Final Exam, Opus 1 Flashcards
(219 cards)
1
Q
General shape of cells in interphase
A
Stretched out
2
Q
General shape of cells in mitosis
A
Rounded up, circular sphere
Change in shape is due to mitosis-related changes in the underlying cytoskeleton, specifically in the microtubules
3
Q
Non-radioactive technique to identify S phase cells
A
Incubate cells with BrdU (bromo-deoxyuridine, an analog of thymidine)
Cells that replicated their DNA during the incubation period can be detected with BrdU antibodies
4
Q
What is flow cytometry used for?
A
To determine cell ploidy, how much DNA is in each cell
5
Q
Flow cytometry technique
A
Cells are fixed with a suspension, labelled with a DNA stain, suspension is run through a cytometer that drips the cells through single file. A laser light source measures 2 things
- Scatter, is it a cell yes or no
- How much fluorescence is coming off the cell
The fluorescence is used to determine how much DNA is in each cell
6
Q
Describe the graph generated by normal cells in flow cytometry
A
Graph has 2 peaks. The valley in the middle represents cells in G1 or G0, undergoing DNA synthesis
7
Q
Describe the graph generated by abnormal (cancerous, failed cytokinesis, other funky stuff) in flow cytometry
A
These cells would have weird ploidy counts, would generate peaks beyond 2C
8
Q
General shape/form of DNA in cells that have just divided (G1 or G0)
A
Each chromosome is a single long piece of DNA
9
Q
General shape/form of DNA in cells just after DNA replication (G2 or M)
A
Two sister chromatids are joined by cohesins, they are condensed into the classic X chromosomes
10
Q
General description of cellular cyclin concentrations over time
A
Concentrations crash just before cleavage
Concentrations are inversely correlated with cleavage time
Cyclin levels cycle up and down over time
11
Q
Are cyclins and CDKs evolutionarily conserved?
A
Yes, so much so that human versions of CDK can “genetically rescue” yeast CDK mutants
12
Q
How many CDKs and cyclins do yeast have?
A
One CDK
Multiple cyclins
13
Q
How many CDKs and cyclins to humans have?
A
Multiple CDKs
Multiple cyclins
14
Q
CDK levels in actively growing cells
A
CDK levels are relatively stable in actively growing cells
15
Q
CDK activity levels in actively growing cells
A
CDK’s activity levels cycle up and down because cyclin levels cycle up and down. CDK requires cyclin to be active, it’s in its name
16
Q
The on/off transitions of CDK activity are much sharper and more abrupt than the changes in cyclin levels are. What does this suggest?
A
Suggests that there are additional levels of regulation involved in CDK’s on/off transitions, more than just cyclin regulates CDK activity
17
Q
The “questions” asked at the G2 to M phase transition
A
Is all DNA replicated?
Is the environment favorable?
18
Q
The “questions” asked at the Metaphase to Anaphase transition
A
Are all chromosomes attached to spindles?
19
Q
What mediates cyclin’s rapid degradation?
A
APC/C + coactivator (either Cdc20 in Mitosis, or Cdh1 in G1)
20
Q
List the 4 different ways CDKs are regulated
A
- APC/C
- CAK
- CKI
- Wee1 and Cdc25
21
Q
What is APC/C?
A
A protein complex that functions alongside E1 and E2 to polyubiquitinate cyclin for destruction in a proteasome.
Selective destruction of cyclin
It cycles up and down in different cell cycle phases, levels are highest in G1 and Mitosis
Must be bound to a coactivator to function (Cdh1 in G1, Cdc20 in Mitosis)
Drives both the metaphase to anaphase transition and the exit from mitosis
22
Q
What coactivator is APC/C bound to in G1 phase?
A
Cdh1
23
Q
What coactivator is APC/C bound to in mitosis?
A
Cdc20
24
Q
How can APC/C drive both the metaphase to anaphase transition and the exit from mitosis?
A
It can do both because it works with two different coactivators, it has E3 ubiquitin ligase activity for different substates depending on which coactivator it’s associated with
25
How does APC/C regulate CDK?
It selectively degrades cyclins, the necessary coactivator for CDK's kinase activity
26
What transitions does APC/C drive?
Metaphase to anaphase
Exit from mitosis
27
What is CAK?
CAK stands for CDK Activating Kinase
It phosphorylates CDK's T-loop, this positive phosphorylation changes CDK's active site and allows CDK to be fully active
28
Activity level -- regular plain CDK
Inactive
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Activity level -- CDK+cyclin, T-loop is not phosphorylated
Partially active
30
Activity level -- CDK+cyclin, T-loop is phosphorylated
Fully active
31
Is CAK's positive phosphorylation rate limiting?
