Gradients in axon guidance Flashcards
What attracts commissural axons to the floor plate?
Netrin in the floor plate
What distance (at least) does netrin diffuse over?
0.5mm
Why doesn’t netrin need to act over the whole DV axis of the neural tube?
BMPs are made in the roof plate - repel axons
What is netrin?
SECRETED chemoattractant molecule
What does netrin associate with?
The ECM
Where is netrin expressed?
Along the MIDLINE
What happens to C axons in MAMMALS as they cross the floor plate?
They turn ROSTRALLY (anteriorly)
What are the possibilities of what causes the neurons to turn anteriorly after crossing the floor plate?
1) Long-range diffusible cue
2) Short-range non-diffusible cue
3) Attractant or repellant
How might a short-range non-diffusible cue still make a gradient?
Cells may MAKE the cue in a gradient
How do we distinguish what type of molecule is responsible for the turning of an axon anteriorly after crossing the floor plate?
Culturing large vs small explants:
- If long range diffusible - will matter how long the explant is
- If short range non-diffusible - will not matter how long the explant is
Why does it matter how big the explants are with a long-range diffusible cue?
- May cut off the source or the
Why does it matter how big the explants are with a long-range diffusible cue?
- May cut off the source
- Cue may diffuse INTO THE GEL (away from the neuron)
What IS the molecule type that causes the neurons to turn and go anteriorly?
Long-range diffusible cue
How can distinguish if the LRDC is an attractant or a repellant?
What did this determine?
Use a large explant with neurons anterior, middle and posterior:
- If attractant, expect anterior most axon to turn posteriorly
- If repellant, posterior most axon turns posteriorly
Posterior and middle turned anteriorly
Anterior most turned posteriorly
This determined it was an attractant
So, what molecule causes neurons to turn anteriorly after crossing the floor plate?
What is the name of this molecule?
LONG-RANGE DIFFUSIBLE ATTRACTANT
Wnt4
How does Wnt4 cause neurons to turn anteriorly once crossing the floor plate?
Wnt4 is expressed in a GRADIENT:
- High anteriorly
How do we know it is Wnt4 that causes axons to turn anteriorly once crossing the floor plate?
It is:
1) Expressed in the right place at the right time
2) It is sufficient
3) It is necessary
How do we know Wnt4 is sufficient?
Transfection with Wnt4 turned axons
Control = empty vector
How do we know Wnt4 is necessary?
KO of frizzled3 (receptor for Wnt4) = confused turns post floor plate
Why KO frizzles to test if Wnt4 is necessary?
Hard to KO Wnt4 ONLY in the floor plate
What 2 ways can cells measure a gradient?
1) Change in concentration over time (TEMPORAL DETECTION)
2) Change in concentration across the cell/growth cone (SPATIAL DETECTION)
What is temporal detection for cells?
Cells must compare the amount/gradient of a ligand at 2 different TIME POINTS
What is spatial detection for cells?
Cells compare amount of ligand at 2 different points on the cell surface
What cells use spatial detection?
Temporal detection?
Spatial - slow-moving eukaryotic cells
Bacteria - temporal
What does the ACCURACY of orientation towards a source depend on?
1) ABSOLUTE concentration of the chemoattractant
2) STEEPNESS of the gradient
In regards to concentration, when is the most accurate response of orientation of the growth cone?
Why?
When closest to the KD (when half of the receptors are full)
Greatest capacity to detect even small increases/decreases
What is gradient expressed as?
% change per micron
When do leukocytes orient with accuracy?
ONLY when the gradient is >0.2 (2% change over 10micron diameter)
What happens to orientation when concentration of cue increases?
Move from RANDOM orientation to being orientated TOWARDS the source
Why do larger diameter cells orient more accurately than smaller diameter cells in shallower gradients?
Due to differences they can detect across the width of the cell
Why must there be a mechanism to enhance the range over which gradients can be detected?
ROSOF found that the growth cone could sense a difference in the gradient of ONE in a thousand NGF molecules across its width
Must be a way of amplifying the difference in receptor occupancy
What are the 2 ways that enhance sensitivity of the GC to gradients?
AMPLIFICATION
ADAPTATION
What is amplification?
The LOCAL ENHANCEMENT of the signal together with the INHIBITION of signal reception in other parts of the cell
What happens in the cell when detect a signal (amplification)?
What does this cause?
CLUSTERING of receptors and/or signalling components in the region where the receptors were activated
By TRANSPORTING component from OTHER parts of the cell
Causes inhibition of the signal being detected elsewhere in the cell
What is the evidence of clustering downstream when a signal is received?
Chemotaxis in Dictyostelium
What happens in Dictyostelium when a cue is encountered?
