Cell Cycle
What are the functions of M-Cyclin?
-Allows cytokinesis to occur -Moves chromosomes to the poles so they can divide. -Establish the cytoskeletal network
The length of the different phases of the cell cycle depends on multiple factors including:
-Availability of cyclins -Chemical signals (hormones) -Death of nearby cells. -Damage. -DNA replication error.
What are the three stages of cell division?
1. Cell growth and chromosome replication 2. Chromosome segregation 3. Cell division
Name 3-4 proteins that are important for the cell cycle
1. Cyclin 2. Cyclin-Dependent Kinases
How is the Cyclin-Cdk complex activated?
1. Cyclin comes into close contact with an inactive CDK with a T-loop and ATP on it 2. Cyclin binds to CDK, moving the T-loop out of the way, partially activating it. 3. Cdk activating kinase (CAK) uses ATP to phosphorylate the T-loop, moving it out of the way, allowing the Cyclin-Cdk complex to become an active kinase
G0 Phase
A nondividing state occupied by cells that have left the cell cycle, sometimes reversibly.
Inducible Pluripotent Stem Cells (iPSCs)
Cells derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of human cell needed for therapeutic purposes.
Senescence
Cells that aren't entering the cell cycle and are stuck in the G1 phase due to the process of aging.
What would happen if Wee1 was deregulated or inhibited?
Cells would be stuck in the G2 phase and wouldn't be able to move on to the M phase. Very rapid division and small cells.
What would happen if Cdk inhibitors were deregulated or inhibited?
Cells would continuously divide, potentially becoming cancerous.
What would happen if Cdc25 was deregulated or inhibited?
Cells would stay in the M-phase.
What goes on in S phase?
Chromosome duplication goes on within the nucleus and cytoplasm.
How would the cells divide if ATP wasn't present?
Glycolysis and oxidation phosphorylation
Why was the frog egg introduced in biochemical studies regarding the cell cycle?
It was hard to do biochemistry in yeast as they're single-cell organisms. With the frog eggs, it was a multicell organism and you had enough product to run biochemical tests.
What goes on in M phase?
Mitosis and Cytokinesis
M-Cyclin
Mitotic regulatory factors. A regulatory protein that binds to mitotic Cdk to form M-Cdk, the protein complex that triggers the M phase of the cell cycle.
How does CAK regulate Cdk?
Moves the T-loop out of the way.
What are the two main phases of cell division?
S phase and M phase
Mitosis
Part of eukaryotic cell division during which the cell nucleus divides
When looking at embryonic or cancer cells in flow cytometry, most cells are either in the ___ or ___ phases.
S; G2
S-Cdk
Protein complex whose activity initiates DNA replication; consists of an S cyclin plus a cyclin-dependent protein kinase (Cdk).
What happens to the cell cycle at permissive (low) temperatures?
Proteins are active, meaning that they can participate in their specific roles of the cell cycle and the cells can divide and grow.
What happens to the cell cycle at restrictive (high) temperatures?
Proteins are inactive. This is because proteins contain multiple bonds (i.e. salt linkages, sulfur bridges, H-bonds) and at a higher temperature they unfold and become inactive. Thus, the cells stay in the G1 phase.
Ctd1
Provides instructions for making a protein that is important in the copying of a cell's DNA before the cell divides
What is the role of cytoskeletal structures?
Pull chromosomes to different parts of the cell.
Cell Cycle Dependent Kinases (Cdk)
Regulatory proteins that are linked to different phases of the cell cycle by binding to cyclin.
Telomeres
Repeated DNA sequences at the ends of eukaryotic chromosomes.
Ubiquination
Targets a protein for degradation by a proteasome.
___ determine how many times a cell can divide.
Telomeres
During the G1 phase, ___.
cells increase in size and synthesize new proteins and organelles getting ready to go into the S phase
During the G2 phase, ___.
cells prepare to undergo cytokinesis.
p27 is an ___.
cyclin-dependent kinase inhibitor.
