Axon Guidance

Question 1

What are growth cones?

Answer 1

The motile sensory tips of growing axons

(and dendrites for that matter)

Question 2

What is a neurite?

Answer 2

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

Question 3

What are the features of growth cones on neurites?

Answer 3

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.

Question 4

Describe the cytoskeleton in growing axons

Answer 4

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.

Question 5

Describe the microtubules in growing axons

Answer 5

Filamentous protein polymerised to form tensile cables - forms the backbone of the nerve process

• can also be referred to as intermediate filaments

Question 6

What is the function of actin in the neurites?

Answer 6

Filamentous protein on the extremity of the growth cones

Question 7

What is the activity of the microtubules in the growth cone?

Answer 7

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.

Question 8

What are the three arbritrary zones of the growth cone?

Answer 8

The central or core region

Transition region

Peripheral region

Question 9

What happens in the transitional region of growth cones?

Answer 9

This is where most of the microtubule actin interactions take place

Question 10

What is the peripheral region largely composed of?

Answer 10

Actin filaments

Question 11

What are the states that actin can exist?

Answer 11

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

Question 12

What are filopodia?

Answer 12

Slender cytoplasmic projections that extend beyond the leading edge of lamellipodia in migrating cells.

Question 13

What do filodopia contain?

Answer 13

They contain actin filaments cross-linked into bundles by actin-binding proteins, e.g. fascin and fimbrin.

Question 14

What is the purpose of filodopia?

Answer 14

Filopodia form focal adhesions with the substratum linking it to the cell surface

Question 15

Define lamellipodia

Answer 15

The lamellipodium (plural lamellipodia) (from Latin lamina, “thin sheet”; pod, “foot”) is a cytoskeletal protein actin projection on the leading edge of the cell

Question 16

What doe lamellipodia contain?

Answer 16

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

Question 17

What are actin filaments composed of?

Answer 17

Bundled actin monomers bound to ATP or ADP molecules

Question 18

Why is ATP actin preferentially added to the distal end of an actin filament?

Answer 18

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

Question 19

When does treadmilling occur?

Answer 19

In the absence of acin binding proteins

Question 20

What is treadmilling?

Answer 20

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.

Question 21

What is the significance of the equilibrium between actin polymerisation and depolymerisation?

Answer 21

The equilibrium between actin polymerisation and depolymerisation is the driving force behind axon elongation and retraction

Question 22

What are microtubules composed of?

Answer 22

Microtubules are also polarised structures made up of repeating units of a-tubulin and b-tubulin dimers.

Question 23

Which dimers add to the distal end and which dimers add to the proximal end?

Answer 23

a/GTP-b-tubulin dimers add to at the distal end and a/GDP-b-tubulin dimers dissociate from the proximal end

Question 24

Again, rapid hydrolysis of GTP to GDP occurs within the tubule.

Question 25

What post-translational modifications might occur to the tubulin as the tubules age and stabilise?

Answer 25

detyrosination or acetylation

Question 26

What follows in the protrusion of filodopia and lamellopodia which stimulates growth?

Answer 26

Influx of microtubules and organelles

(Termed engorgement)

Question 27

What is the process of consolidation?

Answer 27

Cell membrane tightens around the newly formed cable of microtubules resulting in consolidation of the nervous process.

Question 28

What determines the direction of travel of the neurite?

Answer 28

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.’

Question 29

After there is detection of the environmental signal, what prompts the polymerisation and depolymerisation of the cytoskeleton?

Answer 29

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

Question 30

What does it mean if the transduction pathways are convergent?

Answer 30

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

Question 31

What is meant by a divergent downstream signal pathway?

Answer 31

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.

Question 32

What is the role of the Rho family of GTPases?

Answer 32

Pivotal in mediating cytoskeleton dynamics

Question 33

What are the 3 classical members of the Rho family?

Answer 33

Rho

Rac

cdc42

Question 34

What is the role of RhoA?

Answer 34

Usually involved in growth cone collapse (and therefore actin depolymerisation)

But have also known to regulare actin bundling in non-neuronal cells

Question 35

What is the role of rac?

Answer 35

Thought to induce lamellopodial protrusion

Question 36

What is the role of cdc42?

Answer 36

Regulates filopodial formation

Question 37

Give an example of proteins that can be activated or inactivated downstream of GTPases

Answer 37

Actin binding protein ADF/Colifin

Question 38

What regulates the activity of Rho GTPases?

Answer 38

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

Question 39

What are the three categories of growth cone guidance mechanisms?

Answer 39

Chemical

Physical

Electrical

Question 40

What is chemotropism?

Answer 40

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

Question 41

How do chemotropic molecules affect direction of travel?

Answer 41

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.

Question 42

What is the basis of physical axon guidance?

Answer 42

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

Question 43

What are the molecular requirements for physical growth?

Answer 43

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

Question 44

Give examples of inhibitory physical cues

Answer 44

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

Question 45

Where does the ‘electrical’ come from in axon guidance mechanisms?

Answer 45

Minute endogenous electrical currents exist across epithelial cell membranes

Question 46

How do growing neurites orientate themselves in an in vitro physiological electric field.

Answer 46

In vitro a physiological electric field established across a culture dish induces a remarkable orientation of growing neurites towards the cathode, or negative electrode

Question 47

What is a likely mechanism of action underlying the electrical growth cone guidance?

Answer 47

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.

Question 48

Summary

Answer 48

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.

Question 49

How many axons are projected in comparison to how many are maintained?

Answer 49

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.

Question 50

Once contact has been made what are the proposed mechanisms for axon survival?

Answer 50

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.