NO!!!
32
How does CAK regulate CDK?
It changes the shape of CDK's active site via positive phosphorylation, allows CDK to be fully active
33
What is CKI?
A class of proteins that bind to and poke into CDK+cyclin's ATP binding site, physically blocks CDK's kinase activity
34
How do CDKs get around the CKI checkpoints?
More E3 ubiquitin ligases, including SCF, target CKIs for destruction, releasing the checkpoint
35
What protein family is p27 a member of?
CKI
36
What protein family is p21 a member of?
CKI
37
What protein family is SCF a member of?
E3 ubiquitin ligase
38
How do CKIs regulate CDKs?
They bind to and physically block CDK+cyclin's ATP binding site
39
What is Wee1?
An inhibitory kinase. It adds an inhibitory Pi, negative phosphorylation, to block CDK's active site
40
What is Cdc25?
A corrective phosphatase. It removes the inhibitory Pi from CDK's active site placed there by Wee1
41
How does Wee1 regulate CDKs?
It blocks CDK's active site by adding an inhibitory Pi to it
42
How does Cdc25 regulate CDKs?
It removes the inhibitory Pi from CDK's active site placed there by Wee1, allows the CDK's active site to be open again
43
Active CDK+cyclin has a positive forward feedback loop. What does this loop do?
It inhibits Wee1 and promotes Cdc25. It's a mechanism to create a steeper curve of CDK+cyclin activity (once it's going it's going)
44
APC/C drives the cell cycle forwards. Does it do so reversibly or irreversibly?
IRREVERSIBLY!
45
SCF drives the cell cycle forwards. Does it do so reversibly or irreversibly?
IRREVERSIBLY!
46
What does SCF do?
It's an E3 ubiquitin ligase, targets CKIs for destruction
47
Why are the 4+ layers of CDK regulation necessary?
The layers make it possible to do both tight/fast and irreversible cell cycle transitions
48
Best type of regulation for fast transitions
Dephosphorylation
49
Best type of regulation for medium speed transitions
Rapid targeted protein destruction
50
Best type of regulation for slow transitions
Rapid cyclin synthesis
51
Best type of regulation for irreversible transitions
Rapid targeted protein destruction
52
Transition speed of dephosphorylation
Fast
53
Transition speed of rapid targeted protein destruction
Medium
54
Transition speed of rapid cyclin synthesis
Slow
55
What is Myc
A TF that promotes transcription CDK4/6 and Cyclin D
56
How does growth factor drive the transition from G1 to S phase?
Growth factor stimulates RTKS, downstream effects include inducing Myc
Myc promotes transcription of CDK4/6 and Cyclin D, these form active G1-CDK
Active G1-CDK phosphorylates Rb, making it dissociate from E2F
E2F is active and free to promote the transcription of S phase genes
57
Active E2F also creates a positive feedback loop. What does this loop do?
E2F also promotes transcription of G1/S-CDKs and S-CDK, both of which phosphorylate Rb to keep E2F continually active
58
What protein family is Rb a member of?
Tumor suppressor gene
59
What does Rb do?
It's normal job is to block the cell from entering S phase by binding to and inactivating E2F
60
What happens to a cell that has lost both copies of its Rb gene?
Cancer, probably
61
Where is E2F located in the cell?
It's always bound to the DNA, even when it's bound to Rb and no growth factors are present to release it
62
What does E2F do?
TF that promotes transcription of G1/S-CDKs and S-CDKs, also promotes centrosome duplication
63
How do cells get past APC/C at the G1 to S-phase transitions?
G1/S-CDKs phosphorylate Cdh1, an essential cofactor for APC/C, inactivating APC/C's E3 ubiquitin ligase activity
64
True or false: CDK is required to form the complete helicase complex
True
65
How do cells block their DNA from re-replicating?
CDK inactivates ORCs, this ensures DNA replication happens only once per cell cycle
66
Explain the two-fold importance of S-CDKs to DNA replication
ORCs bind to DNA, but nothing happens until S-CDK levels are high and the complete helicase complex forms
S-CDKs also inactivate ORCs after replication is complete, blocks re-replication
67
What is the point of mitosis?
To distribute an equal and identical set of genetic material to the two daughter cells
68
Cohesins
Proteins that form loops around the chromatids, gathers the DNA
Keeps sister chromatids together during replication
69
How are centrosomes/MTOCs replicated?
Semi-conservatively
Initially the centrioles split, then they each grow a sister centriole at a 90 degree angle. Later M-phase kinases activate the two centrosomes/MTOCs
70
Can the two centrioles' semi-conservative replication growth happen at different times?
No. Growth must happen at roughly the same time
71
What drives the beginning of mitosis?
