Exam 4 Lesson 36 Flashcards
How do we study synchronous cells?
We can find natural synchrony or induce it.
How do we study naturally synchronous cells?
We can look at them naturally in the early embryo stage, when it all is synchronous, like Xenopus.
How do we induce synchrony to study cells?
We can a. add inhibitors and cause cell arrest, b. Remove mitogens, or c. limit nutrients
What could we inhibit to make cells synchronous?
DNA synthesis, microtubules, etc.
In what type of cells would we remove mitogens? What do mitogens do?
In animal cells. They promote growth.
In what type of cell could we limit nutrients to induce synchrony?
Yeast
How do we study asynchronous cells?
We can study them a. in situ or b. isolated to analyze at specific phases
How do we study asynchronous cells in situ?
Use BrdU
How do we isolate asynchronous cells to analyze them at specific phases?
We can isolate them on the basis of a. cell size/shape, b. add fluorescent DNA dye, c. express fluorescent proteins that are cell-cycle regulated (which tells you which phase cell is in)
What tools can we use to isolate specific cells?
Flow cytometry or fluorescent activated cell sorting (FACS)
What purpose does flow cytometry serve?
Analysis only. Analyzes cell size and properties and takes quantitative data.
What purpose does FACS serve?
Analysis and collection
How does one study cells in tissue?
Break down cell wall (if plant) with enzymes (in humans, collagenase) FACS+RNA-seq+ proteomics
In what cell cycle phase will one see the largest number of cells with the least DNA percentage?
G1
What regulates cell cycle in yeast (3)? What are they?
Cdc2ts – mitotic promoter and kinase, cdc25ts – mitotic promoter and phosphatase, wee1ts – mitotic inhibitor and kinase
In frog/clam embryos, what was done? Describe experiment.
Preincubate embryos. With 35S methionine (building block for protein), fertilized , SDS page, discovered cyclin, a protein
What is MPF?
Maturation promotion factor
What was discovered about MPF when the cytoplasm liquid and mitotic extract was centrifuged?
MPF is made up of two proteins. Cyclin and kinase. Kinase is cdc2, the same protein that was encoded in yeast.
What kind of kinase is cdc2?
It is a cdk, or cyclin dependent kinase.
What is the role cyclin dependent kinases?
They are at the heart of cell cycle regulation. All eukaryotic cells have cdks.
How is cell cycle controlled?
Via cyclin dependent kinases
Are cyclin dependent kinases always present?
Yes, but not always active
How are cyclin dependent kinases controlled/activated?
By cyclin binding, phosphorylation status
How do we change phosphorylation status of cdk?
a. cyclin activating kinase activates cdks before or after binding cyclin by phosphorylating T-loop. B. phosphatases – like cdc25 for m-cdk, remove inhibitory phosphates.
What proteins do cdks phosphorylate?
ATP substrate
Let’s look at the major cyclins. What happens to cyclins during each cell cycle?
They undergo synthesis and degradation.
What are the four classes of cyclins?
G1/S-cyclin, S-cyclin, M-cyclin, G1-cyclin
G1/S-cyclins
Activates Cdks in late G1 and thereby help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase.
S-cyclins
Bind Cdks soon after progression through Start and help stimulate chromosome duplications. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of the early events.
M-cyclins
Activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin levels fall in mid-mitosis.
G1 cyclins
In most cells, help govern the activities of the G1/S-cyclins, which control progression through Start in late G1.
What are the major cyclin-Cdk complexes of vertebrates and budding yeast?
G1-Cdk, G1/S-cdk, S-cdk, M-cdk
Cyclin and Cdk partner in vertebrate G1-cdk
Cyclin D, cdk4 and cdk6
Cyclin and cdk partner in vertebrate g1/S-cdk
Cyclin E and Cdk2
Cyclin and cdk partner in vertebrate s-cdk
Cyclin A and cdk2, cdk1
Cyclin and cdk partner in vertebrate m-cdk
Cyclin B and cdk1
What are the three D cyclins in mammals?
D1, D2, and D3
Cyclin and Cdk partner in b.yeast G1-cdk
Cln3, cdk1
Cyclin and cdk partner in b.yeast g1/S-cdk
Cln1,2 and cdk1
Cyclin and cdk partner in b.yeast s-cdk
Clb5, 6 and cdk1
Cyclin and cdk partner in b.yeast m-cdk
Clb1, 2, 3, 4 and cdk1
Do concentrations of cdks change during cell cycle?
No
If we want to find cells in early G1, what protein would we look for?
Vertebrates – cyclin D, yeast – cln3
If we want to find cells in late G1, what protein do we look for?
Vertebrates – cyclin E, yeast – cln 1,2
If we want to find cells in S, what protein do we look for?
Vertebrates – cyclin A, yeast, clb5,6
If we want to find cells in M, what protein do we look for?
Vertebrates – cyclin B, yeast, clb 1,2,3,4
Let’s talk about the activation of a cdk with cyclin. What does cdk have that inhibits it?
Inhibitor phosphate
Suppose you add cyclin to cdk with inhibitor phosphate. What happens?
Cyclin binds, but phosphate is still on, so it is inactive.
Suppose you add CAK to cdk with cyclin bound. What happens?
CAK will phosphorylate mitogen promoter and dephosphorylate mitogen inhibitor. The cdk exchanges one phosphate for another and is active.
How does one turn off cdk once it has been activated?
Add polyubiquitin to cyclin via ubiquitin ligase and send cyclin to lysosome. Cyclin leaves cdk, which then is turned off.
We are looking at a graph with the following legend. X is relative amount of DNA per cell and y is number of cells. The dot is high on Y axis but not far on x axis. At what point of cell cycle are we?
We have a lot of cells but not a lot of relative amount of DNA per cell. Cells have just divided, so there is a lot of them, and DNA is split evenly among them, so there is little DNA relative to cell. We are at G1.
How much DNA is in each cell per G2 or M phase?
Twice as much as in G1 because cells have replicated but not divided.
The cells that have an intermediate amount of DNA are in what phase?
S phase
When a cdk is inactive, what blocks the active site?
The t-loop
How can we get t-loop to stop blocking cdk active site?
Bind cyclin to it. It moves the t-loop and partially activates cdk.
How does t-loop react when CAK binds?
Its conformation changes so enzyme can better bind to its protein substrate.
How do Wee1 kinase and cdc25 phosphatase work together to activate and inactivate cdk?
Active cyclin-cdk complex turned off when wee1 phosphorylates two closely spaced sites above the active site. Removal of these phosphates by phosphatase cdc25 activates the cyclin-cdk complex.
How does APC/C control proteolysis?
In mitosis, APC/C is activated by association with cdc20, which recognizes specific amino acid sequences on M-cyclin and other target proteins. With E1 and E2 proteins, APC/C assembles polybiquitin chains on the target protein. The polyubiquitylated target is then recognized and degraded by proteasome.
How does SCF control proteolysis?
The activity of the uniquitin ligase SCF depends on substrate-binding subunits called F-box proteins, of which there are many different types. The phosphorylation of a target protein allows the target to be recognized by a specific F-box subunit.
