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Lecture 19

BIO241 Lecture 19

12 Pages
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Department
Biology
Course Code
BIO120H1
Professor
Jennifer Harris

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Description
Tuesday, March 17, 2009 - If we go back to the question he raised in the first lecture: How do cells control their shapes, their interactions, their numbers to build tissues, organs & organisms? - So far we’re talked about control of cell shape, cell interactions with themselves or with the matrix & now he’d like to turn to the control of cell numbers, both by controlling cell division & cell death & next week we’ll talk more about mitosis & also the link to cancer b/c one of the key things to cancer is the control of cell number. - Next week we’ll discuss more about cancer, but during the lecture today, he’ll also highlight molecules, link to the cell cycle that are also implicated in cancer as well & so we’ll cover that again next week in more detail.  Cell duplicates and segregates its genome - Here is the eucaryotic cell cycle illustrated in a basic way. Here we start off with a single cell and then that cell divides. A key aspect of this is that the material in the cell has to duplicate itself. If the cell is going to divide, a lot of material has to be produced so the cell cycle is important to have cell growth which is illustrated in this simple cartoon. You can see the increased size of this cell, so that’s the increased growth of membranes, increased growth of the cytoskeleton, for example, mitochondria, ER membranes, Golgi membranes so there’s enough material to divide into 2 for the next round. - The duplication and the segregation of the genome is critical because this encodes all the instructions for everything that’s happening inside the cell so if there is a mistake made in the duplication of this genome, during the synthesis of the DNA, that could create mutations which would lead to problems in the cell. If during mitosis, certain chromosomes were lost, then of course that’s going to cause problems as well. So it’s critical for both cells that are dividing & developing organisms that they synthesize & segregate their DNA properly during division so that those cells maintain a proper cell physiology & if they don’t, then that’s often associated with the development of cancers in our bodies. - Also for the proper passage of DNA from one generation to the another to maintain the species, they need to maintain their DNA properly to be able to pass on from generation to generation & this all comes down to the cell cycle & the control of how the cell duplicates & segregates the genome properly. - Here is the cell cycle in more detail, broken into specific phases. So we can start right here – this is just after division has happened so this single cell has just been born from a division & the first phase is called G1 or gap phase 1 and it is a period of cell growth. Here is when the cell is partially doubling its proteins & organelles so that it builds up in size getting ready for a future division itself. So here it’s growing. - The next phase is called S phase which is dedicated to the synthesis of DNA so the replication of the genome. Here we have the replication of the DNA. - This is then followed by a second gap phase, G2, and this is another period of cell growth where the remaining proteins and organelles will duplicate themselves. - Then if everything is in order the cell will then enter M phase where both st the nucleus & the entire cell will divide so 1 you’ll have a nuclear division, then you’ll have a cell division to split all the material of the cell in 2. - So next week we’ll talk about mitosis in much more detail. Today we’re going to talk about the transition b/w these different phases. - You can see from the fact that it has 1 phase after the other, this needs to be temporally regulated & coordinated & there are many processes going on. - So a key thing for this is the cell must ask itself, it must check whether things are going okay during the process. Ex: Are there enough resources for DNA replication? Is this initial growth phase going effectively? Are there enough resources there for me, the cell, to replicate my DNA? Then for example, has the DNA actually replicated before mitosis starts? So before you start trying to split the DNA into two cells, the cell has to be sure it has actually replicated the DNA, that it has 2 copies of the DNA to separate into the 2 cells – if it doesn’t, then one of the cells is going to be missing huge sections of the genome. So these questions are asked at specific phases of the cell cycle. - So if we start again at this growth phase, before the cell goes into the S phase, the cell will ask itself is the environment favourable? Are there enough resources present? Here is a key checkpoint right here, the transition into the synthesis phase of the DNA so if there aren’t enough resources present at that time, the cell will just stay in G1 phase, it will wait until resources are available & then it will replicate its DNA when the conditions are better. - The other key point where there is a checkpoint is here for the entry of mitosis. Here it will ask is all the DNA replicated? Are there 2 copies of the genome to divide b/w the cells & it can also check again if the environment is still favourable. Here is a growth phase right here, the cell can monitor whether it’s growing properly, are there enough resources there? If there aren’t then it can halt again at this stage & wait until the environment is more favourable & then divide. - Then there are other checkpoints during mitosis & we’ll touch on those again next week.  