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Cell Biology > Exam 4 Lesson 36 > Flashcards

Exam 4 Lesson 36 Flashcards

1
Q

How do we study synchronous cells?

A

We can find natural synchrony or induce it.

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2
Q

How do we study naturally synchronous cells?

A

We can look at them naturally in the early embryo stage, when it all is synchronous, like Xenopus.

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3
Q

How do we induce synchrony to study cells?

A

We can a. add inhibitors and cause cell arrest, b. Remove mitogens, or c. limit nutrients

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4
Q

What could we inhibit to make cells synchronous?

A

DNA synthesis, microtubules, etc.

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5
Q

In what type of cells would we remove mitogens? What do mitogens do?

A

In animal cells. They promote growth.

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6
Q

In what type of cell could we limit nutrients to induce synchrony?

A

Yeast

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7
Q

How do we study asynchronous cells?

A

We can study them a. in situ or b. isolated to analyze at specific phases

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8
Q

How do we study asynchronous cells in situ?

A

Use BrdU

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9
Q

How do we isolate asynchronous cells to analyze them at specific phases?

A

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)

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10
Q

What tools can we use to isolate specific cells?

A

Flow cytometry or fluorescent activated cell sorting (FACS)

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11
Q

What purpose does flow cytometry serve?

A

Analysis only. Analyzes cell size and properties and takes quantitative data.

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12
Q

What purpose does FACS serve?

A

Analysis and collection

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13
Q

How does one study cells in tissue?

A

Break down cell wall (if plant) with enzymes (in humans, collagenase) FACS+RNA-seq+ proteomics

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14
Q

In what cell cycle phase will one see the largest number of cells with the least DNA percentage?

A

G1

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15
Q

What regulates cell cycle in yeast (3)? What are they?

A

Cdc2ts – mitotic promoter and kinase, cdc25ts – mitotic promoter and phosphatase, wee1ts – mitotic inhibitor and kinase

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16
Q

In frog/clam embryos, what was done? Describe experiment.

A

Preincubate embryos. With 35S methionine (building block for protein), fertilized , SDS page, discovered cyclin, a protein

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17
Q

What is MPF?

A

Maturation promotion factor

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18
Q

What was discovered about MPF when the cytoplasm liquid and mitotic extract was centrifuged?

A

MPF is made up of two proteins. Cyclin and kinase. Kinase is cdc2, the same protein that was encoded in yeast.

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19
Q

What kind of kinase is cdc2?

A

It is a cdk, or cyclin dependent kinase.

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20
Q

What is the role cyclin dependent kinases?

A

They are at the heart of cell cycle regulation. All eukaryotic cells have cdks.

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21
Q

How is cell cycle controlled?

A

Via cyclin dependent kinases

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22
Q

Are cyclin dependent kinases always present?

A

Yes, but not always active

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23
Q

How are cyclin dependent kinases controlled/activated?

A

By cyclin binding, phosphorylation status

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24
Q

How do we change phosphorylation status of cdk?

A

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.

25
Q

What proteins do cdks phosphorylate?

A

ATP substrate

26
Q

Let’s look at the major cyclins. What happens to cyclins during each cell cycle?

A

They undergo synthesis and degradation.

27
Q

What are the four classes of cyclins?

A

G1/S-cyclin, S-cyclin, M-cyclin, G1-cyclin

28
Q

G1/S-cyclins

A

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.

29
Q

S-cyclins

A

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.

30
Q

M-cyclins

A

Activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin levels fall in mid-mitosis.

31
Q

G1 cyclins

A

In most cells, help govern the activities of the G1/S-cyclins, which control progression through Start in late G1.

32
Q

What are the major cyclin-Cdk complexes of vertebrates and budding yeast?

A

G1-Cdk, G1/S-cdk, S-cdk, M-cdk

33
Q

Cyclin and Cdk partner in vertebrate G1-cdk

A

Cyclin D, cdk4 and cdk6

34
Q

Cyclin and cdk partner in vertebrate g1/S-cdk

A

Cyclin E and Cdk2

35
Q

Cyclin and cdk partner in vertebrate s-cdk

A

Cyclin A and cdk2, cdk1

36
Q

Cyclin and cdk partner in vertebrate m-cdk

A

Cyclin B and cdk1

37
Q

What are the three D cyclins in mammals?

A

D1, D2, and D3

38
Q

Cyclin and Cdk partner in b.yeast G1-cdk

A

Cln3, cdk1

39
Q

Cyclin and cdk partner in b.yeast g1/S-cdk

A

Cln1,2 and cdk1

40
Q

Cyclin and cdk partner in b.yeast s-cdk

A

Clb5, 6 and cdk1

41
Q

Cyclin and cdk partner in b.yeast m-cdk

A

Clb1, 2, 3, 4 and cdk1

42
Q

Do concentrations of cdks change during cell cycle?

A

No

43
Q

If we want to find cells in early G1, what protein would we look for?

A

Vertebrates – cyclin D, yeast – cln3

44
Q

If we want to find cells in late G1, what protein do we look for?

A

Vertebrates – cyclin E, yeast – cln 1,2

45
Q

If we want to find cells in S, what protein do we look for?

A

Vertebrates – cyclin A, yeast, clb5,6

46
Q

If we want to find cells in M, what protein do we look for?

A

Vertebrates – cyclin B, yeast, clb 1,2,3,4

47
Q

Let’s talk about the activation of a cdk with cyclin. What does cdk have that inhibits it?

A

Inhibitor phosphate

48
Q

Suppose you add cyclin to cdk with inhibitor phosphate. What happens?

A

Cyclin binds, but phosphate is still on, so it is inactive.

49
Q

Suppose you add CAK to cdk with cyclin bound. What happens?

A

CAK will phosphorylate mitogen promoter and dephosphorylate mitogen inhibitor. The cdk exchanges one phosphate for another and is active.

50
Q

How does one turn off cdk once it has been activated?

A

Add polyubiquitin to cyclin via ubiquitin ligase and send cyclin to lysosome. Cyclin leaves cdk, which then is turned off.

51
Q

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?

A

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.

52
Q

How much DNA is in each cell per G2 or M phase?

A

Twice as much as in G1 because cells have replicated but not divided.

53
Q

The cells that have an intermediate amount of DNA are in what phase?

A

S phase

54
Q

When a cdk is inactive, what blocks the active site?

A

The t-loop

55
Q

How can we get t-loop to stop blocking cdk active site?

A

Bind cyclin to it. It moves the t-loop and partially activates cdk.

56
Q

How does t-loop react when CAK binds?

A

Its conformation changes so enzyme can better bind to its protein substrate.

57
Q

How do Wee1 kinase and cdc25 phosphatase work together to activate and inactivate cdk?

A

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.

58
Q

How does APC/C control proteolysis?

A

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.

59
Q

How does SCF control proteolysis?

A

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.

Cell Biology (35 decks)

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