M-CDK activation
72
What other kinases work alongside M-CDK?
Aurora kinases and Polo-like kinases (Plk)
73
What kind of kinase are Aurora kinases?
Ser/thr kinase
74
What kind of kinase are Plk kinases?
Ser/thr kinase
75
Active mitotic kinases (M-CDKs, Aurora, Plk) are required for...
Chromosome condensation
Preparation for spindle chromosome interactions (kinetochore assembly, changes in MT dynamics, positioning of microtubule asters)
Nuclear envelope breakdown
76
How do insulin secreting beta pancreatic islet cells know when blood glucose levels are elevated?
GLUT2 is the transporter, has relatively low Km and low transport rate, so it's more sensitive to rising glucose levels. When blood glucose levels are high, GLUT2 is still able to bring more glucose into the cell
Glucose goes straight into glycolysis, immediately turned into pyruvate and ATP
77
What do insulin secreting beta pancreatic islet cells do in response to glucose uptake + glycolysis turning on?
The ATP from glycolysis acts as the ligand to gate K+ channels, closes them. K+ accumulates in the cell when blood glucose levels are high. This changes the voltage across the plasma membrane.
In response to the change in voltage, the voltage-sensitive Ca2+ channel opens, bringing Ca2+ into the cell. Increased calcium uptake causes insulin-containing secretory vesicles to be secreted. These vesicles can't fuse with the plasma membrane and be secreted until they get that calcium signal, calcium-dependent insulin secretion
78
How do fat and muscle cells increase their glucose uptake in response to insulin?
Remember that insulin receptors are an usual RTK, they're always dimerized
Insulin binds to the pre-dimerized insulin RTK receptor, starts a signal cascade that includes adding more GLUT4 uniporters to the plasma membrane
79
Specific process of adding more GLUT4 transporters to the plasma membrane of fat and muscle cells in response to insulin
Insulin signaling activates a protease, which cleaves TUG into two pieces. TUG's N-terminal end is still attached to the GLUT4 storage vesicle, but the C-terminal end is cleaved free. TUG's cleavage allows the GLUT4 storage vesicle to be taken to the plasma membrane via kinesins/MTs
Insulin signal also activates PI-3 kinase, which activates Protein Kinase B (PKB). PKB phosphorylates and inhibits the Rab GAP protein AS160, this causes Rabs to accumulate in its GTP form on GLUT4 storage vesicles, increasing their fusion with the plasma membrane
80
How do fat and muscle cells reset the insulin signaling?
They remove the additional GLUT4 uniporters from their plasma membranes via endocytosis
81
Why do many tumor cells have dead cells in their centers?
Because those cells became hypoxic and died
82
HIF-1(alpha) in high oxygen levels
Is prolyl hydroxylated (OH attached to proline residues), this increases its binding affinity to VHL, an E3 ubiquitin ligase, which polyubiquitinates it for proteasomal degradation
83
HIF-1(alpha) in low oxygen levels
Is not prolyl hydroxylated (no OH groups on proline residues), cannot be targeted for destruction, HIF-1(alpha) levels rise.
It then enters the nucleus and acts as a TF to turn on genes for glycolysis/anaerobic metabolism, angiogenesis, vasodilation, erythropoiesis, and increased breathing
84
VEGF
Vascular Endothelial Growth Factor
Promotes the growth of new capillaries, new offshoots of the vascular system. Essential in development and tissue repair, but bad in cancer
85
Erythropoietin (EPO)
Glycoprotein produced by the kidneys, stimulates RBC production. EPO drugs can treat anemia and are good for cancer patients with low RBC counts caused by chemo treatments.
Banned by WADA in and out of competition
86
Lupus
Autoimmune disease caused by cells not dying via apoptosis, instead they explode their cell contents everywhere, triggers inflammation, causes problems
87
What causes the cellular and biochemical changes to a cell during apoptosis?
Caspases (cysteine aspartate specific proteases)
88
Key amino acid in the catalytic domain of a caspase protease
Cysteine
89
Caspase protease's target
Is next to an aspartate, the target is cleaved on the C-terminal side of an aspartate
90
Caspase 8 and 9
Initiator caspases
Their job is to activate effector caspases
91
Caspase 3 and 7
Effector caspases
Their job is to cleave a variety of cellular targets to set off apoptosis
92
Why are caspases similar to insulin?
They are both synthesized as one large polypeptide before being cleaved into their final forms
For caspases, the initial large polypeptide is called a procaspase
93
In what form are caspases active?
Procaspase is cleaved twice, subunits assemble into an active tetramer with two large subunits and two small subunits.
Tetramer is the active caspase
94
How are caspase cascades similar to kinase cascades?
