Axon Guidance Flashcards
What are growth cones?
The motile sensory tips of growing axons
(and dendrites for that matter)
What is a neurite?
A neurite or neuronal process refers to any projection from the cell body of a neuron. This projection can be either an axon or a dendrite. The term is frequently used when speaking of immature or developing neurons, especially of cells in culture, because it can be difficult to tell axons from dendrites before differentiation is complete
What are the features of growth cones on neurites?
Highly dynamic and motile
They explore the environment seeking guidance cues
They form focal contacts with the substrate that provide a tensile grip in which to establish process elongation.
Describe the cytoskeleton in growing axons
Dynamic
. To change direction an axon must be able to rapidly dismantle its cytoskeleton in one orientation in favour of its establishment in another direction.
Describe the microtubules in growing axons
Filamentous protein polymerised to form tensile cables - forms the backbone of the nerve process
- can also be referred to as intermediate filaments
What is the function of actin in the neurites?
Filamentous protein on the extremity of the growth cones
What is the activity of the microtubules in the growth cone?
The growth cone is the factory in which axon microtubules are stabilised or destabilised, and they in turn respond to interactions with actin, the filamentous proteins of the growth cone extremity.
What are the three arbritrary zones of the growth cone?
The central or core region
Transition region
Peripheral region
What happens in the transitional region of growth cones?
This is where most of the microtubule actin interactions take place
What is the peripheral region largely composed of?
Actin filaments
What are the states that actin can exist?
As a monomer
Diffuse network 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
What are filopodia?
Slender cytoplasmic projections that extend beyond the leading edge of lamellipodia in migrating cells.
What do filodopia contain?
They contain actin filaments cross-linked into bundles by actin-binding proteins, e.g. fascin and fimbrin.
What is the purpose of filodopia?
Filopodia form focal adhesions with the substratum linking it to the cell surface
Define lamellipodia
The lamellipodium (plural lamellipodia) (from Latin lamina, “thin sheet”; pod, “foot”) is a cytoskeletal protein actin projection on the leading edge of the cell
What doe lamellipodia contain?
It contains a quasi-two-dimensional actin mesh; the whole structure propels the cell across a substrate.
It is the re-organisation of these actin structures that lays down the path to be taken by the growing axon, and this path is established by subsequent stabilization of microtubules in the orientation dictated by actin filament assembly and consolidation.
Within the lamellipodia are ribs of actin called microspikes, which, when they spread beyond the lamellipodium frontier, are called filopodia
What are actin filaments composed of?
Bundled actin monomers bound to ATP or ADP molecules
Why is ATP actin preferentially added to the distal end of an actin filament?
ATP-actin and ADP-actin can be bound or released at either end, but the equilibrium constant for ATP-dissociation is greater at the pointed (proximal) end, and so ATP-actin is preferentially added to the barbed (distal) end of an actin filament.
Pointed = proximal
Barbed = distal
When does treadmilling occur?
In the absence of acin binding proteins
What is treadmilling?
ATP-actin is added at the barbed end, hydrolysed rapidly to ADP-Pi-actin and then the phosphate group is slowly dissociated to ADP-actin, which can be released from the pointed end.
What is the significance of the equilibrium between actin polymerisation and depolymerisation?
The equilibrium between actin polymerisation and depolymerisation is the driving force behind axon elongation and retraction
What are microtubules composed of?
Microtubules are also polarised structures made up of repeating units of a-tubulin and b-tubulin dimers.
Which dimers add to the distal end and which dimers add to the proximal end?
a/GTP-b-tubulin dimers add to at the distal end and a/GDP-b-tubulin dimers dissociate from the proximal end
Again, rapid hydrolysis of GTP to GDP occurs within the tubule.
What post-translational modifications might occur to the tubulin as the tubules age and stabilise?
detyrosination or acetylation
What follows in the protrusion of filodopia and lamellopodia which stimulates growth?
Influx of microtubules and organelles
(Termed engorgement)
What is the process of consolidation?
Cell membrane tightens around the newly formed cable of microtubules resulting in consolidation of the nervous process.
What determines the direction of travel of the neurite?
The direction in which these processes occurs is driven by the direction in which the initial filopodial and lamellipodial protrusion takes, which is in turn dictated by environmental signals perceived by membrane receptors on the surface of the growth cone.
