Module 1 Lecture 4: Axon Guidance Flashcards
main characteristic of the growth cone
engine and navigator of axon guidance
what major cytoskeletal systems are essential for dynamics
- microtubules (MTs) (common in cell division - spindle)
- actin (makes up thin filaments in muscles to shorten them)
how are microtubules and actin assembled
from monomers into polymers
what are the two poles of the polymers
fast growing plus and slow growing minus ends
- structurally and kinetically distinct
where does the plus end of the polymer point
toward the leading edge of growth cone
what drives growth cone movement
new polymer assembly
what composes the peripheral domain
- long, bundled actin filaments in filopodia (exploratory structures)
- crosslinked actin networks in veil-like structures between filopodia
- individual, dynamic ‘pioneer’ microtubules which reach along actin bundles
what composes the central domain
- stable, bundled microtubules enter the growth cone from the axon shaft
- organelles, vesicles
- central actin bundles
what is the transition zone in the growth cone composed of
- interface between peripheral domain and central domain
- ‘actin arcs’: contractile actin-myosin structures (similar to muscles); perpendicular to actin bundles of the peripheral domain
treadmilling
internal retrograde flow of actin with no net growth cone movement
how do microtubules contribute to axon guidance
necessary for movement
- no new microtubule polymerization = no forward movement
- dynamic microtubules (pioneers out in peripheral domain) also important for steering
how does actin contribute to axon guidance
necessary for guidance
- growth cone without actin dynamics can move but cannot navigate
- growth cones turn in the direction of net actin assembly
retrograde flow (‘idling engine’)
actin assembled at the leading edge but pulled back at the transition zone by myosin
- no (or little) net movement
what is a clutch
in a car, a mechanical system that connects rotational motion produced by the engine to the wheels
what is the clutch made up of in the growth cone
protein assemblies that physically link the actin cytoskeleton to the substrate
how does the clutch affect growth cone movement
actin assembly pushes the leading edge forward and myosin pulls the rest of the growth cone to follow
- movement! treadmilling stops
what is the adhesive substrate-bound cues (the roadway)
- cell adhesive molecules (CAMs) and cadherins
- extracellular matrix (laminin and fibronectin)
what are the repellent substrate-bound cues (the roadway guard rails)
- slits and ephrins
- chondroitin sulphate proteoglycans
what is the receptor for the substrate-bound, attractive, ECM (laminin and fibronectin)
integrin
integrin receptor function
links extracellular matrix to actin (clutch component)
- growth cones can switch integrin composition through development to alter their responses
what is the receptor for the substrate-bound, repulsive, extracellular matrix (Chondroitin sulphate proteoglycan)
protein tyrosine phosphatase (PTP) sigma
protein tyrosine phosphatase sigma receptor function
Chondroitin sulphate proteoglycan binding to protein tyrosine phosphatase sigma inhibits axon growth through signaling to the actin cytoskeleton
what are the substrate-bound, attractive, cell surface molecules (the surface is another cell, not extracellular matrix)
- cell adhesion molecules (CAMs)
- cadherins
cell adhesion molecules characteristics
- bind other CAMs on other cells
- binding can be with same (homophilic) or different CAM (heterophilic)
- intracellular domain links to cytoskeleton (clutch component)
-eg NCAM, L1-CAM, APCAM
characteristics of cadherins
- Ca2+ dependent adhesion molecule
- homophilic binder
- clutch component
- eg Cadherin, N-Cadherin
what does APCAM coated bead placed on growth cone cause
engagement of clutch
types of axon-axon interactions
fasciculation, defasciculation, selective fasciculation, selective defasciculation
Post Optic Commissure (POC) function
connects two clusters of ventral neurons in the forebrain of zebrafisu
characteristics of pioneers and followers in the POC
pioneers and followers move at the same rate except at the midline (a decision point)
- at the midline, pioneers slow down, followers do not
- pioneers and followers have different shaped growth cones
why do pioneers slow down at the midline
its growth cone samples the guidance cues