Both cascades allow for signal amplification
95
Quick list: cellular targets of caspases
Gelsolin
Lamins
CAD
Flippase+scramblase
96
Caspases and gelsolin deregulation
Gelsolin is normally Ca2+ dependent. Caspases cleave the Ca2+ regulation domain off gelsolin. This deregulates it, causes it to go crazy and cut up the actin cytoskeleton. Results in breakdown of the actin cytoskeleton
This is how apoptotic cells lose their physical integrity/shape, become floppy and "bleb" off
97
Caspases and lamin cleavage
Remember that lamins are the intermediate filaments forming the mesh underneath the interior leaflet of the nuclear membrane.
Lamin cleavage causes nuclear envelope breakdown
98
How does nuclear envelope breakdown occur in cell cycle/division contexts?
Lamins are phosphorylated, causes their mesh to break apart
99
How does nuclear envelope breakdown occur in apoptosis?
Caspases completely chew the lamins up
100
What is CAD?
Caspase Activated DNase
It's a fragmentation factor that chews up DNA into very predictable 200 bp pieces
101
Caspases and CAD
CADs are normally blocked, bound by an inhibitor (I-CAD). Caspase 3 cleaves I-CAD, allows CAD to be active
102
Why is CAD an important tool in recognizing apoptotic cells?
Because CAD chews up DNA into predictable 200 bp pieces, apoptotic cells can be identified by running their chewed up DNA extracts on a gel. Bands form exactly 200 bps apart.
In necrotic cells (dead/dying but messily, sans CAD) there aren't neat bands, just one big schmear on the gel
103
How do macrophages detect exposed phosphatidylserine?
They have both direct and indirect mechanisms, detection signals for them to phagocytose the apoptotic cell
104
How is gelsolin normally regulated?
By Ca2+
Important for apoptosis because Caspase 3 cleaves and removes its Ca2+ dependent regulatory domain, allows gelsolin to cut up actin filaments unregulated
105
TUNEL Assay
Tdt-mediated dUTP nick and labeling
Method to assess cells for apoptosis
Adds chains of labeled dUTP to the 3' OH ends of a cell's DNA fragments. DNA that's been heavily cleaved has more free 3' OH ends, more things to label, produces a brighter signal in the assay
Specifically this is a way to visualize the location of apoptotic cells within a population
106
Key to the intrinsic molecular mechanisms that regulate the caspase cascade
Mitochondria
107
Describe the intrinsic caspase cascade regulation pathway
Could also be the answer to "How is initiator Caspase 9 activated?"
During apoptosis Cytochrome C, which normally sits in the mitochondrial intermembrane space as part of the ETC, is released into the cytosol
It does so through pores formed by Bax and Bak, genes upregulated by p53
In the cytosol Cytochrome C is the activating agent of apoptosomes, combines with Apf1 to create apoptosomes
Apoptosomes recruit and bind to initiator Caspase 9, activating it. Part of the caspase cascade
108
Apaf1
Combines with Cytochrome C to form apoptosomes
Upregulated by p53
109
Bcl2
Anti-apoptotic factor, is normally bound to Bak/Bax and prevents them from assembling into pores
Inactivated by apoptotic stimulus
110
How are Bcl2, Bim, Bak, Bax, and Puma related?
They're all part of the same BH3 superfamily. Similar structures, related proteins
111
How is Bcl2 inactivated? List 3 ways
1. Normally neurotrophic factors (community survival signals) signal for kinases to keep Bad bound to 14-3-3
Without neurotrophic factors, Bad is not phosphorylated, causing it to release from 14-3-3, it's now free to block Bcl2. This allows Bak/Bax pore formation
2. Another way is via cell detachment, this disrupts integrin and promotes the release of Bim, Bim then inactivates Bcl2
3. Another way is Puma, Puma is directly transcribed by p53, it also inactivates Bcl2
112
How do killer T cells initiate apoptosis in another cell? (extrinsic pathway)
FasL on killer T cell exterior surface binds to Fas receptor on target cell, this is the stimulus that activates Initiator Caspase 8, it activates effector caspase 3
This pathway can involve Cytochrome C release, but it doesn't have to
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Caspase 9
Initiator caspase, intrinsic pathway
114
Caspase 8
Initiator caspase, extrinsic pathway
115
Why is p53 the guardian of the genome?
For cells that should NOT enter the cell cycle, p53 promotes the expression of p21, a CKI
116
What is p53?
A transcription factor and tumor suppressor
Especially important in G1 and mitosis
117
What are the most frequently mutated amino acids in p53 in human cancers?
