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17 Flashcards

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1
Q

DNA replication is initiated by____________.

A

s-cdk

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2
Q

M-Cdk complex is activated by_______.

A

dephosphorylation by Cdc25 phosphatase.

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3
Q

CKIs such as p27 ____________.

A

inhibit the cyclin-Cdk complex

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4
Q

Cdk activity is stimulated by phosphorylation of the T-loop by ________.

A

cak

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5
Q

EdU incorporation marks cells in……….

A

s-phase

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6
Q

M-cyclin levels…

A

fall toward the end of M phase as a result of ubiquitylation and degradation.

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7
Q

Which technique can be used to separate cells rapidly based on their fluorescence signal corresponding to their DNA content?

A

flow cytometry

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8
Q

Cdk activity fluctuates during the cell cycle, partly because ________________.

A

cyclin levels change during the cycle

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9
Q

describe Cdc25 phosphatase

A

Cdc25 increases Cdk activity.

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10
Q

One half of a duplicated chromosome at the end of S phase is called

A

Sister chromatid

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11
Q

A key control system crucial for triggering cytokinesis is………….

A

Metaphase to anaphase transition

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12
Q

If cell cycles were repeated with only the S phase and M phases, what is likely to occur?

A

The cells produced would get smaller and smaller.

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13
Q

Which fluorescently-tagged protein can be used to monitor G2 phase in live cells?

A

Geminin

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14
Q

Which factor inactivates Cdks by adding phosphates on specific sites on Cdks?

A

Wee1

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15
Q

Activation of Greatwall kinase………..

A

inhibits PP2A-B55

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16
Q

Mutations in Ensa that prevents its binding to PP2A-B55 will………..

A

keep PP2A-B55 active in early mitosis.

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17
Q

Which mechanism serves as positive feedback to activate M-Cdks?

A

Inhibition of Wee1 by M-Cdk

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18
Q

is CAK-dependent phosphorylation of M-Cdk sufficient to fully activate M-Cdk activity even in the presence of Cdk phosphorylation by Wee1?

A

no

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19
Q

Cdt1 is activated by ____________.

A

APC/C induced destruction of geminin

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20
Q

During metaphase………………

A

chromosomes align at the spindle equator.

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21
Q

During metaphase………………

A

chromosomes align at the spindle equator.

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22
Q

Condensins ________________.

A

compact chromosomes when phosphorylated by M-Cdk

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23
Q

Cdc20–APC and Cdh1–APC complexes are similar because……………………

A

They both inhibit M-Cdk activity.

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24
Q

ORC is phosphorylated in which phase?

A

S phase

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25
Q

DNA helicase deposition on DNA at the replication origins occurs mainly in which phase of cell cycle?

A

g1

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26
Q

At the beginning of S phase in a cycling cell, which of these events are likely to occur?

A

Activation of geminin

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27
Q

In the metaphase to anaphase transition, activation of which of these factors is important?

A

Cdc20-APC/C complex

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28
Q

Which complex keeps M-cyclin levels low in G1 phase of cell cycle?

A

Cdh1–APC/C

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29
Q

S-cdk ensures that replication happens only once per cell cycle- how?

A

It phosphorylates the Cdc6 protein, marking it for destruction.

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30
Q

If a cell in G1 phase and another cell in G0 phase are induced to pass the restriction point by the addition of a signaling molecule, but the signal is then immediately removed, what is likely to occur?

A

Both cells will replicate their DNA.

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31
Q

Disassembly of nuclear envelope marks which phase of mitosis?

A

Prometaphase

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32
Q

APC is also active in ensuring exit from ___

APC is an ubiquitin ligase and promotes ___

APC is phosphorylated by the (mitotic) Cdk1-cyclinB and binds ___

A

APC is also active in ensuring exit from mitosis, APC is an ubiquitin ligase and promotes anaphase by ubiquitin mediated degradation of securin, APC is phosphorylated by the (mitotic) Cdk1-cyclinB and binds its activator Cdc20 to become active in metaphase.

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33
Q

Which protein plays a direct role in anaphase B?

A

Kinesin-5 on interpolar microtubules and dynein on astral microtubules

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34
Q

Which of motor protein is minus-end directed motors that cross link interpolar microtubules and pull the poles together?

A

Kinesin-14

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35
Q

Duplication of centrosomes occurs in ________.

A

s phase

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36
Q

Polar ejection force that pulls chromosomes to the spindle equator is mediated by ______.

A

Kinesin-4

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37
Q

The polar ejection force does which of the following?

A

Pushes the chromosome arms away from the spindle poles with the help of kinesin-4 and 10

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38
Q

The mitotic spindle helps

A

segregate the chromosomes to the two daughter cells.

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39
Q

Some cell types in animals do not have centrosomes and therefore they do not have….

A

Astral microtubules

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40
Q

The plus ends of these microtubules are attached to large protein structures at the centromeres and they are called…………..

A

Kinetochore microtubules

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41
Q

Proper bi-orientation of the sister-chromatid pair on the spindle generates a tension that is sensed by_____.

A

Aurora-B kinase

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42
Q

Degradation of securin is important for ________.

