14 - Signaling Mechanisms Regulating Cytoskeletal Dynamics II Flashcards
What questions can be asked about neuronal development?
- Why only one axon?
- Is the neurite that will become the axon pre-chosen?
- Do the different signaling pathways engage in important crosstalk?
- Is this process based on physical localization, or activation localization?
What are the different branches (signaling pathways) in axonal development?
The cAMP branch, and the PI3K branch
Is it likely that the signaling determinants involved in axonal development are localized in physical space, or localized in activation?
Localized in activation
Why is it more likely that the signaling determinants involved in axonal development are localized in activation?
These signaling determinants are critical for many different functions throughout the cell, thus it is unlikely that they are localized in space
How long does axonal development take?
24-48 hours
What question can be asked about the neurite becoming an axon (during the 24-48 time span)?
What is happening to the neurite during this time?
What question can be asked regarding that only one axon is formed in a neuron?
Do the other neurites not respond to cAMP or PI3K?
True or false: one neurite is unique, which will become the axon
False: at the starts, the neurites are not unique
How can it be shown that one particular neurite is not unique in axon formation?
The signaling determinants (such as PI3K) for axonal development “jump” between the neurites
True or false: every neurite has the potential to become an axon
True: it depends on whether it reaches a critical threshold to engage in the positive feedback loop
Before axon development, what do the signaling determinants do?
Jump around different neurites in a random pattern
What determines whether a particular neurite will become an axon?
Whether it reaches a threshold to engage in the positive feedback loop
What happens if a neurite reaches the threshold to engage in the axonal positive feedback loop?
It is a point of no return (that neurite will become the axon)
Is a global application or a local application better to understand axonal development?
A local application
Why is a local application (as opposed to a global application) better to understand axonal development?
A global application could affect the health of the entire cell, which would give less direct information about axonal development
Which is easier to achieve: a global application or a local application?
A global application
How is a global application achieved?
By putting the stimulant in the media that the cell is growing in
In terms of axonal development, what technologies do scientists want to develop?
How to get a local application (as opposed to a global application)
How many neurites are found on a normal cell?
~5
What conclusion could be drawn from a global application of a PI3K inhibitor?
PI3K could just prevent growth of neurites in the first place (axonal development could be a side effect of this)
Which enzyme sits at the center of the axonal positive feedback loop?
PI3K
What was known about the signaling pathways upstream of PI3K?
Ras activated PI3K
What class of enzymes can activate PI3K?
Small GTPases (Rac-1, Ras, etc.)
What downstream effector of PI3K suggests a positive feedback loop?
Rho GTPases
Why does activation of rho GTPases by PI3K suggest a positive feedback loop?
PI3K can be activated by other GTPases, such as Rac-1 and Ras
What was not known about the signaling pathway upstream of PI3K?
Whether it could activate Ras, and thus create a positive feedback loop
What was the hypothesis proposed about PI3K and Ras?
- Ras was important for axon formation
2. Ras and PI3K are connected in a positive feedback loop
What assumptions were needed for the hypothesis proposing a positive feedback loop between PI3K and Ras?
- PI3K is important for axon formation
- Ras activates PI3K
- Positive feedback is essential for axon development
What is needed to study the hypothesis about a positive feedback loop between PI3K and Ras?
A probe to examine the localized activity of PI3K
What probe was used to examine the localized activity of PI3K?
Akt-PH-GFP
What is Akt-PH-GFP?
Akt with GFP attached to the PH domain
What is the purpose of Akt-PH-GFP?
Use it as a marker of localized PI3K activity
Why was Akt-PH-GFP used as a probe for localized PI3K activity?
Akt is a downstream target of PI3K
What is a PH domain?
A pleckstrin homology domain, where PIP3 binds
What did the experiments with just Akt-PH-GFP show?
That this probe (and consequently PI3K) are localized at the tips of the axons during axonal development
What happens when a PI3K inhibitor is added to neurons with Akt-PH-GFP?
There is no localized pattern of active Akt
What is laminin?
An extracellular cue known to promote axon development
What does laminin do?
Promote axon development
What happens if a neurite is exposed to laminin?
It will differentiate into an axon
How was laminin presented to cells in vitro?
Glass beads coated with laminin
What was the significance of the glass beads coated with laminin?
Ensures that only single neurites are exposed to laminin
What were the results of the experiments that used laminin and Akt-PH-GFP?
The Akt was only activated in the neurites that were exposed to the laminin (promoting axon formation)
Which proteins are probably highly localized in physical space during axon formation?
Scaffolding proteins (PAR-3 and PAR-6)
True or false: axon development is a highly dynamic process
True: there is a lot of proteins moving around
What is the significance of axon development being a highly dynamic process?
Scaffolding proteins (PAR-3 and PAR-6) are trying to become localized within the neurite that will become the axon
True or false: scaffolding proteins can be predictors of neurite fate
True: the neurite that has the localization of scaffolding proteins is very likely to become the axon
What is the consequence of scaffolding proteins localizing to a specific neurite?
There are patches of activity that lead to eventual axon formation
How does Ras activate PI3K?
By directly recruiting its catalytic domain
What do FRET probes do?
They light up when and where they detect protein activity
What FRET probes were used to study axonal development?
Active Ras FRET probes
What was found using the FRET Ras probes?
That Ras was active at the tips of the neurites during axon formation
True or false: low FRET signals for Ras means that there is little Ras in that area of the cell
False: it means that there is low Ras activity at that part of the cell
What was seen with the FRET Ras probes in neurons before axon formation?
Ras was still localized to the tip of the neurite (before the axon was formed)
True or false: Ras was found localized in neurites before the axon formed
True: the activity of Ras was localized before axon formation, signaling that this was a determiner of axon fate
What evidence was needed to support the hypothesis about PI3K and Ras?
The increase of Ras activity required the activity of the downstream target PI3K
If PI3K and Ras are engaged in a positive feedback loop, what assumption can be made?
That they co-localize during axon formation and development
What results were shown when cells had both FRET probes for Ras, and Akt-PH-GFP?
Akt (and consequently PI3K) and Ras co-localized during axon formation and development
What experiments were done to (directly) show that PI3K and Ras were engaged in a positive feedback loop?
FRET Ras probes were used to track Ras when a PI3K inhibitor and activator were presented to the cell
What happened when a FRET Ras probe and an inhibitor of PI3K was used in the cell?
There was declined activity of Ras
What happened when a FRET Ras probe and BDNF was used in the cell?
There was increased activity of Ras
What is BDNF?
A secreted extracellular factor
What does BDNF do?
Activates PI3K, and thus promotes axon formation
How does BDNF activate PI3K?
By binding to an RTK, which can activate PI3K
What happens if Ras is overexpressed in neurons?
The cell has multiple axons
What happens if Ras is knocked out in neurons?
The cell has defective axons
True or false: axon development involves multiple positive feedback loops
True: there are positive feedback loops upstream and downstream of PI3K
Which enzyme is critical for both of the positive feedback loops in axonal formation?
PI3K
What is the purpose of having two positive feedback loops in axonal development?
Have a backup pathway, and amplification of PI3K
True or false: PI3K is only involved in axonal development
False: PI3K is seen in many cell functions
What kinds of functions is PI3K involved in?
Growth, proliferation, development, survival, cell-cell interaction, trafficking, migration, etc.
Why does the cell need complex signaling pathways?
Regulation, specificity, and redundancy
