20 - Signaling in Time and Space Flashcards
1
Q
True or false: simple motifs can display complex dynamics
A
True: these motifs can be arranged into loops and self-regulation
2
Q
True or false: segregation can lead to specificity
A
True: segregating signaling determinants can increase specificity
3
Q
How can diffusion alter the rate of a signaling pathways?
A
Diffusion of substrates into different regions (kinases and phosphatases) can alter the rate of the signaling pathway
4
Q
What happens when activated Erk is added to cells?
A
It can do some nerve-y things, but cannot mimic the full effects of NGF
5
Q
What is the conclusion of adding activated Erk to cells?
A
NGF has branched signaling, where it doesn’t all go through Erk
6
Q
What is another name for MAP kinase kinase kinase?
A
Raf
7
Q
What is another name for MAP kinase kinase?
A
MEK
8
Q
What is another name for MAP kinase?
A
Erk
9
Q
What is another name for Raf?
A
MAP kinase kinase kinase
10
Q
What is another name for MEK?
A
MAP kinase kinase
11
Q
What is another name for Erk?
A
MAP kinase
12
Q
True or false: different aspects of NGF go through different parts of the pathway
A
True: these effects are mediated in different stages of the pathway
13
Q
Which part of the Ras pathway is responsible for the action potential?
A
The receptor
14
Q
Which part of the Ras pathway is responsible for survival?
A
The adaptors and Ras
15
Q
Which part of the Ras pathway is responsible for cell division?
A
Ras
16
Q
Which part of the Ras pathway is responsible for neurites?
A
Erk
17
Q
Which part of the Ras pathway is responsible for neurotransmitter synthesis?
A
Erk
18
Q
What is the effect of the receptor in the Ras pathway?
A
The action potential
19
Q
What is the effect of the adaptors in the Ras pathway?
A
Survival
20
Q
What is the effect of Ras in the Ras pathway?
A
Cell survival and division
21
Q
What is the effect of Erk in the Ras pathway?
A
Neurotransmitter synthesis and neurites
22
Q
How can the steps of the Ras pathway be tested to tease out their effects?
A
Through the use of activated/inhibited forms for each step of the pathway
23
Q
What is one of the alternative pathways found at the same p-Tyr of TrkA for NGF (not PI3K)?
A
Signaling through the Rap/B-raf pathway (which goes into the Erk pathway)
24
Q
What is one of the alternative pathways found at the same p-Tyr of TrkA for NGF (not B-raf)?
A
Signaling through PI3K and Akt
25
What determines which signaling pathway will be activated by a p-Tyr?
Preferential binding and competition
26
What is one alternative pathway found at the different p-Tyr of TrkA for NGF?
The PLC-gamma / PKC pathway
27
True or false: gene expression is constant over time
False: there are early genes and late genes
28
Generally, what do early genes do?
Help transcribe later genes
29
True or false: gene transcription can cluster together over time
True: they can travel and cluster together over time for temporal resolution
30
True or false: there are functional effects to the timing of gene regulation
True: they can lead to varied phenotypes happening at precise times
31
What happens when NGF is added to PC12 cells?
They get neuronal like processes
32
What happens when EGF is added to PC12 cells?
They undergo rapid division
33
What types of changes does NGF lead to?
Long term changes
34
What type of changes does EGF lead to?
Transient changes
35
Which growth factor leads to long term changes?
NGF
36
Which growth factor leads to transient changes?
EGF
37
True or false: NGF and EGF signal through very different pathways
False: both go through a very similar Ras pathway
38
What does NGF and EGF have in common?
They both signal through a very similar Ras pathway
39
What happens to MEK and ERK under NGF activation?
MEK and ERK activation are sustained
40
What happens to MEK and ERK under EGF activation
MEK and ERK activation occurs very quickly in time
41
Why does MEK activation occur ERK activation?
Because MEK is upstream of ERK
42
Which occurs first: MEK activation or ERK activation?
MEK activation
43
Where does MEK/ERK go under NGF activation?
They translocate to the nucleus
44
How much time is needed for MEK/ERK to translocate to the nucleus?
>15-20 min
45
What is the limit of EGF signaling?
15-20 min
46
How come EGF signaling cannot lead to MEK/ERK translocation into the nucleus?
