Axon Guidance 1 Flashcards
What are growth cones?
the sensory organelles of growing axons
What is the role of the growth cone
they translate extracellular information into cellular responses.
has lots of cell surface receptors which instigate a variety of transducer signals that ultimately impinge on the cytoskeletal machinery of the growth cone.
this is where axon microtubules are stabilised or destabilised.
These axon tubules respond to interactions with actin→the filamentous proteins of the growth cone extremity.
What are the three regions of growth cones
Peripheral region - lamellipodia
Transitional region - between both
Central region - central organelles and microtubules
What are the lamellipodia
Outgrowth of growth cone that is made up of actin
What are the filopodia
Dynamic assemblies of filamentous F-actin (finger like projections at periphery) (occurs through polarised filaments)
What is axon pathfinding
The key to axon pathfinding is the dynamic nature of the axon cytoskeleton. To change direction an axon must be able to rapidly dismantle its cytoskeleton in one orientation in favour of its establishment in another direction. A nerve process has a backbone of filamentous protein polymerised together to form tensile cables, commonly known as microtubules or intermediate filaments.
What interacts with filopodia to stabilise them
Microtubules - stabilises them and prevents them from retracting
What do the microtubules become
Highly stabilized in the central region leading to neurite formation
What is the role of actin in the cytoskeleton
Actin monomers can exist as free monomers or diffuse networks of polymerised actin filaments.
They can also be polarised to form rigid filaments that form the backbones of thin processes that protrude from the growth cone body. These are filopodia.
Often, between filopodia veils of cytoplasm (lamellipodia) extend, largely made up of meshes of actin filaments and diffuse actin.
What 4 steps of axon guidance are needed
Neurite initiation
Axon growth and pathfinding
Axon termination
Survival in target tissues
How does neurite initiation work
Actin filaments are distributed evenly around the microtubule rich cell body –> actin localised to tips and microtubules at the backbone of the neurites –> one neurite preferred and becomes axon, the rest resorb into the body or become dendrites
How do actin filaments develop?
Under steady-state conditions, ATP-actin binding occurs and is added to the distal end of an actin filament
ATP –> ADP and the iP leaves and becomes ADP-actin
ADP-actin is released from the proximal end
ATP-actin and release of ADP-actin occurs at the same rate meaning no change in filament length (treadmilling)(same added to distal as is getting removed from proximal end)
if more is added to wrong side- will start to rectract.
The equilibrium between actin polymerisation and depolymerisation is the driving force behind axon elongation and retraction.
How do microtubules develop
Similar to actin filaments, undergo subunit turnover but with alpha/GTP-beta tubulin dimers.
These tubulin dimers are added to the distal end and alpha/GDP-beta is removed at the proximal end
Post-translational tubulin modification (detyrosination or acetylation) ages and stabilises the microtubule
What is ADF cofilin recycling
It severs actin filaments and aids dissociation of ADP-actin
Once ADP-actin is detached, it dissociates from ADP-Actin
When does ADF/cofilin become inactivated
Phosphorylation by LIM kinases and then stabilised by 14-3-3zeta proteins
How is ADF-cofilin reactivated
Phosphatases through the PIP3 receptor
What are Rho GTPases
Cytoskeletal regulating molecules
What is the Rho function, part of the Rho family
A disruptive cytoskeletal regulating molecule that breaks up the actin filaments
What is the Rac function, part of the Rho family
Promotes lamellipodia outgrowth
What is CDC42’s function, part of the Rho family
Promotes filopodial outgrowth
What activates RhoGTPases
Specific GEF’s - guanine nucleotide exchange factors which promote filopodia growth through phosphorylation of Rho-GDP to Rho-GTP
What do GEFs and GAFs do overall
Regulate the cytoskeleton
What is rho GTPases inactivated by
GAPs - GTPase activating proteins They de-phosphorylate Rho GTP and inactivate it
What 3 mechanisms are in play to regulate growth cone development
Chemical
Physical
Electrical
Give some degradation examples by Rho GTPase on the cytoskeleton
Myosin promotes retrograde flow of the actin filament and thus prevents elongation
Rho blocks MLC phosphatase, having the net effect of stimulating myosin and therefore prevents elongation
Cofilin disassembles ADP actin
Give some examples where Rho GTPases promote axon growth
Scar and N-WASP promote filament branching
VASP family and profiling prevent capping of actin filaments which promotes more monomer binding
Rac also inhibits the effects of capping proteins that prevent addition of actin to distal ends
What are the 3 stages of axon elongation
Protrusion -filopodia move forward
Engorgement - organelles follow filopodia
Consolidation - microtubules become stabilised by post-translational modification and a new membrane is formed around the axon
How does chemical axon guidance work
Involves chemotropism - through attraction and repulsion
What is chemotropism and how does it work
Chemotropism involves the biased expression of molecules in a growth cone’s vicinity that influences the turning of that growth cone in one direction or another.
