20 - Signaling in Time and Space Flashcards
True or false: simple motifs can display complex dynamics
True: these motifs can be arranged into loops and self-regulation
True or false: segregation can lead to specificity
True: segregating signaling determinants can increase specificity
How can diffusion alter the rate of a signaling pathways?
Diffusion of substrates into different regions (kinases and phosphatases) can alter the rate of the signaling pathway
What happens when activated Erk is added to cells?
It can do some nerve-y things, but cannot mimic the full effects of NGF
What is the conclusion of adding activated Erk to cells?
NGF has branched signaling, where it doesn’t all go through Erk
What is another name for MAP kinase kinase kinase?
Raf
What is another name for MAP kinase kinase?
MEK
What is another name for MAP kinase?
Erk
What is another name for Raf?
MAP kinase kinase kinase
What is another name for MEK?
MAP kinase kinase
What is another name for Erk?
MAP kinase
True or false: different aspects of NGF go through different parts of the pathway
True: these effects are mediated in different stages of the pathway
Which part of the Ras pathway is responsible for the action potential?
The receptor
Which part of the Ras pathway is responsible for survival?
The adaptors and Ras
Which part of the Ras pathway is responsible for cell division?
Ras
Which part of the Ras pathway is responsible for neurites?
Erk
Which part of the Ras pathway is responsible for neurotransmitter synthesis?
Erk
What is the effect of the receptor in the Ras pathway?
The action potential
What is the effect of the adaptors in the Ras pathway?
Survival
What is the effect of Ras in the Ras pathway?
Cell survival and division
What is the effect of Erk in the Ras pathway?
Neurotransmitter synthesis and neurites
How can the steps of the Ras pathway be tested to tease out their effects?
Through the use of activated/inhibited forms for each step of the pathway
What is one of the alternative pathways found at the same p-Tyr of TrkA for NGF (not PI3K)?
Signaling through the Rap/B-raf pathway (which goes into the Erk pathway)
What is one of the alternative pathways found at the same p-Tyr of TrkA for NGF (not B-raf)?
Signaling through PI3K and Akt
What determines which signaling pathway will be activated by a p-Tyr?
Preferential binding and competition
What is one alternative pathway found at the different p-Tyr of TrkA for NGF?
The PLC-gamma / PKC pathway
True or false: gene expression is constant over time
False: there are early genes and late genes
Generally, what do early genes do?
Help transcribe later genes
True or false: gene transcription can cluster together over time
True: they can travel and cluster together over time for temporal resolution
True or false: there are functional effects to the timing of gene regulation
True: they can lead to varied phenotypes happening at precise times
What happens when NGF is added to PC12 cells?
They get neuronal like processes
What happens when EGF is added to PC12 cells?
They undergo rapid division
What types of changes does NGF lead to?
Long term changes
What type of changes does EGF lead to?
Transient changes
Which growth factor leads to long term changes?
NGF
Which growth factor leads to transient changes?
EGF
True or false: NGF and EGF signal through very different pathways
False: both go through a very similar Ras pathway
What does NGF and EGF have in common?
They both signal through a very similar Ras pathway
What happens to MEK and ERK under NGF activation?
MEK and ERK activation are sustained
What happens to MEK and ERK under EGF activation
MEK and ERK activation occurs very quickly in time
Why does MEK activation occur ERK activation?
Because MEK is upstream of ERK
Which occurs first: MEK activation or ERK activation?
MEK activation
Where does MEK/ERK go under NGF activation?
They translocate to the nucleus
How much time is needed for MEK/ERK to translocate to the nucleus?
> 15-20 min
What is the limit of EGF signaling?
15-20 min
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
What pathway allows for sustained signaling by NGF?
The Frs2/B-raf pathway
What does the Frs2/B-raf pathway do?
Allows for sustained signaling of MEK/ERK by NGF
What pathway allows for transient signaling by EGF?
The Ras/Raf pathway
What happens if B-raf is knocked out?
There will only be transient signaling
How come Frs2 doesn’t recognize the p-Tyr in EGFR?
There is a slightly different structure, which prevents binding of Frs2 to EGFR
How is EGFR down-regulated?
Receptor-mediated endocytosis via clathrin coated pits
What does clathrin do?
Aids in the formation of endocytotic pits
What happens to EGFR endosomes?
They get degraded in lysosomes
What is the structure of the clathrin coated pits (before forming the endosome)?
Similar to a soccer ball
What is a clathrin triskelion?
An individual clathrin molecule that can aggregate to form the clathrin coated pit
What does dynamin do?
Pinches off the clathrin coated pit
What molecule pinches off the clathrin coated pit?
Dynamin
How does dyanmin pinch off the clathrin coated pit?
Through GTP hydrolysis
What type of molecules are Rab proteins?
Small GTP binding proteins
What do Rabs do?
Determine which endosome pathway (lysosome vs MVB is preferred)
What does MVB stand for?