A

separation of sister chromatids

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43
Q

Does The length of a kinetochore microtubule not change much during metaphase because there is no addition or removal of tubulin subunits

A

no

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44
Q

Centrosomes are duplicated

A

once per cell cycle similar to DNA.

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45
Q

Contractile ring is made of ________.

A

actin and myosin filaments

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46
Q

Disassembly of the nuclear envelope ________________.

A

must occur for kinetochore microtubules to form in animal cells.

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47
Q

Kinetochores assemble onto

A

chromosomes during late prophase.

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48
Q

Which event happens before the nuclear envelope is re-assembled in M phase?

A

assembly of the contractile ring

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49
Q

describe drug targeting microtubules

A

Drugs such as colchicine that inhibit microtubule polymerization, inhibit mitosis, Drugs that stabilize microtubules such as Taxol inhibit mitosis, Drugs that stabilize microtubules such as Taxol do not promote mitosis by stabilizing the spindle.

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50
Q

What are mitogens?

A

They are extracellular signals that stimulate cell division.

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51
Q

DNA damage induced checkpoint response in G1 ________________.

A

involves the inhibition of cyclin-Cdk complexes by p21.

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52
Q

UV exposure can cause DNA damage. What would you expect to occur as a result of genomic damage?

Activation of the ___, Activation of the protein kinase ___, Inactivation of the protein phosphatase___, nonBinding of ___ to Mdm2

A

Activation of the protein kinase ATR, Activation of the protein kinase Chk1, Inactivation of the protein phosphatase Cdc25, nonBinding of p53 to Mdm2

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53
Q

Which event contributes to helping the cells progress from G1 to S phase?

Activation of ___, Destruction of CKIs that target ___, Phosphorylation of Rb by ___, ___, and ___

A

Activation of E2F gene expression, Destruction of CKIs that target S-Cdks, Phosphorylation of Rb by G1-Cdk, G1/S-Cdk, and S-Cdk

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54
Q

What is an immediate early gene whose expression is triggered by Ras activation to promote cell cycle entry?

A

Myc

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55
Q

ATM and ATR kinases phosphorylate Ser-15 residue in p53 which________.

A

Stabilizes p53

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56
Q

G1/S Cdk also phosphorylates and inactivates APC/C towards end of G1 phase, which directly helps in accumulation of ………………

A

S-Cyclin

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57
Q

G1/S Cdk also phosphorylates and inactivates APC/C towards end of G1 phase, which directly helps in accumulation of ………………

A

S-Cyclin

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58
Q

Retinoblastoma is caused by excessive proliferation of some cells in retina that is induced by ………………

A

high E2F activity

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59
Q

The Chk1-mediated checkpoint response to DNA damage can arrest cells in G2/M phase by inhibiting which of these factors?

A

Cdc25

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60
Q

What is the purpose of activating cell cycle checkpoint responses to DNA damage?

A

promote cell death if damage is excessive, halt cell cycle to facilitate DNA repair, halt cell cycle to suppress accumulation of mutations,

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61
Q

p53 expression is often lost in early stages of cancer development, and in the absence of p53, which factor serves to initiate key cell cycle checkpoint-response to DNA damage? (see lecture-8 ppt.)

A

Chk1

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62
Q

A regulator critical for enhancing cell growth through stimulation of protein synthesis rates is ………..

A

mTORC1

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63
Q

describe cell division

A
  1. cell growth and replication
  2. chromosome segregation
  3. cell division
  4. repeat
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64
Q

S-phase

A

chromosome duplication (DNA synthesis phase)

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65
Q

M phase

A

mitosis, cytokinesis phase

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66
Q

interphase

A

G1-S-G2

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67
Q

G phase stands for what?

A

Gap phase

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68
Q

stages of cell cycle

A

s-phase then m-phase

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69
Q

describe dna and chromosome relationship

A

its packed into a thread like structure called chromosomes. connected with linker DNA around eight histones (nucleosome). these bead and string like structures are coiled super packedly, producing condense, supercoiled chromatin fibers. these form even great loops, forming a chromosome

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70
Q

Duplication of Eukaryotic chromosome

A

during interphase - a chromosome splits twice - at both replication origins. this creates a replicated chromosome (now 2) that are connected at the centromere.
during mitosis - mitotic spindle pulls them apart at centromere, leading to cell division
back to interphase - duplicated chromosomes in sister daughter cells.

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71
Q

interphase includes

A

s phase - gene expression and chromosome duplication; and results from m phase, with a paternal and maternal chromosome

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72
Q

m phase includes

A

mitosis - chromosomes are pulled apart by mitotic spindle - cell division - results in interphase

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73
Q

explain duplication and segregation of chromosomes in numbers of humans

A

Human- 46 chromosomes (23 pairs- diploid) becomes 92 after DNA replication/S phase. The duplicated chromosomes stay attached to each other and together they are now called sister chromatids

During mitosis, one of the sister chromatid is pulled away from the other by the spindle in opposite directions. So, after cells split into two, we will have 46 chromosomes (23 pairs), go into each daughter cell.