Protein kinases - So the core molecules here are Cdks which are protein kinases & Cdks stand for cyclin dependent kinase. These are protein kinases & they have targets that control the cell cycle so they phosphorylate proteins that will control different stages of the cell cycle. - One key step is that another protein called a cyclin has to bind to these kinases to turn them on, so this is one way to turn on these molecules to pass through the checkpoint. - Diagram; We’ve got the cyclin present here, here is the Cdk – you need both of these components, and more as we’ll see in a moment, to activate the checkpoint.  At different stages of the cell cycle - So to coordinate things in time during this cycle, there are different cyclin- Cdk checkpoints at different stages of the cell cycle. - So if we started off here at G1 right here, the cell is growing, things are favourable, there are lots of resources available, if that’s the case the cell will then signal to produce this S cyclin, the cyclin that will promote the synthesis phase. This will then bind to the S-Cdk & this complex can then phosphorylate machinery involved with DNA replication in the cell so the cell can dedicate all of its machinery to replicating the DNA. - Once that job is done, the cell wants to turn off that machinery since the genome has been replicated, they don’t need all of those enzymes present anymore to duplicate the DNA so now this cyclin is destroyed. So we have a very specific cyclin-Cdk complex to promote a specific phase of the cell cycle & then it’s destroyed after its job is done. - Now once the cell then passes through S phase, it passes the G2 phase, it grows again & then it monitors are there is enough resources still and has the genome been fully replicated? So if both of those things are okay, then it will produce a different cyclin called an M-cyclin which will interact with the Cdk, producing a different cyclin-Cdk complex & this complex promotes the machinery controlling mitosis which we’ll talk about in detail next week. - Once that is done, so this would be machinery like building the mitotic spindles out of microtubules, once that’s job is done & the DNA has been segregated into 2 cells, you don’t need that machinery anymore, so this cyclin is destroyed, the activity of this cyclin-Cdk complex is turned off so then this mitosis machinery is turned off. - So this is a molecular cycle then where the cell can control whether it’s activating DNA replication machinery or mitosis machinery depending on which cyclins are available & these are monitoring the availability of resources & whether the DNA has been duplicated & so on – these are the checkpoints. - Here in this table it shows these different cyclin-Cdk complexes. Here we can see the different phases of the cell cycle – the G1, the G1 to S, going into S & here is the mitosis phase so it is focused around the synthesis phase and the mitosis phase, the control of that DNA replication & segregation into 2 cells. - Now in vertebrates here, we can see the major cyclins that are associated with these checkpoints, so cyclin D, E, A & B & here is the cyclin- dependent kinase partner so we have all these different combinations, one that is specific for each of these checkpoints. - Notice that there is also combinatorial control here & that look at this one right here, the same Cdk partner at both steps here but when it’s bound to cyclin E it has one behaviour, whereas when it’s bound A, it has a different behaviour, it regulates a different checkpoint. Now this combinatorial effect is shown even more so in yeast so here is budding yeast & here are the different cyclins that are expressed or that build up the different checkpoints during the cell cycle, they are all binding to the exact same Cdk partner, so it’s the same Cdk, the same cyclin dependent kinase, all through the cell cycle in yeast & it’s just whether it’s in a combination with a different cyclin that controls its downstream activity, whether it controls the transition into S phase or the transition into M phase so there’s this key combinatorial control bringing cyclin together with the Cdk. - So this activation of the Cdk, this cyclin dependent kinase involves cyclin as we’ve talking about but it also involves a phosphorylation step as well which is highlighted here in this slide. - Here we’ve got our Cdk, it is completely inactive, there’s no cyclin there at all. Then this Cdk, once the cyclin is expressed at a specific stage in the cell cycle, it will bind to the Cdk and now this Cdk will become partly active but this cartoon highlights that there is a loop, a polypeptide loop, within these Cdk that right at this stage here, this is blocking substrates from going to the active site of this kinase so this kinase is about to phosphorylate, for example, the machinery that is going to activate DNA replication, but at this state right here all of those substrates that are in that DNA replication machinery they can’t get into the active site of this kinase b/c of this T loop. So to get this T loop out of the way, there is a Cdk activating kinase that has to act, so this phosphorylates this T loop, opens up the active site so now this kinase can phosphorylate its downstream targets. - So the full activation of the Cdk requires both cyclin binding & then also this activating phosphorylation, the addition of this activating phosphate to this T-loop by Cdk activating kinase. So once this is activated then, now it can start to engage with the machinery that actually controls the structure of the cell so that it can either drive DNA synthesis or drive mitosis. So the Cdks regulate the machinery that directly replicate the cell.  