Amino acids in residues that bind p53 to the DNA
118
MDM2
E3 ubiquitin ligase for p53
Under normal conditions, excess p53 is lethal, so though it's regularly synthesized by the cell it's marked for degradation by MDM2 so it doesn't cause problems
119
How is p53 activated? (how does it get around it's MDM2 block point)
When DNA is damaged, the damage activates DNA-dependent protein kinases. These kinases phosphorylate p53 so that it no longer binds to MDM2
120
What does p21 do?
It's a CKI that inhibits G1 and S phase CDKs
121
Why, under normal conditions, is excess p53 lethal?
Because it's a TF for p21, which inhibits G1 and S phase CDKs, preventing the cell cycle from moving forwards. This is lethal if the cell needs to progress but can't
122
What is p53 a TF for?
p21
Also MDM2 (this suggests that when the DNA damage is repaired, p53 can be turned off again)
When DNA damage is beyond repair, it's also a TF for Puma, Bak/Bax, Apaf1, and other apoptosis genes
123
Why is blocking p53 a cancer risk?
Allows damaged DNA to be replicated, cells proliferate with mutated DNA
124
Why is it advantageous for viruses with a DNA genome to promote cell cycle entry?
Remember that HPV, a DNA virus, inhibits tumor suppressors like Rb and p53
It allows the viral DNA genome to be replicated with the rest of the cell's DNA
125
Definition of cancer
An abnormal growth of cells that tend to PROLIFERATE in an INVASIVE, uncontrolled manner, and in some cases, metastasize
126
List the two types of driver mutations in cancer
1. Mutation in a tumor suppressor gene (lost "brake")
2. Mutation in a proto-oncogene (jammed accelerator)
127
Mutation in a tumor suppressor gene
Loss of both copies of a tumor suppressor gene
Broken or lost checkpoint proteins and/or repair proteins
Brake lines are cut
Recessive
128
Mutation in a proto-oncogene
Acquiring a mutation in a proto-oncogene that causes it to become an oncogene
One gene copy is altered to be always or inappropriately on
Accelerator is jammed on
Dominant
129
What are proto-oncogenes
Genes that you need for growth and development. Without these genes the cell would never grow or proliferate. Loss is always fatal
It's only when they're always on or expressed too much do they become oncogenes
130
Why is E-Cadherin a tumor suppressor?
Part of a cell becoming invasive is its ability to move around, to get up and leave to move to a new location. This requires an invasive cell to lose its cell-cell and cell-ECM attachments
The invasive cell needs this so it can no longer be limited by cell-cell contact, needs to lose contact inhibition
131
Why is pRb a tumor suppressor?
Active (non-phosphorylated) Rb binds to E2F and prevents it from promoting the transcription of G1/S-phase CDK
Without functional Rb, the G1/S cell cycle checkpoint would be bypassed
pRB, phosphorylated Rb, is the inactive form
132
Why is VHL a tumor suppressor?
VHL is the E3 ubiquitin ligase for HIF-1(alpha)
Without functional VHL, HIF-1(alpha) is free to enter the nucleus and act as a TF for many things related to cell growth and proliferation
133
What kinds of things is HIF-1(alpha) a TF for?
Glycolytic enzymes for anaerobic metabolism
VEGF for angiogenesis
i-NOS and HO-1 for vasodilation
EPO for erythropoiesis
Tyrosine hydroxylase for increased breathing
134
Examples of proto-oncogenes
Cell cycle genes (proteins that control the cell cycle, like cyclin and CDK, and TFs for cell cycle proteins and other proteins that promote cell growth)
Growth factors and growth factor receptors
Protein kinases and proteins that activate protein kinases (SRC, Ras, Raf)
Proteins that affect apoptosis (Bcl2)
135
What does too much Bcl2 mean?
Not enough programmed cell death!
136
PDGF (SIS)
Growth factor (example of a proto-oncogene)
137
HER2
Cell surface receptor for epidermal growth factor (example of a proto-oncogene)
138
How do proto-oncogenes become oncogenes? (3 ways)
1. Deletion or point mutation in the coding sequence
2. Gene amplification
3. Chromosome rearrangement
139
More details on how a proto-oncogene becomes an oncogene, via deletion or point mutation in the coding sequence
These types of mutations make the protein itself hyperactive, though it's still synthesized in normal amounts
140
More details on how a proto-oncogene becomes an oncogene, via gene amplification
The gene itself isn't mutated, there's just more copies of it in the genome now. Normal unmutated protein is being overproduced
141
More details on how a proto-oncogene becomes an oncogene, via chromosome rearrangement
Break in the chromosome DNA causes...
