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BIO 317 > 11 - Second Messenger Signaling in Neuronal Development I > Flashcards

11 - Second Messenger Signaling in Neuronal Development I Flashcards

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1
Q

What experiments were done to show that epithelial cells could differentiate alone?

A

In vitro models with manipulation of LKB1

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2
Q

What cells were used to show that epithelial cells could differentiate alone?

A

Intestinal epithelial cells

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3
Q

Why were intestinal epithelial cells used to show that epithelial cells could differentiate alone?

A

Intestinal epithelial cells did not endogenously have LKB1

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4
Q

What were the control and experimental groups of the experiments that showed that epithelial cells could differentiate alone?

A

Control: No LKB1
Experimental: Overexpression of LKB1

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5
Q

In the experiments that showed that epithelial cells could differentiate alone, what was measured and how?

A

The actin cytoskeleton was tracked using a fluorescent label

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6
Q

Why was actin targeted for the experiments that showed that epithelial cells could differentiate alone?

A

Actin is used to create the microvillae structure on the apical domain of the cell

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7
Q

What did LKB1 do to intestinal epithelial cells (in the in vitro experiments)?

A

Causes actin cytoskeleton to localize to the apical domain to form the microvillae structure

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8
Q

What did the time course experiment of intestinal epithelial cells with overexpressed LKB1 show?

A

Over time, the actin cytoskeleton relocated to the apical domain

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9
Q

What is the structure of the actin cytoskeleton in intestinal epithelial cells without LKB1?

A

Uniformly distributed

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10
Q

How long does cytoskeletal reorganization take in intestinal epithelial cells with overexpressed LKB1?

A

24 hours

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11
Q

True or false: there are many processes needed for epithelial cells to polarize

A

True: each of these can be investigated and tracked in experiments

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12
Q

What does ZO1 do?

A

Act as a marker or tight junctions

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13
Q

How was ZO1 used to study epithelial polarization?

A

Can use ZO1 to see how tight junctions change when LKB1 is added to the system

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14
Q

What happened when LKB1 and ZO1 was added to epithelial cells in experiments?

A

The LKB1 caused the ZO1 (tight junctions) to localize

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15
Q

How can cell surface receptors be tracked in cell polarization studies?

A

By using specific markers for a certain domain (a set of markers for apical domain, and another set of markers for basolateral domain)

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16
Q

When happened when cell surface markers were used in conjunction with LKB1 in epithelial cell studies?

A

The markers localized to their respective regions

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17
Q

What is downstream of LKB1?

A

A large family of kinases, and the PAR proteins

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18
Q

What does LKB1 do in neuronal development?

A

Acts as a signal for axon initiation

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19
Q

What is the model system to study LKB1 on axonal development?

A

In vitro hippocampal neurons (isolated)

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20
Q

What is the ultimate goal of a researcher studying neuronal development?

A

Understand how development happens in vivo

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21
Q

True or false: in vivo studies are easier than in vitro studies

A

False: in vivo studies are significantly more expensive and difficult

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22
Q

Why do researchers use in vitro models, when their ultimate goal is to understand development in vivo?

A

Because they can do robust experimentation in a simpler system

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23
Q

What is the simplest experiment that can be done?

A

“Go and take a look”

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24
Q

What does a “go and take a look” study refer to?

A

Observing where certain proteins are localized within the cell

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25
Q

What was shown in observational studies of neuronal development?

A

LKB1 localized to the axons of these cells

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26
Q

How can proteins be labeled to be useful in experiments?

A

By using specific antibodies to tag a specific protein

27
Q

What happens when LKB1 is overexpressed in neurons?

A

Multiple axons form

28
Q

In control conditions, how many axons does each neuron have?

A

One

29
Q

What happens when LKB1 is down-regulated in neurons?

A

Many cells form no axons

30
Q

How was LKB1 down-regulated in experiments studying axonal development?

A

By using siRNA

31
Q

What happens to the neurites when LKB1 was down-regulated?

A

They continued to grow, but did not polarize into an axon

32
Q

True or false: to study LKB1 in vivo, LKB1 needed to be knocked out in the entire organism

A

False: it needed to be localized to the brain

33
Q

How come LKB1 couldn’t be knocked out in the entire organism?