Scientists mutated ___ in budding yeast to make the bud larger than the original cell. The purpose of this was to ___.
cytokinesis (CBC15); where in the cell cycle things were
The cell is deciding whether or not to ___.
enter the cell cycle
Almost all genes that have been identified in yeast ___.
have mammalian counterparts
During G1 and G2 Phase ___.
the cell grows and other structures are duplicated as they are regulatory checkpoints
Explain the steps in DNA Replication
1. Helicase unzips DNA stands. 2. The unbound strands- leading and lagging- are used as templates. 3. DNA polymerase adds matching nucleotides to the leading strand. Before it can do that, RNA prime lays down new strands at the beginning. 4. Since the lagging strand runs in the opposite direction, DNA polymerase cannot synthesize these strands. RNA primes lays down matching nucleotides giving DNA polymerase a starting point (Okazaki fragments) 5. DNA polymerase works backwards along the strand. 6. Another kind of DNA polymerase has to replace all the RNA primers. 7. DNA ligase joins those final strands together.
Explains what happens during S phase in DNA Replication
1. PRC is fully formed 2. S-Cdk Triggers S phase 3. Degradation of phosphorylated Cdc6 4. Inhibition of Ctd1 by geminin 5. Preinitiation complex comes in and binds to the origin of replication (ORC) 6. The ORC gets phosphorylated
How is Flow Cytometry performed?
1. Take cells and attach a protein marker to it (fluorescent). 2. Put cells through a cell sorting tube and make them go in one by one. 3. Shine a laser to the cells. 4. Light goes through the detector based on the DNA/RNA and the marker. 5. The more DNA, the higher the fluorescence and the more specific phase that cell is in.
Explains what happens during the G1 Phase in DNA Replication
1. The origin recognition complex (ORC) binds to the DNA. 2. Cdc6 and Ctd1 bind to the ORC. 3. Mcm (helicase) binds the prereplicative complex (pre-RC).
___ is an anaphase promoting complex which gets rid of the M-Cdk complex by summoning ubiquitin enzymes to degrade it.
APC/C
What is the primary driver of the cell cycle?
ATP, mainly the phosphorylation of different proteins using kinases.
How does Cdc25 regulate Cdk?
Activates the M-phase of the cell cycle.
Which types of cells really aren't dividing?
Adult cells
Telomerase
An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells.
Helicase
An enzyme that untwists the double helix of DNA at the replication forks.
Why was Wee1 named the way it was?
Because the cells replicated very quickly to the point were there were small because they were moving through the cell cycle so quickly they weren't able to grow properly.
Why is looking at cell culture from cancer cells important in regards to the cell cycle?
Because we can evaluate specifically where in the cell cycle the cells are and use drugs to arrest and treat those cells from becoming metastatic.
Cdc6
Binds to the ORC and helps helicase bind
How does cyclin drive the cell cycle?
By partnering with a family of enzymes called the cyclin-dependent kinases (Cdk's). A lone Cdk is inactive, but the binding of a cyclin activates it, making it a functional enzyme and allowing it to modify target proteins by providing kinase activity.
How does Cdk inhibitor regulate Cdk?
Can be bound to the complex to prevent entry into the cell.
What is the role of cell division in the adult?
Cell division in an adult takes place only when signals indicate the need to grow or to replace cells that have been lost, damaged, or worn out.
Explain what happens in the G2/M Phases of DNA Replication
Complete DNA Replication
___ are really specific for different stages of the cell cycle.
Cyclins
Cytokinesis
Division of the cytoplasm during cell division
Budding Yeast
Following mitosis, a yeast cell divides asymmetrically to produce two new cells, one large and one small.
Fission Yeast
Following mitosis, a yeast cell divides evenly to produce two new cells.
Flow Cytometry
Follows cells in the cell cycle in real-time by looking at the number of cells expressing fluorescent markers.