EITHER a nearby regulatory DNA sequence is now next to an unmutated protein, causing the protein to be overproduced
OR a nearby regulatory DNA sequence fuses to an actively transcribed gene, causing the newly fused gene to be greatly overproduced or causes the fusion protein to be hyperactive
142
Example of a proto-oncogene becoming an oncogene: RTKs
Method 1: mutation causes the RTKs to dimerize even in the absence of growth factors (whereas sans mutation they would only dimerize in the presence of growth factors)
Method 2: mutation cuts off the RTK's extracellular domains, leaving the remaining TMD and intracellular regions always dimerized, thus making the mutated RTK a constitutively active kinase
143
Example of a proto-oncogene becoming an oncogene: HER2
Overexpression of the receptor, too many receptors in the plasma membrane
This is bad because if even one gene copy of HER2 is overexpressed, the cell becomes overly sensitive to proliferation stimulus
144
Example of a proto-oncogene becoming an oncogene: Ras
A point mutation in the Ras gene compromises its ability to hydrolyze GTP, makes Ras always on
The point mutation allows it to still bind to GTP, but prevents it from hydrolyzing said GTP. Always on state
145
Most cancers need a suite of mutations or epigenetic changes that lead too...
...misregulated cell growth (nutrient uptake and utilization), misregulated cell proliferation, abnormal disregard of stress and DNA damage, and invasiveness
146
Important step in the G1 to S phase transition
G1/S CDKs inhibit Cdh1, inactivating APC/C's E3 ubiquitin ligase activity so it no longer degrades cyclin
147
Describe the process of chromosome condensation
Another way to ask this is how is the classic X chromosome shape formed?
Compaction occurs via condensins, progressive loop gathering
in G2, cohesin loops are all along the chromosome. Eventually cohesins on the chromosome arms are phosphorylated by Aurora and Plk, making them fall away. Then condensins compact everything further
Cohesins are retained only in the middle, giving the X shaped chromosome
148
Why is chromosome condensation/compaction necessary?
It avoids tangles and breakage
149
How are condensins and cohesins related?
They are molecular cousins, almost identical structures, just different subunits making up those structures
150
Why are only the cohesins on the chromosome arms phosphorylated? How are the cohesins in the middle preserved?
The ones in the middle are protected from phosphorylation by localized phosphatases
151
Can plain chromosomes bind to MTs on their own?
Nope! MTs must bind to a kinetochore platform, they can't bind to the actual chromosome itself
152
When in the cell cycle are kinetochores fully established and the MTs fully engaged?
Only after nuclear envelope breakdown
153
Are mitosis MTs more or less stable than interphase MTs?
Mitosis MTs are much less stable
154
Kinesin-13 activity levels though the cell cycle (what would the graph look like?)
Activity levels remain constant through the cell cycle
155
XMAP215 levels through the cell cycle (what would the graph look like?)
Levels crash during mitosis
156
Stability of interphase MTs
Very stable
157
Stability of mitosis MTs
Very unstable
158
How does MT activity become more dynamic during mitosis?
Increase in gamma-tubulin rings, this creates more MT starts and leads to more dynamic MT activity
Also, MAPs are turned off, meaning there are fewer proteins binding to the sides of the MTs to keep them intact.
Fewer MAPs also means the MTs are shorter
159
What is required for the centrosomes (MT asters) to move towards opposite ends of the nucleus?
Kinesin-5 to push poles apart
Kinesin-14 to cross-link in the midzone
Chromokinesins (4,10) to move arms away from poles
Dyneins to pull poles towards cell cortex
160
When in the cell cycle do the centrosomes (MT) asters move towards opposite ends of the nucleus?
In pro-metaphase, in preparation for metaphase
161
Kinesin-5
MT associated motor protein
Double headed, motors on both ends
Both ends are + end directed, moves to the + end
Pushes poles apart
162
Kinesin-14
MT associated motor protein
Atypical kinesin, single head
Moves to the - end
Cross-link at midzone
163
Chromokinesins (Kinesin 4,10)
MT associated motor protein
Crawls to the + end
Moves arms away from poles
164
Dyneins
MT associated motor protein
"drunken sailors"
Dives into the cell center, moves to the - end
Pulls poles towards cell cortex
Can be either an anchoring force or a shortening force
165
What is the las step in the G2 to Mitosis transition?
Nuclear envelope breakdown. This is the point of no return.
CDKs phosphorylate lamins, makes their mesh structure fall apart
166
What is the point of no return in the road to mitosis?
Nuclear envelope breakdown
167
What must happen in the prometaphase to metaphase transition?