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74
Q

stages of cell cycle in order

A

interphase, prophase, pro metaphase, metaphase, (metaphase-to-anaphase transition), anaphase, telophase/cytokinesis

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75
Q

list ALL parts of m phase

A

prophase, pro metaphase, metaphase, (metaphase-to-anaphase transition), anaphase, telophase/cytokinesis (meaning interphase is the only thing not apart of m phase)

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76
Q

list all parts of mitosis

A

prophase, pro metaphase, metaphase, (metaphase-to-anaphase transition), anaphase, first half of telophase

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77
Q

list all parts of cytokinesis

A

first half of anaphase, telophase

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78
Q

Four Phases of Cell Cycle and Resting Phase- G0

A

G0 (resting phase), G1, S phase (dna replication), g2, m phase (mitosis (nuclear division) and cytokinesis (cytoplasmic division)), back to G0

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79
Q

G0 to G1

A

Cells can stay in G0 or resting phase for years before resuming proliferation or till an organism dies (e.g. terminal differentiated cells). If extracellular signals favor division, cells in G0 or early G1 pass through a commitment phase near the end of G1 called Start (in yeast) or the Restriction Point ( in mammalian cells) where cells are committed to DNA synthesis.

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80
Q

terminal differentiation

A

Terminal differentiation – cell cycle arrest/no proliferation

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81
Q

Cell cycle progression can be studied in many ways

A

Flow Cytometry, microscopy to analyze cell shape or size to correlate with cell cycle phases.

82
Q

microscopy to analyze cell shape or size to correlate with cell cycle phases. ex?

A

example- cell size analysis in Yeast tell you based on the size of the bud what stage of cell cycle they are in.
Cell shape analysis- example- rounded cells indicate mitotic cells (or could be dying cells) in mammalian cell culture.

83
Q

Analysis of DNA content with a flow cytometer

A

1) sample (stained cells in suspension) goes in machine, 2) hydrodynamic focusing cells pass through in single file, 3) light source - flourescence emitted from stained cells detected, forward and side scattered light from all cells detected

84
Q

see graph on slide 15 17A

A

see graph on slide 15 17A

85
Q

Labeling S phase and mitotic cells

A

Microscopy or Flow Cytometry using and fluorescent labeled antibodies that recognize BrdU, an artificial thymidine analog.

Fluorescent antibodies that recognize phosphorylated Histone H3 to mark mitotic cells.

86
Q

Measuring cell cycle timing in live cells

A

geminin in s, g2, and m phase

cdt1 in g1 phase

87
Q

Cell-Cycle Control System

A

Cell Cycle Controls ensure that each phase is properly completed before the cells can cycle into the next phase

88
Q

Trigger anaphase, and proceede to cytokinesis

A

Are all chromosomes attached to the spindle

89
Q

Enter cell cycle and proceed to S phase

A

Is environment favorable

90
Q

Enter mitosis

A

Is all DNA replicated and is the environment favorable?

91
Q

What does not happen in interphase

A

The nuclear envelope breaks down.

92
Q

Progression Through Cell Cycle depends on Cyclin-Dependent Protein Kinases (CDKs)

A

cDK must fine to its regulatory proteins cyclin to become enzymatically active (partial activation).

the activation of the cyclin CDK complex also requires dephosphorylation at specific sites and phosphorylation event at another site

once active the CDK cyclin complex, phosphorylates and regulated substrates.

93
Q

Unlike CDKs, Cyclins concentrations

A

vary in a cyclical manner during the cell cycle.

94
Q

g1/s1 cyclin concentration

A

high during second half of g1

95
Q

s-cyclin concentration

A

High during second half of G1 phase all the way through first half of m-phase

96
Q

m cyclin concentration

A

high through G2 phase through first half of metaphase

97
Q

The G1/S Cdks work with G1/S Cyclins to

A

drive cells through G1 towards S phas

98
Q

the S-Cdks work with S cyclins to

A

help launch the S phase

99
Q

M phase cyclins activate

A

G2 to M transition

100
Q

G1 cyclins- control

A

transit through start in late G1

100
Q

G1 cyclins- control

A

transit through start in late G1

101
Q

The Structural basis of Cdk activation

A

Enzyme is inactive with no cyclin bound and the active site is blocked by a region of the protein called T Loop

binding of cycling causes the Tloop to move out of the active site, causing partial activation of the CDK2

phosphorylation of CDK2 by a CAK kinase in a Tloop residue changes shape of tloop and further activates the enzyme (fully active -depends on the loss of an inhibitory enzyme)

102
Q

Other phosphorylation and dephosphorylation events also affect Cyclin-CDK activity

A

Wee1 kinase opposes Cdc25 phosphatase

103
Q

wee1 kinase does what and what does cdc25 phosphatase do in relation to this

A

causes inactive cyclin-cdk… by placing an inhibitory phosphate

cdc25 phosphatase gets rid of it and allows disposal of inhibitory p and activates the other phosphate, causing active cyclin-cdk

104
Q

The activity of Cdk-cyclin can be blocked by

A

binding of a Cdk inhibitor such as p27 & p21

105
Q

Progression through the cell cycle requires a cyclin to bind to a Cdk because _________.