Multivesicular body
What type of endocytosis does EGFR go through?
Clathrin dependent endocytosis
What type of endocytosis does Trk go through?
Clathrin independent endocytosis
Where is Ras found in the cell?
At the cell surface
Where is Rap found in the cell?
At the endosomes
Where is B-raf found in the cell?
At the endosomes
What does Rab7 do?
Brings endosome into the lysosome
Would Rab7 be expected in EGFR endosomes or Trk endosomes?
EGFR endosomes, since it signals for degradation
How does the MVB of Trk lead to sustained signaling?
Through stabilization and retrograde transport to the cell body
What does Rab5 and Rab1 do?
Lead to sustained signaling of the MVB
Would Rab5 and Rab1 be expected to remain in EGFR endosomes or Trk endosomes?
Trk endosomes, since it signals for sustained activation of ERK
What is the signaling endosome hypothesis?
Endosomes were retrogradely transported to the cell body for signaling
True or false: the signaling endosome hypothesis is fairly old
False: it is a fairly recent hypothesis
Before the signaling endosome hypothesis, what was the prevailing function of endosomes?
Degradation and recycling (not signaling)
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
What happens when endocytosis of EGFR is blocked?
The cell will experience prolonged EGF signaling
How can endocytosis of EGFR be blocked?
By blocking dynamin
How come blocking dynamin prolongs the EGF response?
The receptor stays at the cell surface, and is not down regulated by endocytosis
What is the shape of PC12 cells?
Small round tumor cell balls
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
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
How does a signaling endosome increase spatial resolution?
It is a mobile signaling unit
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
What does Huntingtin do?
Mediates the retrograde transport of BDNF-TrkB signaling endosomes in dendrites of striatal neurons
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
How come neurons degenerate without the TrkB-BDNF signaling endosome?
No more sustained survival signaling
How are in vivo studies hard to manipulate?
In terms of the context of other cells
How are in vitro studies hard to manipulate?
In terms of space (no orientation, random processes)
What is a Campenot chamber culture?
A device with a greasy teflon divider, and scratches of collagen in the middle
What is the purpose of a Campenot chamber culture?
To separate the cell body from the dendritic and axonal compartments
What is the purpose of the collagen scratches in the Campenot chamber culture?
Place to grow the cell
What is the purpose of the greasy teflon divier in the Campenot chamber culture?
Prevent material in one compartment to diffuse into another compartment
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
What is the more recent, sophisticated form of a Campenot chamber culture?
Microfluidic chambers
How do microfluidic chambers work?
They maintain a pressure difference to prevent material from diffusing between different compartments
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)
What is dynein?
A motor protein that moves vesicles from the periphery to the cell body
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
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
How can middle axons be examined in a microfluidic system?
By placing “dividers” between the cell bodies, the middle axons, and the distal axons
How can endosomes be tracked in the cell?
By using domain swapping
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
What is QDot605?
A fluorescent tag that can be added to EGF
What happens when neurons are cultured with EGFR and EGF in the microfluidic chamber?
They die (no sustained ERK signaling)
What happens when neurons are cultured with EGFR/TrkA chimeric receptor and EGF in the microfluidic chamber?
They survive (sustained ERK signaling)
In the microfluidic chamber, where is ETrkB expressed?
All over the cell body
In the microfluidic chamber, where is qEGF expressed?
In specific portions of the cell body
In the microfluidic chamber, where is pERK expressed?
At the places where qEGF is expressed
Why is ETrkB expressed all over the cell body in the microfluidic chamber?
The adenovirus inserts the gene, so it is expressed everywhere
Why is pERK expressed only where qEGF is expressed?
The receptor bound to the ligand is needed to lead to pERK signaling
Where did the qEGF and pERK come from in the microfluidic chamber?
Retrogradely transported signaling endosomes
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
After 2 hours, what happened to the qEGF/pERK endosomes in the microfluidic chamber?
They formed multivesicular bodies
True or false: the EGFR endosome is not retrogradely transported
False: it is also retrogradely transported like the Trk endosome
Where are EGFR endosomes retrogradely transported to?
Lysosomes
Where are Trk endosomes retrogradely transported to?
MVBs (not lysosomes)
What does MVB stand for?
Multivesicular body
What do Trk endosome MVBs also contain?
Rab5
What type of protein is Rab5?
A small GTPase
What does Rab5 do?
Help determine where in the endosomal pathway a particular endosome will go
What are the spatial effects of neurotrophin signaling?
They can signal at different places for different effects (dendritic branching, neurite growth, etc.)
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
What is the signal for soma derived TrkA receptors to be stimulated?
Phosphorylation by NGF endosomes
What is kinesin?
A motor protein that moves endosomes from the cell body to the periphery
Which motor protein aids in anterograde transport?
Kinesin
Which motor protein aids in retrograde transport?
Dynein