Why is the APC/C complex needed?
If the Cyclin-Cdk complex is there, it will bind, therefore, it would need to be degraded before that can happen.
What would be an advantage of p27?
If you got an organ or a tissue that is already big enough, you don't need to grow it even further. Therefore, p27 allows it to minimize and even stop cell division.
What is the difference between the G0 phase and senescence?
In the G0 phase, cells can re-enter the cell cycle, while in senescence, cells cannot re-enter the cell cycle.
Geminin
Inhibits Ctd1
How does Wee1 regulate Cdk?
Inhibits the M-phase of the cell cycle.
What is the role of p27?
It binds to the Cyclin-Cdk complex and inhibits it by blocking its entry into the cell cycle.
How does temperature control the cell cycle?
It controls the rate and speed of the cell cycle.
Explain the role of Wee1
It puts another phosphate on the Cyclin-Cdk complex to inactivate it. This inhibits the S phase.
Explain the role of Cdc25 Phosphatase
It takes one phosphate off of the Cyclin-Cdk complex, reactivating the kinase, causing the protein to arrest the cell in S phase. This causes chromosomes not to be replicated.
What happens when the DNA is not completely copied correctly?
Mutations, which can then lead to cancer.
How does cyclin regulate Cdk?
No cyclin, no activity.
Cyclin
One of a family of closely related proteins that regulate the cell cycle in eukaryotic cells
What is the purpose of the G2/M Phase checkpoints?
Some cells like yeast are dependent on nutrients that are available, while mammalian cells are dependent on growth factors. Therefore, the G2 checkpoint determines whether the DNA has been replicated completely, and the M phase checkpoint determines whether the nutrients and other factors are there to complete replication.
___ are cancer targets.
Stem cells
Pluripotent Stem Cells
Stem cells that can become almost all types of tissues and cells in the body.
What would happen if CAK was deregulated or mutated?
The T-loop on the Cyclin-Cdk complex would stay there, meaning that the complex would stay in its partially active state. There could be two possible outcomes. One would be that some of the cells would move onto the next phase of the cell cycle slowly, meaning it'll take a longer time to reach specific checkpoints. Or, the cells wouldn't move onto the next phase and die.
What is the purpose of cell division?
The generation of new cells for growth and repair.
What does flow cytometry measure?
The physical and chemical characteristics of a cell.
What would happen if cyclin was deregulated or mutated?
There wouldn't be any activity in the cell cycle. Cells would be stuck in one specific phase and eventually die because they didn't have the factor that was needed to proceed into the next cell cycle phase.
Why do cancer cells depend entirely on glycolysis?
They don't have an oxygen source.
What is happening to telomeres every time a cell divides?
They get shorter, this is called aging.
How was the cell cycle discovered?
Through fission and budding yeast.
Temperature-Dependent Mutants
Variants of genes that allow normal function of the organism at low temperatures, but altered function at higher temperatures.
What would be the consequence if Wee1 was mutated?
We would lose regulation into mitosis and the cell is replicated very quickly.
Why is ATP added to the test tube during the generation of frog eggs for biochemical studies?
When you take the nuclei from a frog sperm, mix it with the cytoplasm from a frog egg, and add ATP, the mixture will undergo a cell-free mitotic cycle for 40-60 minutes and you'll end up with chromosomes and DNA. ATP is added because it is the energy source, where it'll transfer the phosphates from ATP to another protein via kinases for activity. This ultimately tells us that this process is energy consuming.
What is the advantage of using yeast to study the cell cycle?
You could tell where you were within the cell cycle.
What are some advantages of using frog eggs for biochemical studies BESIDES the amount of product?
You get quick division without much growth, meaning that you can isolate proteins and determine what makes the cell grow.
Both fission and budding yeast are used as a ___.
model system.
___ sets up a senescence situation such that a cell can't divide.
p27
