Lecture 11 Flashcards
Contact of the growth cone with a cue is thought to first arrest actin before dragging the microtubules in T or F
F - Microtubules accumulate to a guidance cue before actin responds there is still retrograde actin transport at this point. Hence the idea that cue first arrests the actin before dragging microtubules is wrong and the microtubules in fact go in first. The first response of the growth cone is thus to uncouple the microtubules from F-actin. This is then followed by filopodial invasion by microtubules
What is thought to be the purpose of initial microtubule invasion following cue contact
Invading microtubules may bring important signal transduction elements and/or factors required for membrane protrusion into area of contact with cue. This includes RhoGEFs which are known to associate with microtubule + end Tracking Proteins (+TIPs). Other componets such as monomeric actin and collapsin response mediator protein (CRMP) may also be recruited
What is the name of the molecule that is secreted by the floor plate to direct axon growth towards it
Netrin
What type of axon is guided by the floor plate
Commissural sensory relay neurons – interneurons that carry sensory information from the dorsal root ganglia up to targets in the thalamus
What is the name of the chemorepellent secreted by the roof plate to direct axon growth away from it
BMP7
Chemorepellents can induce growth cone collapse T or F
T
Give examples of early patterning molecules that are later used to direct axon growth
BMP7 initially involved in dorsalisation of the neural tube later directs commissural sensory relay neurons away from the roof plate. Shh used to specify ventral neuronal fates in the embryo is also sued to guide neurons to the floor plate
What are the major limitations that guidance signals need to overcome
Diffusion/transport barriers the extracellular matrix removal from the sink and saturation of the receptors
What is behaviour is exhibited by the commissural axons of mammals once they cross the floor plate
They turn and project rostrally
What two possibilities were proposed for how the commissural axons changed behaviour after crossing the midline
Possibility of an A-P gradient of either an attractant or a repellent acting as a long range diffusible cue. Or similarly the action of a short range non-diffusible cue
Explain how the nature of the signals governing the directional change of commissural axons was determined by experimentation
Different size explants of spinal cord tissue were taken and compared by culturing in a collagen matrix. If you take a large spinal cord explant a long-range diffusible cue would take longer to diffuse out than it would if it was a smaller explant. By contrast a short range non-diffusible cue would remain stable in both large and small spinal cord explants. With this rationale the two spinal cord explants were taken and prepared in an open book configuration and injected with DiI. The lipophilic DiI was taken up into the cells and transported along the axon. In the larger explants the anterior/rostral turn made by the commissural axons as they crossed the floor plate could be seen however this is consistent with either a long or short-range cue. In contrast in the smaller explants it was found that the axons got knotted or stalled at the floor plate. This indicates that it’s a long-range diffusible cue and that the cue is diffusing out in the small explant and can’t cause commissural axons to change direction. This is because the diffusion of the molecule will be much faster in the collagen matrix than in the explant itself
Describe how the nature of this long-range diffusible signal governing the change in direction of commissural axons was determined
DiI was injected into a spinal cord explant anteriorly centrally and posteriorly. Unusually this revealed that the posterior and middle axons turned anteriorly as would be expected for a chemoattractive signal however the anterior axons turned posteriorly. If the signal was a repellent secretion from a posterior source would lead to the diffusion of the molecule out of the explant near the edges hence shifting the highest point of the gradient towards the middle. If the signal was an attractant then the secretion from an anterior source would again lead to the diffusion of the molecule out of the explant near the edges. This would result in the highest point of the gradient shifting towards the middle too. Its this shift of the highest point of the gradient seen in vitro that accounts for the fact that the anteriormost axons turn caudally after crossing the floor plate and hence the signal is a chemoattractant
What is the nature of the chemoattractant molecule responsible for the anterior turning of the commissural axons after cross the floor plate
Wnt4 is expressed in an anterior-posterior gradient in the floor plate and acts as the chemoattractant signal responsible for the commissural axons turning anteriorly
How was the nature of the chemoattractant signal responsible for the change in commissural axon direction determined
Wnt4 was found to attract axons in vitro. In addition frizzled 3 knockout results in confusion when the commissural axons reach the floor plate
Over what sort of range were the chemoattractant signals directing commissural axon direction found to be acting
Distances of at least several millimetres
What two methods are there for detecting a gradient
Temporal detection - change in concentration over time where cells compare the amount of ligand at two different time points. Spatial detection – where cells compare the amount of ligand at different points on its surface
Which type of gradient interpretation mechanism is used by neurons
Spatial detection
At what point is the accuracy of orientation of a growth cone towards its source most accurate
The absolute concentration of the chemoattractant is most accurate closest to the KD of the receptor
What level of gradient is most suitable for growth cones to respond accurately to
2% change in ligand concentration over 10µm (0.2% per micron)
Larger diameter cells orient more accurately that small cells in shallow gradients T or F
T
Amplification mechanisms are required for a growth cone to respond accurately to small differences in receptor occupancies. What is required of these amplification mechanisms
Amplification requires a local enhancement of the signal together with inhibition of signal reception in other parts of the cell
How is it suggested that amplification of guidance cues is achieved
Clustering of receptors and/or signalling components in regions where receptors activated by transporting components from other parts of the cell
Describe how signal amplification occurs in Dictyostelium
Chemoattractants in leukocytes and Dictyostelium activate PI3-K via a GPCR. This chemoattractant is detected by a GPCR which activates PI3-K which in-turn phosphorylates PIP2 to PIP3 . This provides a docking site for proteins such as Akt which contains PH domains. This creates a localised signalling domain on the membrane. PI3-K activity is antagonised by PTEN phosphatase which acts to ensure signals are highly localised and labile dependent on continued external activation
Amplification mechanisms involve receptor clustering and sequestration of components T or F
T
As well as amplification of guidance cues describe the need for adaptation by the growth cone
If growth cones operate most discriminatively when the chemoattractant concentration is closest to the receptor KD then the receptors will saturate as concentration increases. Even the shallowest gradients required to turn cells (0.2% per µm) will result in 90% receptor occupancy just 250µm from the optimal ligand concentration. Hence a way to modulate the response of the growth cone to different concentrations of the cue is essential
Outline the experiments of Μu-Ming Poo in implying the need of axons to adapt to guidance cue concentration
Axons of neurons isolated from early embryonic Xenopus spinal cord were found to respond to netrin pulsed into a dish from a pipette. This pulsed netrin forms a (exponential) gradient which is revealed by including a fluorescent marker in the pipette. Growth cones responding to such a gradient initially exhibit a zig-zagging trajectory which implies axons are sensitising and de-sensitising as they go along
How was the de-sensitisation and re-sensitisation of axons later proven
To determine whether growth cones adapt to increasing attractant concentrations axons were pre-treated with differing concentrations of netrin. Immediately (30min) after treatment growth cones no longer responded to the guidance cue. However after a longer period (90mins) growth cones became resensitized. This resensitization was dependent upon MAPK activation and local protein synthesis in the growth cone