A

the binding of a cyclin to Cdk is required for Cdk enzymatic activity.

106
Q

Structure of PP2A

A

catalytic subunit (c), scaffold protein, and regulatory subunit B

107
Q

Structure of PP2A

A

catalytic subunit (c), scaffold protein, and regulatory subunit B

108
Q

Regulation of PP2A

A

PP2A is active and phosphorylates a CDK substrate (which goes off to mitosis) with the help of active MCDK, which then causes great wall kinase to be phosphorylated and active, which then activates Ensa, which is then phosphorylated and causes PP2A to be inactive

109
Q

Substrate specificity is highly dependent on which of these subunits of PP2A phosphatase?

A

regulatory subunit

110
Q

Positive feedback in the activation of M-Cdk. first and second route via inhibition of pp2a

A

cdk-M-cyclin is inactive when bound. Then active wee1 and active CAK put two phosphates on m-CDK… one being inhibitory and one is an activating one…

  1. (early mitosis) with the help of CDC 25, it takes away the inhibitory phosphate, which then allows active MCDK, which pushes positive feedback to inhibit active PP2A. this dephosphorylates cdc25, but with positive feed back from active m-cdk, is allows cdc25 to be phosphorylated and active.
  2. (anaphase) with the help of CDC 25, it takes away the inhibitory phosphate, which then allows active MCDK, which pushes positive feedback to inhibit active PP2A. this dephosphorylates Wee1, making it active, but with positive feed back from active m-cdk, is allows cdc25 to be dephosphorylated and inactive.
111
Q

M-Cdk is active in

A

early mitosis and inhibits PP2A

112
Q

In anaphase, M-Cdk is

A

inactive and PP2A becomes active

113
Q

Cdc25 activity is further stimulated by M-CDK phosphorylation, which

A

creates a positive feedback for M-CDK activity.

114
Q

Wee1 activity is inhibited by M-CDK phosphorylation, which creates a

A

positive feedback for M-CDK activity.

115
Q

C- Activation of M-CDK

A

inactivates PP2A-B55.

116
Q

The Anaphase-promoting Complex/Cyclosome (APC/C) Triggers the

A

Metaphase-to-Anaphase Transition.

117
Q

The Cell-Cycle Control System Triggers the

A

Major Events of the Cell Cycle

118
Q

Cyclin concentrations are regulated by transcription and protein degradation

A

Active cyclin CDK complex is ubiquitylated by APC and then is destructed by the proteasome

119
Q

Abrupt disruption of S and M phase cyclins depends on

A

the Anaphase Promoting Complex (APC), that ubiquitinates the Cyclins that are then directed to proteosomal degradation.

120
Q

Control of Cyclin proteolysis by Ubiquitin Ligase- APC/C

A

APC/C (Anaphase Protein Complex/Cyclosome) is activated in metaphase by association with Cdc20, which recognizes specific amino acid sequences on M-cyclin and other target proteins.

With the help of two additional proteins called E1 and E2, the APC/C assembles polyubiquitin chains on the target protein. The polyubiquitylated target is then recognized and degraded in a proteasome.

121
Q

Sequential activation of APC/C by Cdc20 and Cdh1

A

MCDK phosphorylates APCC and CDH1, both still inactive and then CDC 20 binds to APCC (anaphase onset), which then causes MCDK inactivation, which causes dephosphorylation of APCC & CDH1. CDC 20 leaves a PCC and CDH1 binds to APCC

122
Q

Overview of the Cell Cycle Control System

A

scyclin binding to s-cdk and inhibition of s-cdk via dna damage causes start of s-phase

unreplicated dna and dna damage causes in hiition of m-cdk, causing inhibition of dna re-replication, leadings to start of m phase

chromosome unattached to spindle inhibits APC/C which leads to (likely start of anaphase?)… middle of m phase

123
Q

Which complex of APC/C is active in early G1?

A

APC/C-Cdh1 - Cdh1 is phosphorylated by Cdks and kept inactive from late G1 till anaphase.

124
Q

Activation of APC/C is important for:

A

Metaphase to Anaphase transition

125
Q

S PHASE – DNA SYNTHESIS

A

S-Cdk Initiates DNA Replication Once Per Cell Cycle, Chromosome Duplication Requires Duplication of Chromatin Structure, Cohesins Hold Sister Chromatids Together

126
Q

Replication

A

At replication origin, double helix is opened with help from helicase which leads to single-stranded DNA templates ready for DNA synthesis

127
Q

Pre-replicative complexes

A

prereplicative complexes (preRC) containing inactive helicases (MCM-Helicases) assemble at origins (Ori).

Origins cannot be re-used till next a new preRC is assembled for the next replication cycle.

S-Cdk Initiates DNA Replication Once Per Cycle as it phosphorylates ORC and Cdc6 and inhibits their activity

127
Q

Pre-replicative complexes

A

prereplicative complexes (preRC) containing inactive helicases (MCM-Helicases) assemble at origins (Ori).

Origins cannot be re-used till next a new preRC is assembled for the next replication cycle.