The limit of EGF signaling is 15-20 min, which is the time needed for MEK/ERK translocation into the nucleus
47
What pathway allows for sustained signaling by NGF?
The Frs2/B-raf pathway
48
What does the Frs2/B-raf pathway do?
Allows for sustained signaling of MEK/ERK by NGF
49
What pathway allows for transient signaling by EGF?
The Ras/Raf pathway
50
What happens if B-raf is knocked out?
There will only be transient signaling
51
How come Frs2 doesn't recognize the p-Tyr in EGFR?
There is a slightly different structure, which prevents binding of Frs2 to EGFR
52
How is EGFR down-regulated?
Receptor-mediated endocytosis via clathrin coated pits
53
What does clathrin do?
Aids in the formation of endocytotic pits
54
What happens to EGFR endosomes?
They get degraded in lysosomes
55
What is the structure of the clathrin coated pits (before forming the endosome)?
Similar to a soccer ball
56
What is a clathrin triskelion?
An individual clathrin molecule that can aggregate to form the clathrin coated pit
57
What does dynamin do?
Pinches off the clathrin coated pit
58
What molecule pinches off the clathrin coated pit?
Dynamin
59
How does dyanmin pinch off the clathrin coated pit?
Through GTP hydrolysis
60
What type of molecules are Rab proteins?
Small GTP binding proteins
61
What do Rabs do?
Determine which endosome pathway (lysosome vs MVB is preferred)
62
What does MVB stand for?
Multivesicular body
63
What type of endocytosis does EGFR go through?
Clathrin dependent endocytosis
64
What type of endocytosis does Trk go through?
Clathrin independent endocytosis
65
Where is Ras found in the cell?
At the cell surface
66
Where is Rap found in the cell?
At the endosomes
67
Where is B-raf found in the cell?
At the endosomes
68
What does Rab7 do?
Brings endosome into the lysosome
69
Would Rab7 be expected in EGFR endosomes or Trk endosomes?
EGFR endosomes, since it signals for degradation
70
How does the MVB of Trk lead to sustained signaling?
Through stabilization and retrograde transport to the cell body
71
What does Rab5 and Rab1 do?
Lead to sustained signaling of the MVB
72
Would Rab5 and Rab1 be expected to remain in EGFR endosomes or Trk endosomes?
Trk endosomes, since it signals for sustained activation of ERK
73
What is the signaling endosome hypothesis?
Endosomes were retrogradely transported to the cell body for signaling
74
True or false: the signaling endosome hypothesis is fairly old
False: it is a fairly recent hypothesis
75
Before the signaling endosome hypothesis, what was the prevailing function of endosomes?
Degradation and recycling (not signaling)
76
How does NGF binding to TrkA in the axon lead to sustained ERK signaling in the nucleus?
The endosome is transported from the axon to the cell body
77
What happens when endocytosis of EGFR is blocked?
The cell will experience prolonged EGF signaling
78
How can endocytosis of EGFR be blocked?
By blocking dynamin
79
How come blocking dynamin prolongs the EGF response?
The receptor stays at the cell surface, and is not down regulated by endocytosis
80
What is the shape of PC12 cells?
Small round tumor cell balls
81
What is the advantage of a signaling endosome?
No longer dependent on ligand binding at the cell surface for signaling, allowing for more efficient long-term signaling
82
True or false: signaling endosomes allow for more efficient long-term signaling
True: they no longer depend on the ligand being constantly presented at the cell surface
83
How does a signaling endosome increase spatial resolution?
It is a mobile signaling unit
84
Why is a signaling endosome used over a random walk?
A signaling endosome takes significantly less time to reach the nucleus compared to a random walk
85
What does Huntingtin do?
Mediates the retrograde transport of BDNF-TrkB signaling endosomes in dendrites of striatal neurons
86
What is the effect of Huntingtin mutation (as seen in Huntingtin's disease)?
It prevents retrograde transport of the BDNF-TrkB signaling endosome, leading to degradation of the striatal neurons
87
How come neurons degenerate without the TrkB-BDNF signaling endosome?
No more sustained survival signaling
88
How are in vivo studies hard to manipulate?
In terms of the context of other cells
89
How are in vitro studies hard to manipulate?
