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BIO 317 > 14 - Signaling Mechanisms Regulating Cytoskeletal Dynamics II > Flashcards

14 - Signaling Mechanisms Regulating Cytoskeletal Dynamics II Flashcards

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1
Q

What questions can be asked about neuronal development?

A
  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?
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2
Q

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

A

The cAMP branch, and the PI3K branch

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3
Q

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

A

Localized in activation

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4
Q

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

A

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

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5
Q

How long does axonal development take?

A

24-48 hours

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6
Q

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

A

What is happening to the neurite during this time?

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7
Q

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

A

Do the other neurites not respond to cAMP or PI3K?

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8
Q

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

A

False: at the starts, the neurites are not unique

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9
Q

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

A

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

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10
Q

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

A

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

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11
Q

Before axon development, what do the signaling determinants do?

A

Jump around different neurites in a random pattern

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12
Q

What determines whether a particular neurite will become an axon?

A

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

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13
Q

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

A

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

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14
Q

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

A

A local application

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15
Q

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

A

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

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16
Q

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

A

A global application

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17
Q

How is a global application achieved?

A

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

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18
Q

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

A

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

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19
Q

How many neurites are found on a normal cell?

A

~5

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20
Q

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

A

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

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21
Q

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

A

PI3K

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22
Q

What was known about the signaling pathways upstream of PI3K?

A

Ras activated PI3K

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23
Q

What class of enzymes can activate PI3K?

A

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

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24
Q

What downstream effector of PI3K suggests a positive feedback loop?

A

Rho GTPases

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25
Q

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

A

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

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26
Q

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

A

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

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27
Q

What was the hypothesis proposed about PI3K and Ras?

A
  1. Ras was important for axon formation

2. Ras and PI3K are connected in a positive feedback loop

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28
Q

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

A
  1. PI3K is important for axon formation
  2. Ras activates PI3K
  3. Positive feedback is essential for axon development
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29
Q

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

A

A probe to examine the localized activity of PI3K

30
Q

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

A

Akt-PH-GFP

31
Q

What is Akt-PH-GFP?

A

Akt with GFP attached to the PH domain

32
Q

What is the purpose of Akt-PH-GFP?

A

Use it as a marker of localized PI3K activity

33
Q

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

A

Akt is a downstream target of PI3K

34
Q

What is a PH domain?

A

A pleckstrin homology domain, where PIP3 binds

35
Q

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

A

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

36
Q

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

A

There is no localized pattern of active Akt

37
Q

What is laminin?

A

An extracellular cue known to promote axon development

38
Q

What does laminin do?

A

Promote axon development

39
Q

What happens if a neurite is exposed to laminin?

A

It will differentiate into an axon

40
Q

How was laminin presented to cells in vitro?

A

Glass beads coated with laminin

41
Q

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

A

Ensures that only single neurites are exposed to laminin

42
Q

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

A

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

43
Q

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

A

Scaffolding proteins (PAR-3 and PAR-6)

44
Q

True or false: axon development is a highly dynamic process

A

True: there is a lot of proteins moving around

45
Q

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

A

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

46
Q

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

A

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

47
Q

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

A

There are patches of activity that lead to eventual axon formation

48
Q

How does Ras activate PI3K?

A

By directly recruiting its catalytic domain

49
Q

What do FRET probes do?

A

They light up when and where they detect protein activity

50
Q

What FRET probes were used to study axonal development?

A

Active Ras FRET probes

51
Q

What was found using the FRET Ras probes?

A

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

52
Q

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

A

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

53
Q

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

A

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

54
Q

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

A

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

55
Q

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

A

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

56
Q

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

A

That they co-localize during axon formation and development

57
Q

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

A

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

58
Q

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

A

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

59
Q

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

A

There was declined activity of Ras

60
Q

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

A

There was increased activity of Ras

61
Q

What is BDNF?

A

A secreted extracellular factor

62
Q

What does BDNF do?

A

Activates PI3K, and thus promotes axon formation

63
Q

How does BDNF activate PI3K?

A

By binding to an RTK, which can activate PI3K

64
Q

What happens if Ras is overexpressed in neurons?

A

The cell has multiple axons

65
Q

What happens if Ras is knocked out in neurons?

A

The cell has defective axons

66
Q

True or false: axon development involves multiple positive feedback loops

A

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

67
Q

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

A

PI3K

68
Q

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

A

Have a backup pathway, and amplification of PI3K

69
Q

True or false: PI3K is only involved in axonal development

A

False: PI3K is seen in many cell functions

70
Q

What kinds of functions is PI3K involved in?

A

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

71
Q

Why does the cell need complex signaling pathways?

A

Regulation, specificity, and redundancy

BIO 317 (27 decks)

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