S-Cdk Initiates DNA Replication Once Per Cycle as it phosphorylates ORC and Cdc6 and inhibits their activity

128
Q

Control of the initiation of DNA replication- overview

A

Replication origin is bound by the origin recognition complex (ORC) throughout the cell cycle, but ORC functions only in late mitosis and early G1 when it associates with Cdc6.

ORC–Cdc6 binds the Mcm helicase.

Mcm helicase also associates with a protein called Cdt1, which stimulates DNA binding and helicase activity of Mcm. Using ATP hydrolysis, the ORC and Cdc6 proteins load two copies of the Mcm helicase around the DNA next to the origin.

At the onset of S phase, S-Cdk stimulates the assembly of several accessory proteins, including Cdc45 and GINS, on each Mcm helicase. Another protein kinase, DDK, phosphorylates subunits of the Mcm helicase. The result is a large protein complex called the CMG helicase (for Cdc45–Mcm–GINS), which unwinds the DNA at the origin.

The ORC is displaced by the replication machinery, but new ORCs bind to both replication origins after their replication. Control of the initiation of DNA replication- overview

129
Q

One of the mechanisms that prevents origin
firing /MCM loading more than once per cell
cycle is through Cdt1 regulation.

A

APC/C-Cdh1 triggers Geminin destruction

Geminin inhibits free Cdt1

Cdt1 at replication forks targeted for degradation

130
Q

Activation of what inhibit MCM2 loading onto DNA?

A

S-Cdk

131
Q

Cohesins hold sister chromatids together

A

Cohesin complex has four subunits. Two subunits- Smc1 and Smc3 are coiled-coil proteins, and two other subunits are Scc1 and Scc1 are required for cohesin loading onto DNA at one end. SMC-Structural Maintenance of Chromosomes. SCC- Sister Chromatid Cohesion protein. Cohesin forms a ring holding the two sister chromatids together.

DNA catenation – intertwining of sister DNA during replication (disentangled by Topoisomerase II between S
phase and early mitosis)

132
Q

Cohesins Hold Sister Chromatids Together

A

Cohesins assemble along the length of each chromatid as the DNA is replicated and prevent them from coming apart until rings are broken late in mitosis.

133
Q

Mitotic phases

A

The division of a cell into two daughters occurs in the m phase of the cell cycle. m phase consist of nuclear division, mitosis, and cytoplasmic division, cytokinesis. mitosis is divided into five stages: prophase, prometaphase, metaphase, anaphase, and telophase. mitosis is between metaphase and anaphase, and cytokinesis is after telophase.

134
Q

interphase - most specific

A

During interphase, a cell increases in size. the DNA of a chromosome is replicated in a centrosome is duplicated.

135
Q

prophase - most specific

A

At prophase, the replicated chromosomes, each consisting of two closely associated sister chromatids, condense. Outside the nucleus, the mitotic spindle assembles between the two centrosomes, which are propagated and move apart.

136
Q

prometaphase - most specific

A

Prometaphase start abruptly from the breakdown of the nuclear envelope. chromosomes can now attach to spindle microtubules via their kinetochores and undergo active movement.

137
Q

metaphase - most specific

A

At metaphase the chromosomes are aligned at the equator of the spindle, midway between the spindle poles. the kinetochore microtubules attach sister chromatids to opposite poles of the spindle.

138
Q

anaphase - most specific

A

At anaphase, the sister chromatids synchronously separate to form two daughter chromosomes and each is Paul, slowly towards the spindle pole it faces. the kinetochore microtubules get shorter, and the spindle poles also move apart; both processes contribute to chromosome segregation

139
Q

Telophase - most specific

A

During telophase, the two sets of daughter chromosomes arrive at the poles of the spindle, and decondense. a nuclear envelope resembles around each set, completing the formation of two nuclei, and marking the end of mitosis. the division of the cytoplasm begins with a contraction of the contractile ring

140
Q

cytokinesis - most specific

A

During cytokinesis, the cytoplasm is divided in two by a contractile ring of actin and myosin filaments (this creates a cleavage furrow), which pinches the cell and two to create two daughters, each with one nucleus

141
Q

MITOSIS - basic rundown

A

Spindle Assembly in Animal Cells Requires Nuclear-Envelope Breakdown

Mitotic Chromosomes Promote Bipolar Spindle Assembly

Kinetochores Attach Sister Chromatids to the Spindle

Bi-orientation Is Achieved by Trial and Error

Multiple Forces Act on Chromosomes in the Spindle

The APC/C Triggers Sister-Chromatid Separation and the Completion of Mitosis

Unattached Chromosomes Block Sister-Chromatid Separation: The Spindle Assembly Checkpoint

Chromosomes Segregate in Anaphase A and B

Segregated Chromosomes Are Packaged in Daughter Nuclei at Telophase

142
Q

At the end of DNA replication, the sister chromatids are held together by the ______.

A

cohesins.

143
Q

Condensins form a ring structure that encircles loops of DNA

A

When the cell enters M phase the duplicated chromosomes condense with the help of condensins becoming visible under the microscope.