Environmental signals - detected by growth cone receptors - lemallipodial and filopodial protrusion
‘But the direction in which an axon grows is ultimately determined by the spatial distribution of the environmental signals perceived by the growth cone, or the effect of the environment on distributing key signalling molecules within the growth cone.’
After there is detection of the environmental signal, what prompts the polymerisation and depolymerisation of the cytoskeleton?
transduction of a series of signals largely mediated by the sequential phosphorylation of a chain of intracellular proteins that ultimately impinge on molecules involved in mediating the polymerisation or depolymerisation of the cytoskeleton
What does it mean if the transduction pathways are convergent?
The same downstream signals may be activated by pathways originated by different surface receptors
Different surface receptors result in the activation of the same downstream signals
What is meant by a divergent downstream signal pathway?
Some downstream signals are activated in preference to others and may in fact induce some collateral inhibition, or silencing, of other pathways
In this case the response of the growth cone would depend on the comparable strengths of the initial signals received by the different surface receptors.
What is the role of the Rho family of GTPases?
Pivotal in mediating cytoskeleton dynamics
What are the 3 classical members of the Rho family?
Rho
Rac
cdc42
What is the role of RhoA?
Usually involved in growth cone collapse (and therefore actin depolymerisation)
But have also known to regulare actin bundling in non-neuronal cells
What is the role of rac?
Thought to induce lamellopodial protrusion
What is the role of cdc42?
Regulates filopodial formation
Give an example of proteins that can be activated or inactivated downstream of GTPases
Actin binding protein ADF/Colifin
What regulates the activity of Rho GTPases?
The rho GTPases are themselves activated or inactivated by a number of specific proteins, termed guanine nucleotide exchange factors (GEFs – exchange GDP for GTP, activating the enzymes) and GTPase activating proteins (GAPs – deactivate the enzymes by dephosphorylation of GTP).
Thus, the activity of rho GTPases, and the consequent state of cytoskeletal organization, is determined by the relative activity of GEFs and GAPs within the growth cone
What are the three categories of growth cone guidance mechanisms?
Chemical
Physical
Electrical
What is chemotropism?
Chemotropism involves the biased expression of molecules in a growth cone’s vicinity that influence the turning of that growth cone in one direction or another
How do chemotropic molecules affect direction of travel?
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 or away from the source.
What is the basis of physical 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
What are the molecular requirements for physical growth?
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
Give examples of inhibitory physical cues
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
Where does the ‘electrical’ come from in axon guidance mechanisms?
Minute endogenous electrical currents exist across epithelial cell membranes
How do growing neurites orientate themselves in an in vitro physiological electric field.
In vitro a physiological electric field established across a culture dish induces a remarkable orientation of growing neurites towards the cathode, or negative electrode
What is a likely mechanism of action underlying the electrical growth cone guidance?
Movement of charged cell surface receptors within the fluid lipid bilayer, creating a bias of receptor signalling that results in growth towards the negative electrode
In addition, key second messenger molecules within the growth cone may differentially distribute themselves according to charge, creating a gradient of signalling molecules that induce cytoskeletal polymerisation in the direction of the cathode.
Summary
the environment must induce a bias of growth cone signalling that favours axon polymerisation in one direction over another. Cell surface receptor activation leads to a sequence of phosphorylation of a number of intracellular proteins that typically render them active, and which ultimately leads to actin polymerisation and microtubule consolidation. Thus, if a gradient of phosphorylation exists within the growth cone, a bias in axon growth will ensue.
How many axons are projected in comparison to how many are maintained?
Generally, far many more axons project in development than are maintained after target contact has been made. The excess axons die back. This has long been conceived 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.
Once contact has been made what are the proposed mechanisms for axon survival?
In order to survive, axons require neurotrophins such as NGF, NT3, or BDNF. In many cases these are provided by the target cells, and one theory is that there is only enough produced to maintain a certain number of axons. Subsequent axons do not receive adequate amounts of neurotrophin and therefore do not survive. Another theory is that, once synaptic connectivity has been established, electrical activity of neurons is initiated, and this activity also promotes neuronal survival. Both mechanisms are likely to be important in deciding which axons survive and which are excessive to needs.