Chromosomal alignment, bipolar kinetochore attachment
168
Astral MTs
Connected to the centrosome, reaching out towards the cell membrane
Spiders
- end at the MTOC, + end out and away
169
Kinetochore MTs
Reach from the centrosome/MTOC to kinetochores on chromatids
- end at the MTOC, + end out and away
170
Interpolar MTs
Reach out from the chromosome/MTOC to each other
Opposing interpolar MTs line up next to each other with motor proteins
- end at the MTOC, + end out and away
171
Why are mitosis MTs less stable than interphase MTs?
Because many MAPs have been turned off. This makes the MTs shorter, and also means there are fewer proteins binding to the sides of the MTs to keep them intact
172
Why is it important that Kinesin 5 and Kinesin 14 are opposing forces?
Allows for fine motor movement of chromosome alignment, precision
173
Process of chromosome catching
Initial capture is usually lateral/side attachment
Motors move the caught chromosome towards the pole
As it gets closer to the MTOC, MT density increases, and eventually a good attachment directly on the front of the kinetochore forms
Lateral attachments destabilize, front-on attachments stabilize
Eventually opposite end MTs attach, forming the bipolar attachments
174
How did we learn that sans kinetochores, chromosome arms go towards the + ends away from the MTOCs?
Also answers how did we learn that chromosome arms with kinetochores go towards the - end towards the MTOCs
Experiment using a laser focused through a microscope lens to cut chromosomes moving on a spindle pole towards an MTOC
175
Without kinetochores, in what direction do chromosome arms go?
Towards the + end, away from the MTOCs
176
With kinetochores, in what direction do the chromosome arms go?
Towards the - end, towards the MTOCS
Happens due to coordinated MT depolymerization
177
Why are MTs more stable when they are localized to/near the chromosomes?
B/c of Ran-GTP
Because Ran-GEF is bound to chromatin, Ran-GTP is regionally elevated, even when the nuclear envelope has broken down
Ran-GTP disassembles import complexes and releases cargo from importin.
XCTK2 (a crosslinking kinesin) and TPX2 (a MT nucleation factor) both bind to importin. Ran-GTP releases them near the chromosomes, allowing their concentrations to accumulate locally to the chromosomes, so they can stabilize MTs near the chromatin
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Where is Ran-GEF found?
Bound to chromatin
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Is Ran-GTP still regionally elevated near DNA even when the nuclear envelope has broken down?
Yes
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XCTK2
A crosslinking kinesin
Binds to importin, released by Ran-GTP near the chromosomes, concentrations accumulate locally to the chromatin
XCTK2-MT binding is spatially regulated by Ran-GTP
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TPX2
An MT nucleation factor
Binds to importin, released by Ran-GTP near the chromosomes, concentrations accumulate locally to the chromatin
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High Ran-GTP levels
High MT binding, facilitates crosslinking. Slow turnover
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Decreasing/medium Ran-GTP levels
Importin binding to XCTK2's tail inhibits MT binding
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Low Ran-GTP
Tail MT binding is inhibited. Fast turnover
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Do kinetochore MTs continue to undergo treadmilling (MT flux) even with "end-on" bipolar attachments?
YES
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How does the + end of the MT remain attached to the kinetochore while it's adding new tubulin heterodimers (treadmilling/flux)?
The MT + end is attached to the kinetochore via Ndc80 (the pincher claw basket)
Individual Ndc80s detach and reattach as the MT + end grows and shrinks, as new heterodimers are lost and gained
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What would happen to a cell with loss of function mutation in its Ndc80s?
MTs would essentially be blocked from attaching to kinetochores
Fatal mutation, DNA wouldn't be properly sorted/organized into daughter cells
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Relative stability of free MT ends
Least stable
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Relative stability of monopolar MT attachments
Medium stability
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Relative stability of bipolar MT attachments (with tension!)
Most stable
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Kinetochore-MT attachments are regulated by tension. But how is the tension sensed?
The kinetochore has layers. Aurora B kinase is bound to an inner kinetochore layer, stretches out towards the outer kinetochore.
Chromosomes that are NOT under tension have the kinase regions of Aurora B hanging out around Ndc80, keeps Ndc80 phosphorylated, reducing MT binding affinity
Once the chromosome is bi-oriented (under tension), the tension force is enough to increase the distance between Aurora B's kinase head and the Ndc80s in the outer layer, preventing Ndc80 phosphorylation and increasing MT binding affinity
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General rule: in what direction do kinesins move?
Towards the + end
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General rule: in what direction do dyneins move?
Towards the - end
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Anaphase A
Sister chromatids move apart to poles via kinetochore motors and MT shortening/depolymerization
Chromatid cohesins release
For the chromosomes to separate, the cohesive forces must be degraded (via APC/C + Cdc20)
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Anaphase B
Poles stretch apart, interpolar MTs slide apart via bipolar + end directed kinesin motors
Astral MTs shorten/depolymerize
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Why is the metaphase to anaphase transition irreversible?