Condensin is a five-subunit protein complex that has SMC2 and SMC4 with ATPase head domains held together by three additional subunits

  1. condesin non-SMC subuntis bind dna tightly
  2. nearby dna interacts with hinge
  3. atp-dependent motor activity extends dna loop
144
Q

The concentration of M cyclin ________________.

A

falls toward the end of M phase as a result of ubiquitylation and degradation.

145
Q

Metaphase mitotic spindle in an animal cell

A

centrosome - poles towards the outskirts of the separating cell

astral microtubules - coming from the centrosome.

kinetochore microtubes - attached to the kinetochore of the sister chromatid chromosomes

non-kinetochore microtubules - not connected to centrosome, but participated in metaphase

motor proteins - helping to separated sister chromatids along the microtubules

146
Q

A bipolar mitotic-spindle is formed by the selective stabilization of interacting microtubules

A

New microtubules grow out in random directions from the two centrosomes. The minus end is anchored in the centrosome. The free plus ends are dynamically unstable that results in rapid growth/shrinking of microtubules that extend in all directions in Prophase.

At some point when the microtubules from opposite centrosomes overlap, motor proteins and other associated proteins help to crosslink and stabilize these interpolar microtubules by stabilizing plus ends and decreasing the probability of depolymerization. This forms the framework of the mitotic spindle. The two centrosomes that gave rise to these microtubules are now called spindle poles.

147
Q

centrosome - microtubule directions

A
  • end microtubule facing the centrosome, + end growing out
148
Q

Centrosome Cycle

A

G1 phase - cell-cycle entry, and procentriole site selection

S phase - centriole duplication

end of G2 - tether removal, centriole maturation (entry into mitosis)

M-phase - centrosome maturation, centrosome separation (entry into mitosis)

G1 phase (right after m-phase) - central disengagement, centriole-to-centrosome conversion

149
Q

Centrosome in an interphase duplicates to form the two poles of the mitotic Spindle

A

In most animal cells in interphase, a centriole pair (dark green bars) is associated with the centrosome matrix (light green), which nucleates microtubule outgrowth. Centrosome Duplications begins at the start of S phase and is complete by the end of G2.

Initially the two centrosomes remain together but in early M phase, they separate, and each nucleates its own aster of microtubules.

The centrosomes then move apart , and the microtubules elongate preferentially to form the mitotic spindle with an aster at each pole. When the nuclear envelope breaks down, the spindle microtubules are able to interact with the duplicated chromosomes.

This whole process of centrosome duplication and separation is called centrosome cycle.

150
Q

Consider the centrosome duplication cycle and in relation to replication, is centrosome duplication:

A

Centrosome duplication is semiconservative. The paired centrioles in the centrosome separate, and each serves to nucleate the assembly of a new centriole. As a consequence, each new centrosome consists of one old and one new centriole. Thus, centrosome duplication is analogous to DNA replication, in which the new double helix consists of one old DNA strand and one newly replicated DNA strand.

151
Q

Microtubule-dependent motor proteins govern spindle assembly and function

A

kinesin 5 - move towards + end on non-kinetochore microtubule

kinesin 14 - moves toward + end on non-kinetochore microtubule

dyenin - moves towards - end directed on non-kinetochore microtubule; also connect to the plasma membrane (on astral tubules)

kinesin 4/10 are attached to the sister chromatids and non-kinetochore microtubules and are + end directed

151
Q

Microtubule-dependent motor proteins govern spindle assembly and function

A

kinesin 5 - move towards + end on non-kinetochore microtubule

kinesin 14 - moves toward + end on non-kinetochore microtubule

dyenin - moves towards - end directed on non-kinetochore microtubule; also connect to the plasma membrane (on astral tubules)

kinesin 4/10 are attached to the sister chromatids and non-kinetochore microtubules and are + end directed

152
Q

Local microtubule stabilization is promoted by Ran-GTPase

A

Activation of Ran-GTPase is highest around
chromosomes between spindle poles

153
Q

Spindle self-organization by motor proteins

A
  • nucleation
  • antiparallel cross-linking by kinesin- 5
  • outward push by kinesin-4,10
  • focusing of pole say dynein and kinesin 14
154
Q

Spindle self-organization by motor proteins

A
  • nucleation
  • antiparallel cross-linking by kinesin- 5
  • outward push by kinesin-4,10
  • focusing of pole say dynein and kinesin 14
155
Q

Kinetochores attach

A

sister chromatids to the spindle

156
Q

Microtubule attachment sites in the kinetochore

A

inner kinetochore - attached to centromeric nucleosome (chromosome)
outer kinetochore - ndc80 complex attached to actual microtubule and inner

157
Q

Chromosome attachment to the mitotic spindle

A

prophase - begins

early prometaphase - lateral kinetochore attachments, chromosome arms pushed outward

mid pro metaphase - end-on attachment

metaphase: bi-orientation (creates tension) (achieved by trial and error)

158
Q

Tension sensing mechanism depends on

A

Aurora-B kinase - senses by bi orientation is achieved

159
Q

Microtubule flux in the metaphase spindle

A

depolymerization at the plus ends generate poleward force that pulls kinetochore and attached chromatid towards spindle poles. Ndc80 complex is still attached to the sides of the microtubules even as the plus end depolymerizes as attachments between Ndc80 and microtubules are constantly breaking and reforming at new sites even as microtubule depolymerizes.

