14 - Signaling Mechanisms Regulating Cytoskeletal Dynamics II

Question 1

What questions can be asked about neuronal development?

Answer 1

1. Why only one axon?
2. Is the neurite that will become the axon pre-chosen?
3. Do the different signaling pathways engage in important crosstalk?
4. Is this process based on physical localization, or activation localization?

Question 2

What are the different branches (signaling pathways) in axonal development?

Answer 2

The cAMP branch, and the PI3K branch

Question 3

Is it likely that the signaling determinants involved in axonal development are localized in physical space, or localized in activation?

Answer 3

Localized in activation

Question 4

Why is it more likely that the signaling determinants involved in axonal development are localized in activation?

Answer 4

These signaling determinants are critical for many different functions throughout the cell, thus it is unlikely that they are localized in space

Question 5

How long does axonal development take?

Answer 5

24-48 hours

Question 6

What question can be asked about the neurite becoming an axon (during the 24-48 time span)?

Answer 6

What is happening to the neurite during this time?

Question 7

What question can be asked regarding that only one axon is formed in a neuron?

Answer 7

Do the other neurites not respond to cAMP or PI3K?

Question 8

True or false: one neurite is unique, which will become the axon

Answer 8

False: at the starts, the neurites are not unique

Question 9

How can it be shown that one particular neurite is not unique in axon formation?

Answer 9

The signaling determinants (such as PI3K) for axonal development “jump” between the neurites

Question 10

True or false: every neurite has the potential to become an axon

Answer 10

True: it depends on whether it reaches a critical threshold to engage in the positive feedback loop

Question 11

Before axon development, what do the signaling determinants do?

Answer 11

Jump around different neurites in a random pattern

Question 12

What determines whether a particular neurite will become an axon?

Answer 12

Whether it reaches a threshold to engage in the positive feedback loop

Question 13

What happens if a neurite reaches the threshold to engage in the axonal positive feedback loop?

Answer 13

It is a point of no return (that neurite will become the axon)

Question 14

Is a global application or a local application better to understand axonal development?

Answer 14

A local application

Question 15

Why is a local application (as opposed to a global application) better to understand axonal development?

Answer 15

A global application could affect the health of the entire cell, which would give less direct information about axonal development

Question 16

Which is easier to achieve: a global application or a local application?

Answer 16

A global application

Question 17

How is a global application achieved?

Answer 17

By putting the stimulant in the media that the cell is growing in

Question 18

In terms of axonal development, what technologies do scientists want to develop?

Answer 18

How to get a local application (as opposed to a global application)

Question 19

How many neurites are found on a normal cell?

Answer 19

~5

Question 20

What conclusion could be drawn from a global application of a PI3K inhibitor?

Answer 20

PI3K could just prevent growth of neurites in the first place (axonal development could be a side effect of this)

Question 21

Which enzyme sits at the center of the axonal positive feedback loop?

Answer 21

PI3K

Question 22

What was known about the signaling pathways upstream of PI3K?

Answer 22

Ras activated PI3K

Question 23

What class of enzymes can activate PI3K?

Answer 23

Small GTPases (Rac-1, Ras, etc.)

Question 24

What downstream effector of PI3K suggests a positive feedback loop?

Answer 24

Rho GTPases

Question 25

Why does activation of rho GTPases by PI3K suggest a positive feedback loop?

Answer 25

PI3K can be activated by other GTPases, such as Rac-1 and Ras

Question 26

What was not known about the signaling pathway upstream of PI3K?

Answer 26

Whether it could activate Ras, and thus create a positive feedback loop

Question 27

What was the hypothesis proposed about PI3K and Ras?

Answer 27

1. Ras was important for axon formation | 2. Ras and PI3K are connected in a positive feedback loop

Question 28

What assumptions were needed for the hypothesis proposing a positive feedback loop between PI3K and Ras?

Answer 28

1. PI3K is important for axon formation 2. Ras activates PI3K 3. Positive feedback is essential for axon development

Question 29

What is needed to study the hypothesis about a positive feedback loop between PI3K and Ras?

Answer 29

A probe to examine the localized activity of PI3K

Question 30

What probe was used to examine the localized activity of PI3K?

Answer 30

Akt-PH-GFP

Question 31

What is Akt-PH-GFP?

Answer 31

Akt with GFP attached to the PH domain

Question 32

What is the purpose of Akt-PH-GFP?

Answer 32

Use it as a marker of localized PI3K activity

Question 33

Why was Akt-PH-GFP used as a probe for localized PI3K activity?

Answer 33

Akt is a downstream target of PI3K

Question 34

What is a PH domain?

Answer 34

A pleckstrin homology domain, where PIP3 binds

Question 35

What did the experiments with just Akt-PH-GFP show?

