11 - Second Messenger Signaling in Neuronal Development I Flashcards
What experiments were done to show that epithelial cells could differentiate alone?
In vitro models with manipulation of LKB1
What cells were used to show that epithelial cells could differentiate alone?
Intestinal epithelial cells
Why were intestinal epithelial cells used to show that epithelial cells could differentiate alone?
Intestinal epithelial cells did not endogenously have LKB1
What were the control and experimental groups of the experiments that showed that epithelial cells could differentiate alone?
Control: No LKB1
Experimental: Overexpression of LKB1
In the experiments that showed that epithelial cells could differentiate alone, what was measured and how?
The actin cytoskeleton was tracked using a fluorescent label
Why was actin targeted for the experiments that showed that epithelial cells could differentiate alone?
Actin is used to create the microvillae structure on the apical domain of the cell
What did LKB1 do to intestinal epithelial cells (in the in vitro experiments)?
Causes actin cytoskeleton to localize to the apical domain to form the microvillae structure
What did the time course experiment of intestinal epithelial cells with overexpressed LKB1 show?
Over time, the actin cytoskeleton relocated to the apical domain
What is the structure of the actin cytoskeleton in intestinal epithelial cells without LKB1?
Uniformly distributed
How long does cytoskeletal reorganization take in intestinal epithelial cells with overexpressed LKB1?
24 hours
True or false: there are many processes needed for epithelial cells to polarize
True: each of these can be investigated and tracked in experiments
What does ZO1 do?
Act as a marker or tight junctions
How was ZO1 used to study epithelial polarization?
Can use ZO1 to see how tight junctions change when LKB1 is added to the system
What happened when LKB1 and ZO1 was added to epithelial cells in experiments?
The LKB1 caused the ZO1 (tight junctions) to localize
How can cell surface receptors be tracked in cell polarization studies?
By using specific markers for a certain domain (a set of markers for apical domain, and another set of markers for basolateral domain)
When happened when cell surface markers were used in conjunction with LKB1 in epithelial cell studies?
The markers localized to their respective regions
What is downstream of LKB1?
A large family of kinases, and the PAR proteins
What does LKB1 do in neuronal development?
Acts as a signal for axon initiation
What is the model system to study LKB1 on axonal development?
In vitro hippocampal neurons (isolated)
What is the ultimate goal of a researcher studying neuronal development?
Understand how development happens in vivo
True or false: in vivo studies are easier than in vitro studies
False: in vivo studies are significantly more expensive and difficult
Why do researchers use in vitro models, when their ultimate goal is to understand development in vivo?
Because they can do robust experimentation in a simpler system
What is the simplest experiment that can be done?
“Go and take a look”
What does a “go and take a look” study refer to?
Observing where certain proteins are localized within the cell
What was shown in observational studies of neuronal development?
LKB1 localized to the axons of these cells
How can proteins be labeled to be useful in experiments?
By using specific antibodies to tag a specific protein
What happens when LKB1 is overexpressed in neurons?
Multiple axons form
In control conditions, how many axons does each neuron have?
One
What happens when LKB1 is down-regulated in neurons?
Many cells form no axons
How was LKB1 down-regulated in experiments studying axonal development?
By using siRNA
What happens to the neurites when LKB1 was down-regulated?
They continued to grow, but did not polarize into an axon
True or false: to study LKB1 in vivo, LKB1 needed to be knocked out in the entire organism
False: it needed to be localized to the brain
How come LKB1 couldn’t be knocked out in the entire organism?
This would lead to embryonic death (before neuronal development start)
How did researchers get around the issue of knocking out LKB1 in the entire organism?
By only knocking out LKB1 in the neurons of the brain, and not the rest of the organism
What happened when LKB1 was knocked out in vivo?
There were almost no axons present in the brain slices of the cortex
True or false: LKB1 is a crucial mediator in both epithelial and neuronal cells
True: experiments in both of these cells show how important LKB1 is for cell polarization
What is upstream of PAR-1?
LKB1
What is SAD-A and SAD-B?
Mammalian homologs of PAR-1
What happened in neurons when PAR-1 was knocked out?
Continuous growth of neurites, without the differentiation of becoming an axon
Considering that A is important for axonal development, and B is an upstream activator of A, what questions should be considered regarding this relationship?
- Over-expression of which protein might rescue the deletion of the other?
- Does it matter at what time during neuronal development the protein is over-expressed or deleted to affect axon development?
- How will the deletion of A or B affect dendrite development?
How does LKB1 become activated?
It is phosphorylated by PKA
How does PKA become activated?
By high levels of cAMP expression
How can cAMP levels be elevated?
By activating AC, or inhibiting PDE
What gets phosphorylated on LKB1 to activate it?
Ser-431
What does LKB1 phosphorylate?
PAR-1
How was it determined that Ser-431 could be a phosphorylation site for PKA?
Based on observation of surrounding amino acid sequence
True or false: cAMP is a protein
False: it is a small molecule
What are the targets of cAMP?
- PKA
- Cyclic nucleotide-gated ion channels
- EPACs
What does EPAC stand for?
Exchange protein activated by cAMP
What technique was used to study PKA effects on LKB1?
Western blot
What did the western blot do in the experiments that studied PKA effects on LKB1?
Measured the levels of phosphorylated LKB1 (antibody tags)
In the studies of PKA and LKB1, what were the western blots on the control cells and why?
Low levels of phosphorylated LKB1, because the system was not stimulated
In the studies of PKA and LKB1, what were the western blots on the cells with just forskolin and why?
High levels of phosphorylated LKB1, because PKA was stimulated by the increase in cAMP
In the studies of PKA and LKB1, what were the western blots of the cells with an alanine substitute and why?
Low levels of phosphorylated LKB1, because the phosphorylatable serine (431) could not be phosphorylated by PKA when it was replaced with alanine
What experiments can be done to show that Ser-431 is necessary for activation?
Mutate it to an alanine, and observe the effects
What was the phenotype of the axon when it expressed LKB1 with a mutated Ser -> Ala change?
Abnormal phenotype of many growing neurites with no axon
What experiments can be done to show the role of cAMP in axonal formation?
Use a patterned substrate to selectively expose cAMP to different parts of the neuron
How was cAMP selectively presented to the neuron?
By using a patterned substrate (strips of cAMP)
True or false: to study the effect of cAMP on neuronal development, pure cAMP was added to different regions of the neuron
False: the cAMP needed to be modified to be membrane soluble
Why was membrane soluble cAMP needed to study its role on axon formation?
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
How does localized cAMP affect the neuron phenotype?
It induces axon formation in the places where cAMP is localized
Where is LKB1 localized during neuronal development?
In the axon (one single neurite)
True or false: LKB1 is indicative of axonal fate
True: the neurite that is localized with LKB1 is marked to become the axon
Besides LKB1, what protein localizes in the axon during neural development?
Kinesin-1 (after jumping around the cell)