A

This would lead to embryonic death (before neuronal development start)

34
Q

How did researchers get around the issue of knocking out LKB1 in the entire organism?

A

By only knocking out LKB1 in the neurons of the brain, and not the rest of the organism

35
Q

What happened when LKB1 was knocked out in vivo?

A

There were almost no axons present in the brain slices of the cortex

36
Q

True or false: LKB1 is a crucial mediator in both epithelial and neuronal cells

A

True: experiments in both of these cells show how important LKB1 is for cell polarization

37
Q

What is upstream of PAR-1?

A

LKB1

38
Q

What is SAD-A and SAD-B?

A

Mammalian homologs of PAR-1

39
Q

What happened in neurons when PAR-1 was knocked out?

A

Continuous growth of neurites, without the differentiation of becoming an axon

40
Q

Considering that A is important for axonal development, and B is an upstream activator of A, what questions should be considered regarding this relationship?

A
  1. Over-expression of which protein might rescue the deletion of the other?
  2. Does it matter at what time during neuronal development the protein is over-expressed or deleted to affect axon development?
  3. How will the deletion of A or B affect dendrite development?
41
Q

How does LKB1 become activated?

A

It is phosphorylated by PKA

42
Q

How does PKA become activated?

A

By high levels of cAMP expression

43
Q

How can cAMP levels be elevated?

A

By activating AC, or inhibiting PDE

44
Q

What gets phosphorylated on LKB1 to activate it?

A

Ser-431

45
Q

What does LKB1 phosphorylate?

A

PAR-1

46
Q

How was it determined that Ser-431 could be a phosphorylation site for PKA?

A

Based on observation of surrounding amino acid sequence

47
Q

True or false: cAMP is a protein

A

False: it is a small molecule

48
Q

What are the targets of cAMP?

A
  1. PKA
  2. Cyclic nucleotide-gated ion channels
  3. EPACs
49
Q

What does EPAC stand for?

A

Exchange protein activated by cAMP

50
Q

What technique was used to study PKA effects on LKB1?

A

Western blot

51
Q

What did the western blot do in the experiments that studied PKA effects on LKB1?

A

Measured the levels of phosphorylated LKB1 (antibody tags)

52
Q

In the studies of PKA and LKB1, what were the western blots on the control cells and why?

A

Low levels of phosphorylated LKB1, because the system was not stimulated

53
Q

In the studies of PKA and LKB1, what were the western blots on the cells with just forskolin and why?

A

High levels of phosphorylated LKB1, because PKA was stimulated by the increase in cAMP

54
Q

In the studies of PKA and LKB1, what were the western blots of the cells with an alanine substitute and why?

A

Low levels of phosphorylated LKB1, because the phosphorylatable serine (431) could not be phosphorylated by PKA when it was replaced with alanine

55
Q

What experiments can be done to show that Ser-431 is necessary for activation?

A

Mutate it to an alanine, and observe the effects

56
Q

What was the phenotype of the axon when it expressed LKB1 with a mutated Ser -> Ala change?

A

Abnormal phenotype of many growing neurites with no axon

57
Q

What experiments can be done to show the role of cAMP in axonal formation?

A

Use a patterned substrate to selectively expose cAMP to different parts of the neuron

58
Q

How was cAMP selectively presented to the neuron?

A

By using a patterned substrate (strips of cAMP)

59
Q

True or false: to study the effect of cAMP on neuronal development, pure cAMP was added to different regions of the neuron

A

False: the cAMP needed to be modified to be membrane soluble

60
Q

Why was membrane soluble cAMP needed to study its role on axon formation?

A

Because cAMP could be selectively presented on the petri dish outside of the cell, and so it had to move inside the cell through the cell membrane

61
Q

How does localized cAMP affect the neuron phenotype?

A

It induces axon formation in the places where cAMP is localized

62
Q

Where is LKB1 localized during neuronal development?

A

In the axon (one single neurite)

63
Q

True or false: LKB1 is indicative of axonal fate

A

True: the neurite that is localized with LKB1 is marked to become the axon

64
Q

Besides LKB1, what protein localizes in the axon during neural development?

A

Kinesin-1 (after jumping around the cell)

BIO 317 (27 decks)

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