Three different fusions led to three basic ideas: 1) If one fuses a cell in mitosis with a cell in any other stage of the cell cycle, the chromosomes

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1 Section Notes The cell division cycle presents an interesting system to study because growth and division must be carefully coordinated. For many cells it is important that it reaches the correct size before divinding, otherwise cells can become too large or too small after repeated divisions. Also, it is important to ensure that chromosomes are fully replicated before segregation to prevent chromosome breakage or mis- segregation. And lastly, cells must maintain a certain growth and division rate or they can proliferate out of control as in cancer. These are just a few of the types of problems the cell cycle must solve. The basic cell cycle can be divided into distinct steps where one step must finish before the next begins. These transitions are coordianted and controlled. The figure below outlines the cell cycle steps as they are often divided. G1 is where growth after division occurs and the transition from G1 to S is one place where growth and divison can be regulated. S phase is where DNA replication occurs and then there is a G2 phase of more growth before mitosis is triggered. When trying to understand a process such as the cell cycle, it is important to decide what types of questions you are interested in answering. In lecture this was described as the level of abstraction. You could take a big picture approach where you try to understand the basic rules that govern a process and general requirements that must be met before steps can occur. Or you can zoom in to the molecular level and try to understand specifically which molecules trigger certain changes and how they are regulated. Each level of abstraction can help contribute to understanding a problem and it is not necessary to understand the zoomed in level before tackling the big picture. For the cell cycle, an early set of cell fusion experiments revealed some basic rules for the cell cycle. The experiment involved fusing cells at different stages of the cell cycle together and seeing what happens to the chromosomes.

2 Three different fusions led to three basic ideas: 1) If one fuses a cell in mitosis with a cell in any other stage of the cell cycle, the chromosomes from the non- mitosis cell replicate and enter mitosis. This indicates that mitosis dominates. 2) If ones fuses a G1 cell with a cell in S phase, the G1 nucleus also enters S phase. This indicates that there is some sort of signal present that can trigger DNA replication. 3) However, fusing an S phase cell with a G2 cell does not induce the G2 nucleus to synthesize its DNA again. This suggests that the cell has a mechanism for determining that the DNA has already been synthesized and there are ways to keep the cell from re- copying it s DNA. When addressing any scientific question, it is important to determine what type of system you want to study. Oftentimes it is helpful to study specialized cells because there aren t as many extraneous things to obstruct what you want to study. For example, eggs are specialized to divide rapidly and the cell cycle is simplified to S and M, or replication and division. Xenopus eggs are a particularly good system because they are large and fertilized externally, thus easy to access. Fully grown oocytes are arrested until they receive a signal (progesterone) that triggers the divisions associated with meiosis I and II. After meiosis II, the cells arrest again until fertilization after which the cells begin to divide without any growth in between the divisions, resulting in small cells after each round.

3 The cell fusion experiments suggested that there is a mitosis promoting factor (MPF) that can trigger mitosis in cells. There are pulses in the levels of MPF that correlate with each entry into mitosis and the reduction in MPF levels seems to regulate the exit from mitosis. Experiments also showed that MPF pulses do not actually depend on the DNA at all as the eggs still show pulses of MPF even after the removal of the nucleus. This suggests that the pulses are not dependent on new DNA transcription since they occur for many cycles even in the absence of the DNA. Even though the DNA is not needed for these pulses, it is known that protein synthesis is required for embryonic mitoses. This is possible in the absence if DNA because of maternal mrnas that are deposited in the egg. An experiment involving sea urchin eggs eventually revealed the peptide responsible for the oscillations. Because maternal proteins are also deposited in an egg, it can be difficult to distinguish proteins that are newly synthesized from those

4 that are maternal. In order to tell them apart, one must label (usually with radioactive methionine) new proteins. If you add 35 S- methionine to the eggs after they have been isolated and fertilized, any newly synthesized proteins will contain 35 S, which can be detected later. Then at different times points (to see what proteins are present at different points in the cell cycle), you remove samples and disrupt the egg membranes with SDS. This releases the proteins which you can then run on an SDS- PAGE gel which separates them purely based on molecular weight. The gel will show you all of the proteins present in the egg, both newly synthesized and maternal, so you then expose it to film which can detect only the radioactive proteins. This experiment revealed a protein that was synthesized and then degraded as the cells progressed through the cycle. Called cyclin, the levels of this protein peak at mitosis and subsequent degradation triggers the transition to interphase. At this point, the hypothesis was that cyclin synthesis drives cells into mitosis and its destruction pushes them out. An experiment with frog egg cytoplasm shows that cyclin is indeed the protein responsible for this transition and that it is the only factor required. This entire cycle of mitosis and interphase can be replicated in a test tube with cytoplasm removed from frog eggs. After removal, you treat the cytoplasm with RNase which destroys any existing mrnas present. Since there is also no DNA present, there is no way for new protein synthesis. You then block the RNase and add in fresh cyclin mrna. Since it is the mrna present, you know that any protein synthesized will be cyclin. You can then watch and see if the cytoplasm generates cycles of interphase and mitosis, which it does. This shows that cyclin is sufficient to trigger this cycle.

