axon guidance

Question 1

anatomy of growth cone

Answer 1

lamellipodia: diffuse networks of actin

filopodia: dynamic assemblies of F-actin. finger-like projections

microtubules: stabilise filopodia, become highly stabilised in central region of growth cone leading to neurite formation

Question 2

microtubulin post-translational modifications

Answer 2

acetylated Mts more stable. found in central region growth cone and do not interact with filopodia

tyrosinated MTs occupy body of growth cone, interaction with actin at proximal ends of filopodia

Question 3

neurite initiation requires cytoskeletal re-arrangement

Answer 3

before initiation, actin filaments are distributed evenly around MT-rich cell body.
once neurites grow out, actin filaments localise to the tips of growing neurites

Question 4

Actin filaments

Answer 4

barbed (distal) end and pointed end

ATP-actin added to barbed end, ATP hydrolysed to ADP + Pi, ADP-actin filament released from pointed end

recycling actin filaments all the time

if faster addition than dissociation = growth, vice versa = retraction

Question 5

microtubules

Answer 5

alpha-GTP-beta-tubulin dimers added to plus end (distal)
alpha-GDP-beta-tubulin dimers removed from minus (proximal) end

Question 6

ADF/Cofilin Recycling

Answer 6

ADF/cofilin rapidly dissociates ADP-actin

is inactivated when phosphorylated by LIM kinases
re-activated by variety of phosphates

activation and inactivation regulated by distinct pathways stimulated by receptor-activted PIP3

phosphatase sling-shot is activated via Akt1, which will then activate cofilin

PIP3 can activate specific regulators of RhoGTPases to induce LIM-kinase mediated inactivation

Question 7

RhoGTPases and axon growth

Answer 7

Rho - destructive
Rac - good for lamilopodial outgrowth
Cdc42 - good for filopodial outgrowth

activated by GEFs, inactivated by GAPs

Question 8

process of axon elongation

Answer 8

protrusion: filopodia move forwards
engorgement: organelles and dynamic MT follow filopodia
consolidation: MTs stabilise via post-translational modification. new membrane made around axon

Question 9

mechanisms of axon guidance

Answer 9

chemotropism: chemoattraction & chemorepulsion

physical
- guidepost cells
- supporting cells
- tram lines

electrical - cathodal orientation

Question 10

electrical guidance cues

Answer 10

EFs detected using non-invasive vibrating probe

detect currents rostrocaudally at rostral plate, across neural plate, across neural folds.

EFs influence neurite orientation in vitro

Question 11

possible mechanisms underlying EF-induced axon guidance

Answer 11

1. receptors in lipid-bilayer redistribute themselves to cathode side - interact with guidance molecules at that side
2. intracellular signalling molecules become redistributed in gradients under EF. e.g. Calcium and Erk

Question 12

models for what happens when axon gets to destination

Answer 12

signalling endoscope model: ligand(NGF)-receptor complex internalised by endocytosis and transported to necleus where instigate survival signals

domino model: NGF binds leading to ligand-independent propagation of TRK phosphorylation back to cell body

retrograde effector model: messenger molecules activated in growth cone transported back to cell body where they initiate further signal transduction

Question 13

what kinds of molecules are involved in guiding axons

Answer 13

cell adhesion molecules (good for growth)
ECM molecules: laminin (good), fibronectin (good), tenascin (bad)
netrins: good embryonically, bad as mature
Slits/robo receptors: inhibitory
semaphoring: usually inhibit through Rho, can be good by activating LIM kinase
ephrins: inhibit
MAIs

Question 14

cAMP receptors

Answer 14

when less cAMP (adults) below threshold Epac activation, so PKA is activation –> inhibition