Second poleward force is microtubule flux- where microtubules themselves are pulled towards the spindle poles by depolymerization at the minus ends. In metaphase addition of new tubulin at plus end compensates for loss at tubulin at minus end and so, microtubule length remains constant despite movement of microtubules towards the spindle poles

160
Q

Opposing forces may drive chromosomes to the metaphase plate

A

Third force is a polar ejection force or polar wind that pushes chromosomes away from spindle poles and towards the spindle equator. It is mediated by plus-end directed kinesin-4 and kinesin-10 motor proteins on chromosome arms that transport chromosomes away from the spindle poles (could help align chromosomes in metaphase). The the poleward forces generated by depolymerization at the kinetochore together with microtubule flux- pull chromosomes towards the poles

161
Q

For microtubule (spindle) organization in mitosis, chromosomes are not as important as centrosomes

A

false

162
Q

For microtubule (spindle) organization in mitosis, chromosomes are not as important as centrosomes

A

false

163
Q

The initiation of sister chromatid separation by the Anaphase Promoting Complex (APC)

A

Destruction of securin by APC/C allows separase (protease) to cleave cohesion subunit-Scc1.

Phosphorylation by Cdks also inhibits separase. Inactivation of Cdks also promotes separase activation.

APC/C is activated by increase in Cdc20 levels in mitosis and phosphorylation/activation of APC/C by M-Cdk and other kinases.

164
Q

Cleavage of cohesion subunit- Scc1…

A

separates sister chromatids

165
Q

Spindle assembly checkpoint pathway

A

if kinetochore is not attached/tension is absent, MAD2 and BubR1 will bind to Cdc20-APC complex and inhibit anaphase onset

if kinetochore is attached/tension is present, Mad2 and BubR1 won’t attach to anything; Securin and heparin will bind to Cdc20-APC complex. ubiquitin will attach to the securing, causing its degradation, separin leaves, and triggers anaphase onset via breaking SCC1 in the chromosomes

166
Q

Separase is kept inactive due to:

A

Binding of securin

167
Q

Anaphase- A and B

A

A - chromosomes are pulled poleward - kinetochores microtubules shorten, dragging chromosomes toward their spindle pole

B - poles are pushed and pulled apart - a pulling force at the cell cortex drags the two poles apart. a sliding force between antiparallel microtubules at the spindle center pushes the poles apart. microtubule growth at plus end of microtubules helps push the poles apart.

168
Q

Overlapping array of actin and myosin contract to

A

generate the force that divides the cytoplasm into two

169
Q

Midbody

A

After the contractile ring disappears, the plasma membrane of the cleavage furrow forms the midbody

170
Q

Actin and myosin filaments are also main components of the mitotic spindle. T or F

A

False

171
Q

Actin and myosin filaments are also main components of the mitotic spindle. T or F

A

False

172
Q

What inhibits M-Cdk activity in anaphase?

A

activation of APC/C- Cdc20

173
Q

Mitogens Stimulate Cell Division

A

Extracellular Signals

Mitogens – cell division/ proliferation (increase in cell number)

Growth factors- cell growth (increase in cell size)

Survival factors – promote cell survival

174
Q

Mitogens Stimulate G1-Cdk and G1/S-Cdk Activities

A

Mitogen binds to mitogen receptor on the plasma membrane which activates Ras, which then activates Map kinase, which then leads to the activation of transcription regulatory proteins, which then goes form the cytosol to the nucleus, activates early gene expression, which leads to activation of a MYC a transcription regulatory protein

This causes delayed response of gene expression, which then leads to active phosphorylated G1-CDK, which causes active RB proteins to be phosphorylated and to fall off inactivated E2F protein,

This leads to inactivated RB protein, and active E2F proteins. This leads to s-phase transcription, which leads to G1/S-cyclin (cyclin E) and s-cyclin (cyclin A) to be active. Now S-cdk is active (within S-phase) and G1/S-cyclin is active (within G1 phase), which leads to positive feedback of Rb protein leaving E2F protein.

E2F gives itself positive feedback to stay off RB.

Active S-CDK leads to dna synthesis.

175
Q

Rb- Retinoblastoma

A

identified initially in rare childhood eye tumor. Rb is a tumor suppressor

176
Q

DNA damage blocks cell cycle- Cell Cycle Checkpoints

A

DNA damage- UV, radiation, chemicals, replication
errors - affects checkpoint pathways p53 and Chk1and inhibits Cdk/cyclin

STOP at G1 to S, S to G2, or G2 to M

177
Q

majority of cancers have mutations in

A

p53- a tumor suppressor

178
Q

DNA Damage Blocks Cell Division: p53-p21 response

A

DNA damage can temporarily halt progression through G1 phase

p53- Checkpoint protein and a tumor suppressor

process:

DNA damage leads to ATM/ATR kinase activation which leads to CHK1/CHK2 kinase activation, which leads to the phosphorylation of P53, which causes the unending of Mdm2 from P53. Active P53 binds to regulatory region of P21. gene transcription occurs, leading to P21 mRNA. translation occurs, which leads to P21 (CDK inhibition proteins), which binds to active G1/SCDK and SCDK which makes it inactive

179
Q

Activation of Checkpoint Kinase 1 (Chk1) by DNA damage stalls the cell cycle

A

in S or G2 Phase by inhibiting the Cdc25 phosphatase

180
Q

Which of these is a CDK inhibitor, whose transcription is induced by p53?