why do followers not slow down at the midline
- more compact growth cone does not engage as many environmental cues
- receives additional guidance cues directly from pioneer axon
what is another possibility for the different rates of pioneers and followers
may be intrinsic (molecular) differences between pioneers and followers that make pioneers more capable pathfinders
what are types of substrate-bound, repulsive, secreted or cell surface molecules
slits, and ephrins
slit characteristics
secreted, but remain surface bound (minimal diffusion)
- receptor: Robo (short for roundabout)
ephrin characteristics
cell surface molecule
- receptor: Eph
what does signaling through Robo and Eph receptors effect
cytoskeletal dynamics
- not clutch components
what does bath application of slit cause
growth cone collapse and retraction
what are the diffusible chemotropic cues (the road signs)
- classic guidance molecules (netrins and semaphorins)
- morphogens and growth factors (Wnt, Shh, BMP, adnBDNF)
- neurotransmitters
- secreted transcription factors
netrin characteristics
attractive cue
- receptor: DCC
semaphorin characteristics
repulsive cue
- receptor: Plexin
what does signaling through DCC and semaphorin receptors affect
cytoskeletal dynamics
what does bath application of Sema cause
growth cone collapse and retraction
what does Netrin application from the left cause
growth cone turning to the left
are commissural axons guided exclusively by FP-derived netrin 1 gradient?
no
how does Netrin affect commissural axons in dorsal spinal cord explants
they will orient toward a cell culture aggregate expressing netrin (in vitro)
how does Netrin affect isolated growth cones
will turn toward a source of soluble Netrin (in vitro)
what does a floor plate graft cause
causes commissural axons to exit the spinal cord and grow toward it (in vivo, gain of function experiment)
where is Netrin (mRNA) expressed
both at the floor plate and in the ventricular zone
how does genetic ablation of the floor plate affect ventricular zone netrin
does not affect it
is netrin in floorplate crucial?
no, it is dispensible
-ventricular zone netrin is sufficient to support midline crossing
characteristics of netrin in wild-type
netrin is produced by progenitor cells in the ventricular zone
-those progenitor cells deposit netrin protein to the pia surface
- commissural axons use this surface bound netrin path to find their way down to the plate
characteristics of FP-netrin1-/-
does not cause an overt phenotype wrt midline crossing
characteristics of VZ-netrin1-/- (while keeping floorplate source)
leads to disorganization of commissural axons at the dorsal spinal cord and significant reduction in midline crossing
what happens if a growth cone is co-expressing the DCC and UNC5receptors
Netrin becomes repulsive
commissural neuron path
- move away from roof plate
- move ventrally
- find the floor plate and cross
- do not cross back
- move anteriorly toward brain
what cue does the commissural neuron depend on when moving away from roof plate
BMP (from roof plate)
- receptor: BMPR1B
- repulsive
what cue does the commissural neuron dependon when moving ventrally
Netrin (follows pia bound netrin)
- receptor: DCC
- attractive
what cue does the commissural neuron depend on when finding the floor plate and crossing
Netrin, Shh (Shh from floor plate important for crossing; local Netrin activity may still be required for error-free crossing)
- receptor: DCC, BOC
- attractive
what cue does the commissural neuron dependon when not crossing back
slit (floor plate expresses slit. pre-crossing axons unresponsive; become responsive post-crossing)
- receptor: Robo
- repulsive
what cue do the commissural neuron depend on when moving anteriorly toward brain
Wnt4 (anterior (high) – posterior (low) gradient in the floor plate. different Wnt and direction compared to AP patterning)
- receptor: Fz3
- attractive
what do the Drosophila midline glia express
Netrin and SLit
when do commissural neurons respond to Netrin
with receptor Frazzled (DCC homolog) requires contacting surface-bound Netrin
when is Robo active vs downregulated
- downregulated precrossing –> no repulsion
- active post- crossing –> no turning back
Commissureless (Comm) function
down-regulates pre-crossing robo; Comm itself is down-regulated post-crossing
what happens with no Comm
no crossing
what happens with no robo
go round and round the midline (roundabout)