In terms of space (no orientation, random processes)
90
What is a Campenot chamber culture?
A device with a greasy teflon divider, and scratches of collagen in the middle
91
What is the purpose of a Campenot chamber culture?
To separate the cell body from the dendritic and axonal compartments
92
What is the purpose of the collagen scratches in the Campenot chamber culture?
Place to grow the cell
93
What is the purpose of the greasy teflon divier in the Campenot chamber culture?
Prevent material in one compartment to diffuse into another compartment
94
In a Campenot chamber culture, if a substrate was placed in the distal neurite area, and is found in the cell body, what conclusion can you make?
The substrate was endocytoses, and moved retrogradely along the neurite back towards the cell body
95
What is the more recent, sophisticated form of a Campenot chamber culture?
Microfluidic chambers
96
How do microfluidic chambers work?
They maintain a pressure difference to prevent material from diffusing between different compartments
97
In a microfluidic chamber, what ensures that the media given to the distal neurites will not diffuse to the cell body?
A pressure gradient prevents the diffusion of the media (driving force in one direction)
98
What is dynein?
A motor protein that moves vesicles from the periphery to the cell body
99
What happens if dynein is blocked, and NGF is added to the distal neurites?
The cell dies, because it did not receive those survival signals
100
How come blocking dynein causes neuron death, even if NGF is added to the distal neurites?
Dynein is needed to transport the NGF-TrkA endosome back to the cell body for sustained ERK / survival signaling
101
How can middle axons be examined in a microfluidic system?
By placing "dividers" between the cell bodies, the middle axons, and the distal axons
102
How can endosomes be tracked in the cell?
By using domain swapping
103
How does domain swapping work (to track the endosome)?
Can stimulate it with one ligand, and see where the endosome (based on the intracellular domain) goes
104
What is QDot605?
A fluorescent tag that can be added to EGF
105
What happens when neurons are cultured with EGFR and EGF in the microfluidic chamber?
They die (no sustained ERK signaling)
106
What happens when neurons are cultured with EGFR/TrkA chimeric receptor and EGF in the microfluidic chamber?
They survive (sustained ERK signaling)
107
In the microfluidic chamber, where is ETrkB expressed?
All over the cell body
108
In the microfluidic chamber, where is qEGF expressed?
In specific portions of the cell body
109
In the microfluidic chamber, where is pERK expressed?
At the places where qEGF is expressed
110
Why is ETrkB expressed all over the cell body in the microfluidic chamber?
The adenovirus inserts the gene, so it is expressed everywhere
111
Why is pERK expressed only where qEGF is expressed?
The receptor bound to the ligand is needed to lead to pERK signaling
112
Where did the qEGF and pERK come from in the microfluidic chamber?
Retrogradely transported signaling endosomes
113
What is the conclusion of the microfluidic chamber experiments with qEGF?
The Trk receptor needs to bind to a ligand and be retrogradely transported back to the cell body for sustained ERK signaling
114
After 2 hours, what happened to the qEGF/pERK endosomes in the microfluidic chamber?
They formed multivesicular bodies
115
True or false: the EGFR endosome is not retrogradely transported
False: it is also retrogradely transported like the Trk endosome
116
Where are EGFR endosomes retrogradely transported to?
Lysosomes
117
Where are Trk endosomes retrogradely transported to?
MVBs (not lysosomes)
118
What does MVB stand for?
Multivesicular body
119
What do Trk endosome MVBs also contain?
Rab5
120
What type of protein is Rab5?
A small GTPase
121
What does Rab5 do?
Help determine where in the endosomal pathway a particular endosome will go
122
What are the spatial effects of neurotrophin signaling?
They can signal at different places for different effects (dendritic branching, neurite growth, etc.)
123
How can NGF endosomes be used in somal signaling?
They can phosphorylate soma derived TrkA receptors, which signals for them to be transported to the gowth cone
124
What is the signal for soma derived TrkA receptors to be stimulated?
Phosphorylation by NGF endosomes
125
What is kinesin?
A motor protein that moves endosomes from the cell body to the periphery
126
Which motor protein aids in anterograde transport?
Kinesin
127
Which motor protein aids in retrograde transport?
Dynein