Promoting DNA replication  To start mitosis - So here we have our Cdk over here, if this is a cyclin Cdk complex that is driving the transition into S phase, to synthesize the DNA, this kinase, its phosphorylation inhibits an inhibitor of the origin recognition complex. So if this complex is no longer inhibited then DNA replication can occur so it’s directly engaging with the machinery that is replicating the DNA & it’s turning that machinery on. - Here is this is a cyclin-Cdk complex that’s promoting transition into M phase or mitosis, this cyclin will phosphorylate components associated with mitosis so this will phosphorylate a lamin, the intermediate filament that’s associated with the nuclear envelope & this will lead to breakdown of the nuclear envelope so the chromosomes can be separated to the 2 separate cells. They’ll also regulate & phosphorylate proteins that are required for the condensation of chromosomes into small packages so they can be separated into separate cells & they also phosphorylate microtubule regulators & this will create this very specialized structure, the mitotic spindle, to separate the chromosomes & these aspects of mitosis we’ll talk about in much more detail next week. - The key thing here is this is what is downstream of these regulators. The actual machinery that is controlling what the cell is doing, right at the level of microtubules, at the level of DNA & so on. - So how are these guys regulated here? So what turns Cdks on and off? The one key thing is the availability of cyclins. Another thing is the presence or absence of proteins that can inhibit the Cdks & also there are phosphorylation events that can inhibit the Cdks. So let’s walk through these 3 examples. - We’ve seen that the production of a cyclin can promote the activation of a second cyclin Cdk complex and then the destruction of this will then turn that cyclin Cdk complex off. - So as we’ll see in a couple of examples later, the production of these cyclins is often controlled by transcription so the cyclin gene will be transcribed & translated when conditions in the cell are good for progressing through that stage of the cell cycle so that will produce that cyclin here, activate the complex & the destruction mechanism typical of the cyclins is illustrated in the next slide.  The completion of mitosis - So this is an example right here about the destruction of a cycle by the anaphase promoting complex & so the APC targets M-cyclin, so the cyclin of mitosis & this allows the completion of mitosis. So anaphase is a later phase of mitosis, when the cell recognizes that it’s progressed through mitosis, it will activate this complex to start completing the mitosis process. - Now the regulation here involves regulated degradation of the M cyclin & this is through, here we can start off with the inactive APC, here’s our cyclin Cdk complex right here so this one needs to work on this one. - The 1 step is the activation of the APC, then once the APC is activated, it will recruit ubiquitination enzymes to the complex & add the small molecule ubiquitin onto the cyclin molecule so you’ll have this chain of ubiquitin on this cyclin & that chain then is a recognition site for the proteasome so proteins that are polyubiquitinated in this way are recognized by the proteasome, they are targeted to this structure for destruction so here we can see that the ubiquitination of these cyclins will target the cyclin for degradation at the proteasome. - So this is the process that occurs up here at mitosis & a similar process will occur during the S phases as well with the regulated degradation of these cyclins. - Now more control is needed on top of this. - Here is one way – this is by the cyclin, cdk inhibitor proteins, so in this case, we have our active cyclin cdk complex, here it has its activating phosphate group on it so it is all ready to go and activate downstream targets. But if this protein p27 is present, it will bind to this complex and hold it in an inactive state. So now there are two things that have to happen to progress through the cell cycle when p27 is active. You have to receive a signal to activate the complex and the cell also has to receive a signal to inactivate p27. - The cell cycle is critical that it is done properly and so the cell builds in these multiple layers of regulation. Two things must be done for passage through the cell cycle. - Another example of that is regulation by phosphorylation. - Here we have our active cyclin cdk complex but this complex, even though it has the activating phosphate on the bottom site, another kinase called Wee1 can phosphorylate it at another site and inactivate it. Now we have an inactivating phosphate. Here even though this one is all ready to go, if it gets phosphorylated by Wee1, it cannot activate downstream targets and can’t promote the cell cycle. - The cell needs to express or activate the protein Cdc25 which is a phosphatase which specifically removes this inhibitory phosp
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