immediately rise and it will act. This protein Mdm2 normally targets p53 for degradation if there is DNA damage. DNA damage will activate some pathways, don’t worry about the specifica about the pathways, but the cell can recognzie that there is DNA damage, and the patways will lead to the phosphorylation of p53. so when p53 is phosphoryalted p53 will recognzie its phopshorylation -? Expel mdm2 from its complex so now it is no longer targeted by the proteasome. Levels are going to build up and now p53 can act in response to the DNA damage. BIO230 Page 18 What p53 does is, it is a TF. It drives the expression of a gene called p21. it is similar to is one of the Cdk inhibitory proteins. When you have the damage you will express P21 and it will express a protein, and wraps up whatever Cyclin Cdk complex present in the cell and stops it from phosphorylating the downstream targets. That means that when the cell experiences DNA damage now the signals that are activating the DNA synthesis machinery are shut down. And then DNA repair macinery will try to fix the problem. And then after the problem is fixed p53 is defgraded and then cell goes back into the cell cycle. We can see p53 protects cell. Loss pf p53 leads to cancer. If we lose all the p53 then we no longer have this protection. So the cell can no longer respond to DNA damage. Cancerous cells can build up further mutations as they are no longer responsding to DNA damages. Sp p53 is a tumor supressor BIO230 Page 19 So we can use these pathways. We can orgnaize them like this. Advice: use this as a study guide so you can pick out and make a list of all the cell cycle regulators and test yourself. Can you remember which players were in this arm of ther egulatory circuit. Lecture 11 - Apoptosis Important mechanism to control cell numbers. We are talking about induction of programmed cell death. Also called apoptosis. Programmed cell death is important in development and physiology. Example: is the general strategy used during development that an organism makes more cells than it needs and then kills some cells off to make the final structure. Talking about the formation about the limb. Ew talked about how Shh acts like an orgnaizer next to the limb bud and then the limb bud becomes a cell proliferating cell tissue. To create all the cells that are needed to make a limb. The Shh is used to determine all the digits in the hand but what typically happens is that, this is the case where it starts off with more cells in the limb than needed and the cells in the digits are pruned away by apoptosis. So the little tiny dots you can see in the first picture in the tissue are single cells that are going to be killed off so the digits are going to be sepertated in the two hands. This is a pruning idea during development. We can also think that in the life cycles of certain orgnaism there is one stage in it where the body parts will be sued, and then that body part is removed in the enxt life cycle. Eg: tadpoles have tails to swim in one stage and then the tail is removed. This is removed by apoptosis in adult frog. BIO230 Page 20 In addition to that apoptosis also plays a protective role. In our bodies all the time apoptosis is killing off dangerous cells. The danger off this cells is they can lead to cancer. P53 protects our cells and it can recongize the damage to DNA and stops the cell cycle to the cell can repair the DNA. So if it was not
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