Because it involves E3 ubiquitin ligases and protein degradation
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What specifically does APC/C + Cdc20 do?
Marks securin for destruction
Securin is normally bound to separase, inhibiting it. When securin is degraded, separase is free to cut both cohesin's Scc1 subunit and chromokinesin (kinesin-4,10)
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What does too early anaphase cause?
Chromatid segregation errors
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Separase
Cleaves both chromokinesin (kinesin-4,10) and cohesin's Scc1 subunit
Normally bound and inhibited by securin. Is released and active when securin is degraded by APC/C + Cdc20
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Mitotic Checkpoint Complex (MCC)
4 subunits: BUB3, BUBR1, MAD2, and Cdc20
Associates with and binds to kinetochores.
Because Cdc20 is part of its complex, the MCC prevents APC/C from being active until chromosomes are properly aligned at the metaphase plate
After MT attachment to kinetochores, dyneins carry MCCs away towards the spindle poles. This frees Cdc20 to join APC/C, this is how the anaphase checkpoint is passed through
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Anaphase checkpoint
APC/C doesn't have its Cdc20 subunit until chromosomes are properly aligned at the metaphase plate
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To achieve Anaphase A (aka for chromosomes to move towards the spindle poles), what must happen?
Chromokinesins must be suppressed and cohesins must be clipped
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To achieve Anaphase B (aka for spindle poles to separate), what must happen?
Slide-apart motors must be ON
Slide-together motors must be OFF
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True or false: APC/C + Cdh1 indirectly promotes cyclin destruction and facilitates the exit of mitosis
False. APC/C + Cdh1 DIRECTLY promotes cyclin destruction and facilitates the exit of mitosis
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Telophase
The beginning of the biological reset
M-phase cyclins are destroyed, M-CDKs deactivated
Chromosomes decondense
Nuclear envelope reforms, lamins are dephosphorylated
Spindles disassemble
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Meiosis
A single round of DNA replication, followed by two rounds of chromosome segregation and divison
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Process of Meiosis
Meiosis I:
Prophase I
Metaphase I
Anaphase I
Telophase I
Meiosis II:
Metaphase II
Anaphase II
Telophase II
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What are the two ways genetic diversity is generated in meiosis and when do they occur?
Crossing-over recombination occurs during meiotic prophase
Independent assortment of chromosomes occurs in Meiosis I
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What are the two broad categories of genetic disorders/syndromes
1. Alteration of a specific gene's DNA sequence, resulting in an altered amino acid sequence and a new distinct disease-causing allele
2. Errors in meiotic chromosome segregation. These errors can happen in either division in Meiosis I or II. Such errors lead to gametes with abnormal ploidy counts. This type of disorder is almost always severe
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Timing and cell division patterns in oocytes
Meiosis I occurs during oocyte maturation, during gestation/fetal development
Meiosis II occurs only after fertilization
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Timing and cell division patterns in sperm
Mitotic and meiotic divisions occur in adult testes
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Rate of human embryo loss pre-implantation
10-40%
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Total human embryo loss from fertilization to birth
40-60%
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KASH5
Spans the nuclear envelope, reaches out to the cytoplasm where it interacts with cytosolic motor proteins dynein and myosin
The dynein and myosin help manipulate chromosome organization mid-meiotic prophase I, like marionette puppetry
This process is conserved, found in many organisms
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At what stage do human oocytes arrest?
In diplotene, a stage in meiotic prophase before the fetus's birth
In diplotene, the synaptonemal complexes have just disassembled, so the only thing holding the homologs together are the repaired dsDNA breaks surrounded by cohesins
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Chiasmata
The bridge that bolds resolved recombinant dsDNA breaks together
Keeps homologous chromosomes connected in meiotic metaphase I, recombination and the resulting chiasmata necessary for meiosis
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3 differences in oocyte and sperm cell meiosis
1. Oocyte meiosis yields one functional gamete and three polar bodies, spermatogenesis yields four functional gametes
2. Oocyte chromosomes are segregated via unique acentriolar anastral spindles, no astral MTs involved
3. During spermatogenesis, each new generation of gamete divisions is connected by intracellular bridges
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How does oocyte meiosis yield one functional gamete and three polar bodies?
It's advantageous for the egg to be as big as possible
The other half of the chromos not used in the functional gamete are thrown away in a polar body
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Describe the intracellular bridges seen connecting each generation of sperm gametes together
Bridge connections occur in mitosis
Later primary spermatocytes undergo meiosis, and those new generations are also held together with intracellular bridges
All chromosomes are in cytoplasmic connection until almost the very end, the final step is the transition into individual cells