Such cues might be discrete, in that a given molecule or protein is detected on one side of the growth cone and not the other, or they might be laid down in gradients, such that a growth cone can detect on which side a given signal is stronger, and therefore can decide whether to turn towards (chemoattraction) or away (chemorepulsion) from the source.
How do guidepost cells regulate axon guidance
Physical axon guidance relies on axonal contact with structures en route to its target cell. These may take the form of individual intermediate cells in the axon’s pathway, or groups of cells or cellular processes that effectively form tram lines for the axon to grow along. Physical cues would also include extracellular matrix structures that are generally permissive for axon growth.
Give an example of physical guidepost axon guidance
Guide post cells in grasshopper embryos -
In the femur, til cells contact with an intermediate pre-axonogenic neuron to keep it on course.
When this neuron interacts with the trochanter-coxa boundary, it turns 90 degrees and continues growing
A further turn is seen at the coxa region
What are the three types of physical axon guidance
Discrete guidance cells (pre-axonogenic neurons)
Intermediate guiding cells also support some outgrowth with their own short processes which the neuron supports itself on
Continuous chains of cells such as epithelial cells.
What promotes physical axon guidance
The molecular requirements for physical growth support involve a relatively uniform permissive substrate for axons to grow on, such as laminin, fibronectin or collagen laid down in matrix or fibre-like structures, or cell adhesion molecules such as NCAM, L1 or axonin-1.
What inhibits physical axon guidance
physical cues may also be inhibitory, since contact with cells that express inhibitory ligands such as membrane-bound semaphorin or myelin proteins such as Nogo prevent axon growth and may induce branching or a change in direction. Such growth or growth inhibition is contact-mediated, and therefore requires physical contact with the cells or structures expressing the permissive or inhibitory molecules.
Why can electrical axon guidance work
Becomes electric fields can be detected across the whole embryo and within specific regions.
Different current flows occur as a result of different tissues possessing different resistance/thicknesses.
Why is electrical axon guidance controversial
Because scientists believe it manipulates the process too much to be a natural phenomenon.
Does electrical axon guidance actually work
Yes, go towards negative electrode in vitro.
Can definitely manipulate it, whether it’s involved originally or not it is hard to say.
What 2 mechanisms may underpin electrical axon guidance
Asymmetrical protein distribution - charged receptor proteins orientate themselves with one side of the growth cone and react with growth factors with that side of the growth cone and grow outwards towards it.
Key signalling molecules from the cytoplasm are distributed in gradients towards the cathode.
Give an example of key signalling molecules from the cytoplasm moving towards the cathode.
GFP-labelled Akt moves towards the negative electrode in epithelial cells.
Why do we grow more axons than what is needed
as a good way of ensuring that all necessary contacts are made with target tissues and cells, rather than relying on the exact number of axons to project without error.
What are the TrK signalling mechanisms used for
Many Axons grow however not all will survive. There is only a certain amount of growth factor present and once it reaches its destination some axons will die off to save excess materials for other more important areas.
How does TrK signalling mechanisms work
NGF (nerve growth factor) binds to tyrosine receptor kinase (TrK) when NGF binds the whole ligand-receptor complex is endocytosed into the cell and it transports back to the cell body and this promotes cell survival.
What is the domino model
TrK binds to the growth cone leading to a ligand-independent propagation of TRK phosphorylation back to the cell to turn on signalling molecules to enter the nucleus and turn on genes for cell survival.
What is the retrograde effector model
Messenger molecules activated in the growth cone may be transported back to the nucleus to turn on genes for cell survival
What other method is used to promote neuron survival other than the TrK signalling mechanisms
Synaptic connectivity and electrical activity of the neurons is initiated - promotes neuronal survival
What is the role of F-actin
Wider reading
F-actin retrograde flow, driven by myosin II contractility in the transition (T)-zone and F-actin bundle treadmilling, keeps the growth cone engine idling and responsive to directional cues. Growth cone receptor binding to an adhesive substrate leads to the formation of a complex that acts like a molecular `clutch’ mechanically coupling receptors and F-actin to stop retrograde flow and driving actin-based forward growth cone protrusion.
What mediates axon growth inhibition
Wider reading
RTN4R - Nogo receptor 1
What was seen during p75 knockout
Wider reading - wang et al
Outgrowth inhibition was absent
What is cethrin treatment and how does it work
WIDER READING - Baptistie - 2006
Used for neurite growth by applying to the site of nerve injury.
Works by inhibiting ROCK, which is known to phosphorylate proteins that inhibit neurite outgrowth.
What role does the NgR (Nogo-66 receptor) have in mice brains
WIDER READING - Mcgee 2005
It limits visual cortex plasticity - mutant mice with non-functional NgR resulted in enhancement of visual cortex plasticity after the critical period into adulthood, such that adult plasticity in the mutant mice resembled normal visual plasticity in juvenile mice brains
This is being researched as a possible treatment for amblyopia (lazy eye)