Answer 35

That this probe (and consequently PI3K) are localized at the tips of the axons during axonal development

Question 36

What happens when a PI3K inhibitor is added to neurons with Akt-PH-GFP?

Answer 36

There is no localized pattern of active Akt

Question 37

What is laminin?

Answer 37

An extracellular cue known to promote axon development

Question 38

What does laminin do?

Answer 38

Promote axon development

Question 39

What happens if a neurite is exposed to laminin?

Answer 39

It will differentiate into an axon

Question 40

How was laminin presented to cells in vitro?

Answer 40

Glass beads coated with laminin

Question 41

What was the significance of the glass beads coated with laminin?

Answer 41

Ensures that only single neurites are exposed to laminin

Question 42

What were the results of the experiments that used laminin and Akt-PH-GFP?

Answer 42

The Akt was only activated in the neurites that were exposed to the laminin (promoting axon formation)

Question 43

Which proteins are probably highly localized in physical space during axon formation?

Answer 43

Scaffolding proteins (PAR-3 and PAR-6)

Question 44

True or false: axon development is a highly dynamic process

Answer 44

True: there is a lot of proteins moving around

Question 45

What is the significance of axon development being a highly dynamic process?

Answer 45

Scaffolding proteins (PAR-3 and PAR-6) are trying to become localized within the neurite that will become the axon

Question 46

True or false: scaffolding proteins can be predictors of neurite fate

Answer 46

True: the neurite that has the localization of scaffolding proteins is very likely to become the axon

Question 47

What is the consequence of scaffolding proteins localizing to a specific neurite?

Answer 47

There are patches of activity that lead to eventual axon formation

Question 48

How does Ras activate PI3K?

Answer 48

By directly recruiting its catalytic domain

Question 49

What do FRET probes do?

Answer 49

They light up when and where they detect protein activity

Question 50

What FRET probes were used to study axonal development?

Answer 50

Active Ras FRET probes

Question 51

What was found using the FRET Ras probes?

Answer 51

That Ras was active at the tips of the neurites during axon formation

Question 52

True or false: low FRET signals for Ras means that there is little Ras in that area of the cell

Answer 52

False: it means that there is low Ras activity at that part of the cell

Question 53

What was seen with the FRET Ras probes in neurons before axon formation?

Answer 53

Ras was still localized to the tip of the neurite (before the axon was formed)

Question 54

True or false: Ras was found localized in neurites before the axon formed

Answer 54

True: the activity of Ras was localized before axon formation, signaling that this was a determiner of axon fate

Question 55

What evidence was needed to support the hypothesis about PI3K and Ras?

Answer 55

The increase of Ras activity required the activity of the downstream target PI3K

Question 56

If PI3K and Ras are engaged in a positive feedback loop, what assumption can be made?

Answer 56

That they co-localize during axon formation and development

Question 57

What results were shown when cells had both FRET probes for Ras, and Akt-PH-GFP?

Answer 57

Akt (and consequently PI3K) and Ras co-localized during axon formation and development

Question 58

What experiments were done to (directly) show that PI3K and Ras were engaged in a positive feedback loop?

Answer 58

FRET Ras probes were used to track Ras when a PI3K inhibitor and activator were presented to the cell

Question 59

What happened when a FRET Ras probe and an inhibitor of PI3K was used in the cell?

Answer 59

There was declined activity of Ras

Question 60

What happened when a FRET Ras probe and BDNF was used in the cell?

Answer 60

There was increased activity of Ras

Question 61

What is BDNF?

Answer 61

A secreted extracellular factor

Question 62

What does BDNF do?

Answer 62

Activates PI3K, and thus promotes axon formation

Question 63

How does BDNF activate PI3K?

Answer 63

By binding to an RTK, which can activate PI3K

Question 64

What happens if Ras is overexpressed in neurons?

Answer 64

The cell has multiple axons

Question 65

What happens if Ras is knocked out in neurons?

Answer 65

The cell has defective axons

Question 66

True or false: axon development involves multiple positive feedback loops

Answer 66

True: there are positive feedback loops upstream and downstream of PI3K

Question 67

Which enzyme is critical for both of the positive feedback loops in axonal formation?

Answer 67

PI3K

Question 68

What is the purpose of having two positive feedback loops in axonal development?

Answer 68

Have a backup pathway, and amplification of PI3K

Question 69

True or false: PI3K is only involved in axonal development

Answer 69

False: PI3K is seen in many cell functions

Question 70

What kinds of functions is PI3K involved in?

Answer 70

Growth, proliferation, development, survival, cell-cell interaction, trafficking, migration, etc.

Question 71

Why does the cell need complex signaling pathways?

Answer 71

Regulation, specificity, and redundancy