A

p21

181
Q

Stimulation of cell growth by extracellular growth factors and nutrients

A

Growth factor binds to now activated growth factor receptor and interacts with amino acids, which eventually causes activation of mTORC1, which can lead to protein synthesis, lipid synthesis of proteins turnover.

182
Q

if Tloop is in active site, then CDK is:

A

Inactive

183
Q

CDK alone is:

A

Inactive

184
Q

CDK/Cyclin bound to p27 or p21 is:

A

Inactive

185
Q

how do Greatwall and Ensa collaborate to turn off PP2A-B55

A

Greatwall phosphorylates Ensa, which then binds to PP2A-B55, turning it off.

186
Q

Which one of the following possibilities would give the most rapid activation of Mcdk

A

M-Cdk phosphorylates Wee1 and Cdc25, inactivating Wee1 and activating Cdc25

187
Q

g1-Cdk promotes synthesis of what cyclins?

A

G1/S-cyclin and S-cyclin

188
Q

g1-Cdk promotes synthesis of what cyclins?

A

G1/S-cyclin and S-cyclin

189
Q

Entry into mitosis signals an abrupt change in the cell’s microtubules. During prophase, and particularly in prometaphase and metaphase, the average lifetime of a microtubule increases dramatically, giving rise to the stable metaphase spindle that is so prominent in the microscope.
A. True
B. False

A

false

Entry into mitosis signals an abrupt change in the cell’s microtubules. During prophase, and particularly in prometaphase and metaphase, the average lifetime of a microtubule decreases dramatically—particularly the lifetime of non-kinetochore microtubules, which exist for only 15 to 30 seconds. This increase in microtubule instability, coupled with the increased ability of the spindle to nucleate microtubules, results in a remarkably dense and dynamic array of spindle microtubules. Microtubules outside the spindle typically have a lifetime measured in minutes.

190
Q

You have obtained an antibody to myosin that prevents the movement of myosin molecules along actin filaments. If this antibody were injected into cells, would you expect the movement of chromosomes at anaphase to be affected? How would you expect antibody injection to affect cytokinesis?

A

Chromosome movement would be unaffected; cytokinesis would be inhibited.

191
Q

Chromosomes are pulled to the spindle pole at anaphase like fish being reeled in on a line. t or f

A

false

192
Q

Which factors are important to ensure that the Mcm helicases are loaded onto origins of replication in G1 phase?

A

Cdt1, orc, cdc6

193
Q

What is the term for the stage of mitosis in which the spindle poles move apart?

A

anaphase b

194
Q

Microtubule flux is a type of force that pulls:

A

kinetochore microtubules to the spindle poles due to minus end depolymerization.

195
Q

Degradation of Geminin by APC/C in G1, keeps Cdt1 levels

A

high.

196
Q

When kinetochores on sister chromatids attach to the same spindle pole, the arrangement is unstable (see the figure) and normally is rapidly rectified so that each sister chromatid attaches to an opposite pole. The abnormal attachments, however, can be stabilized if a fine glass needle is used to gently pull the chromosome away from the spindle pole to which it is attached.
what explains this?

A

Stability requires that the attachment between the microtubules and kinetochores be under tension.

197
Q

to test whether the yeast Scc1 gene is required for sister-chromatid cohesion, you use a temperature-sensitive (ts) mutant in a strain that carries a special DNA sequence on chromosome V to which a green fluorescent fusion protein will bind. To test the effects of mutant Scc1 on sister-chromatid cohesion, you isolate unbudded cells from wild-type and Scc1ts cells that were grown at 25°C, and grow them at 37°C—the restrictive temperature—for various times. Examples of small-budded cells in S phase and large-budded cells that have passed the metaphase-to- anaphase transition are shown for both strains in the figure. For each yeast strain, the same cells are shown as they appear by light microscopy and by fluorescence microscopy. At which phase of the cell cycle do sister chromatids separate in the Scc1ts strain?

A

sphase

Sister chromatids normally separate at the metaphase-to-anaphase transition; thus, they are separated
at anaphase. Sister chromatids behave as expected in wild-type cells: they are stuck together in small-
budded cells (in S phase) and separate into the mother and daughter cells at anaphase. The single spot
of fluorescence in the small-budded cell in the figure likely results from two sites of binding that are
close together on the paired sister chromatids. In the Scc1 ts strain, the spots are already clearly
separated in one of the small-budded cells (in S phase). Thus, Scc1 ts must act during S phase.
Clicker- Explanation

Cell Biology (3 decks)

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