1) Detected by a GPCR (7TM)
2) Activates PI3K - phosphorylates PIP2 –> PIP3
3) PIP3 provides a docking site for pleckstrin homology (PH) domains such as Akt
4) Akt docking creates a localised signalling domain on the membrane
What antagonises PIP3K activity?
How?
PTEN phosphatase
By PIP3 –> PIP2 (opposite to PI3K) - loss of docking site
Why is it important that PI3K and PTENp antagonise each other?
To be able to turn signals off
In order to detect CHANGES
And to ensure that signals are LOCALISED, LIABLE and dependant on CONTINUED external activation
What happens when a chemotaxing cell comes into contact with a chemoattractant? (PTENp, PI3K)
Why is this important?
PTENp and PI3K constantly antagonising eachother and turning on/off
Important to maintain the RESPONSIVENESS of the cell
Where is the PH domain in a chemotaxing cell?
Around the leading edge
What happens to growth cone turning when treated with PI3K inhibitor?
Disrupts growth cone turning
What happens to PH in a growth cone?
Localised to the growth cone
What happens when quantify PH in the growth cone overtime in response to a chemoattractant? Why?
How is the PH quantified?
As a ratio (one side of the GC compared to the other)
Ration FLUCTUATES up and down - constantly renewing the detection system
What happens to vesicles in the axon in response to a cue?
How is this seen?
Vesicles transport towards the cue:
- Using FM1 membrane dye (to visualise vesicles)
- Load GC with calcium and use a light flash to unload calcium from cage
- Vesicles transported towards the calcium source
How may the vesicles be transported to the GC?
By MT as they are closely associated with MT
So, how does amplification give sensitivity to gradients?
1) Receptor clustering
2) Sequestration of components (PH domain clustering, asymmetric vesicle transport)
What is the idea of amplification consistent with?
1) A LIMITING component model
2) Positive feedback pathways (in the establishment of neuronal polarity) - Involves PI3K
How do neurons deal with the fact that eventually receptors will become saturated in response to a guidance cue?
They sensitise and desensitise synaptic transmission
In the synaptic transmission, what does signal saturation lead to?
Desensitisation and RESETTING of thresholds
In the synaptic transmission, what does a fall in signal lead to?
Re sensitisation
Why is it important to desensitise when receptors saturate and re sensitise when signal falls?
GC operate most discriminatively (to make the right decision) when receptors are at Kd (half receptors occupied)
Keep receptors in a range where they are always sensitive to cues - optimal response of receptor
What is the evidence for adaptation in responses to gradients?
What does this suggest?
Xenopus spinal cord neuron turning assay (Mu-Ming Poo):
- Neurons turn in response to netrin
- PULSED netrin forms an EXPONENTIAL gradient in the dish - revealed by fluorescent marker
- Growth cones respond to the gradient of netrin by a ZIG-ZAG trajectory towards the source of netrin (pipette)
Suggests:
- Alternative attractive and repulsive turning
- Cycles of desensitisation and re sensitisation of the growth cone to netrin
How can you test if there is desensitisation of the growth cone as you go up the gradient of netrin?
Should be able to bath apply a low concentration of netrin to get desensitisation and then test with netrin:
- 30 mins after treatment: growth cone didn’t react
- 90 mins after treatment: growth cone now reacts to netrin (resensitise)
What is the re sensitisation of the growth cone dependant on?
MAP kinase activation and local protein synthesis in the GC
Over what distance could growth cone be guided by gradients?
Depends upon the SHAPE of the gradient, which depends on how the gradient is SET up:
1) DIFFUSIBLE molecules (netrin, wnt)
2) NON-DIFFUSABLE molecules (ephrins)
What are the 2 different types of gradient?
1) Point source
2) Substrate-bound
Describe the point source gradient
- Exponential gradient
- Has a THEORETICAL MAXIMUM range of 1mm
What 2 things does a point source depend on?
1) A limited diffusion rate - diffuse quickly = no gradient
2) A mechanism for removing the guiding molecule (a ‘sink’) = otherwise gradient would flatten over time
How is a substrate-bound gradient different to a point source gradient?
- More LINEAR in shape
- Higher theoretical maximum (1cm)
What is the difference between netrins in the mouse and in the chick?
Mouse - 1 netrin:
- NOT a point source
- Also expressed in the VS
Chick - 2 netrins:
- Netrin 1 = point source (FP)
- Netrin 2 = low levels throughout
Where is netrin found in the spinal cord? How?
In the basement membrane
Has a similar structure to laminin - can incorporate into the ECM
Describe Wnt4 expression
Graded along the neural tube but NOT a point source
Is MADE in a gradient