5 Once there was a basic understanding of the cell cycle at a high level of abstraction, scientists began to zoom in and look at the molecular level. They wanted to understand the genes responsible for cell cycle control and how they relate to each other. To do this, one uses genetics through the isolation of mutants that exhibit a phenotype of interest. Again, it is important to choose the organism you which to study carefully. In this case, scientists chose yeast because they have well established genetic tools, can replicate sexually or asexually, and also replicate quickly. It can be difficult to study essential genes because, by definition, the cells are dead/non- functional if you remove their function through mutation. One way to tackle this problem is to use temperature sensitive mutants, which means that the cells containing the mutation in the essential gene can grow at one temperature (permissive temp), but will express the phenotype if you switch them to a different temperature. Often the permissive temperature is cooler (such as 20 C) and the restrictive temperature is 37 C though this does not necessarily have to be the case. You find these conditional mutants through a process known as replica plating. You mutagenize the cells (induce mutations with UV radiation or a chemical) and then plate them at 20 C where all of the cells should grow even if they have a temperature sensitive mutation. You then transfer some cells from each colony using velvet to a new plate, maintaining spatial orientation. This just allows you to know which colony corresponds to which on the two plates. You then grow the new plate at the restrictive temperature of 37 C. Most of the colonies from the 20 C plate will also grow at 37 C because they are not temperature sensitive. Any colonies that contain temperature sensitive mutations though will not be present on the 37 C plate. Since these are the mutants you are interested in, you save them from the 20 C plate and discard the rest. Most of the temperature sensitive mutants isolated will have mutations in genes unrelated to the cell cycle, so how do you find the ones you are interested in? You

6 can take populations of cells, both wild type and mutant and when you grow them at the permissive temperature and look at them under the microscope, they will be at all different stages of the cell cycle. When you move the wild type cells to the higher temperature, they are unaffected and will still contain cells at all different stages of the cell cycle. The same thing will occur for any mutants that have mutations in genes unrelated to the cell cycle. However, cell cycle mutants will arrest at a certain stage of the cell cycle when shifted to the restrictive temperature. Each mutant may arrest at a different stage depending on what the mutated gene regulates, but you can identify interesting genes by finding the mutants that have cell cycle arrest at the higher temperature. In this case, there were 148 cdc mutants identified and they affected a total of 32 different genes. The number of genes is lower because you can have different mutations in the same gene. By making double mutants (through mating) and analyzing the results, you can reveal a logic map of the cell cycle. Certain processes must finish before the next step can begin (finish replication and spindle body duplication before mitosis) and some parts don t depend on others (mitosis and DNA replication occur independent of budding or cytokinesis). Haploid yeast can remain haploid and reproduce indefinitely, but they also can mate with yeast of the opposite mating type (a and α) whereby the two cells fuse their nuclei creating a diploid. If you mate cells with two different temperature sensitive mutations, you have a diploid with both mutations. In the diploid stage, you can determine if mutations are recessive or dominant because the cell has one copy of the wild type gene and one with the mutation. Through starvation, you can trigger the diploid cells to sporulate. The products of sporulation are four haploid cells and they are equivalent to the products of meiosis in animals (ex. sperm). There are several combinations of ways the mutations can be transferred to the haploid spores (think meiosis). In some instances, you can have a spore that has received both temperature sensitive mutations. Since it is haploid, both mutations will be expressed and you can investigate the interaction between the two genes.

7 Studying the cdc mutants revealed a logic map of how the different steps of the cell cycle interact and depend on each other (shown below). This map shows that budding, DNA replication, and spindle body duplication are all independent of each other. Meaning that if you have a mutation (ex: cdc24 ) that blocks bud formation, you can still get DNA replication. Mitosis though depends on the successful completion of both DNA synthesis and spindle body duplication, but not budding. So a mutation in cdc7 that blocks DNA replication would result in the cell failing to pass through a checkpoint (in this case at the G2 stage) and thus will not enter mitosis. Early in the cell cycle, there are multiple pathways a yeast cell can take: commit to replicating DNA and entering mitosis, mating or sporulating and arresting due to starvation. If a cell starts to replicate its DNA and enter another round of the cell cycle, it is committed to finishing that round of division. This point where the cell has choices is considered the start point of the cell cycle. One mutant discovered in the screen, cdc28, can block all early cell cycle events (no replication, budding or spindle body duplication).

8 Combining the results obtained from the earlier experiments with frog embryos and the genetic crosses in yeast gives a molecular picture of the feedback loop that generates the cyclin oscillation responsible for entry to and exit from mitosis (shown below). In this scenario, cyclin is continuously produced at a constant rate. Initially, after mitosis, cyclin levels are low and as cyclin is produced it is complexed with Cdk1 to form an active kinase (a protein that can phosphorylate other proteins, altering their activity). Because the activity of Wee1 is very high at this point, almost all of the Cyclin:Cdk1 complex is phosphorylated by Wee1 which inactivates it. As the cyclin levels rise, the amounts of Cyclin:Cdk1 (active) and Cyclin:Cdk1:P (inactive) also rise. Even though the ratio of inactive to active complex is very high, the overall levels of active complex are increasing due to the increasing levels of cyclin. The active Cyclin:Cdk1 complex can inactivate Wee1, halting the inactivation of the Cyclin:Cdk1 complex. It also activates Cdc25 which converts inactive complex into active complex. This creates a powerful positive feedback loop which very quickly shifts the balance towards active Cyclin:Cdk1 complex. This is what triggers mitosis and is associated with increasing cyclin levels. At the same time, the active complex activates the APC complex (through phosphorylation) and this complex, once active, can promote the degredation of cyclin through ubiquitination. Thus the more active complex you have, the more degredation of cyclin you get which then reduces the level of cyclin in the cell and pushes you out of mitosis. Because cyclin is constantly synthesized, the cycle starts again.

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