Lecture 18- Axon guidance I Flashcards
What do neurons start off as?
-neurons start off as a non-polar cell but during development it extends an axon
What are the variations in length of axon projections?
-local interneurons: short axons -projection neuronse: long axons -lenth of axons is highly variable (e.g. corticospinal neurons= from motor cortex to lumbar spine, the axon is over a meter long)
What are the steps in the development of neurons?
- Formation of neural progenitors 2. Proliferation 3. Differentiation 4. Migration 5. Neurotransmitter specification (neuron must synthesise the right neurotransmitter for its function) 6. Axon growth and navigation (also called axon guidance or axon path-finding) 7. Target selection 8. Synapse formation 9. Development of mature electrical properties
When do developing neurons extend their axons?
-variable, but some very early and some after birth - e.g. some at 3 days already extending axons -different types of cells extend axons at different times -many do it after birth, that is why a baby doesn’t walk as doesn’t have the circuitry
What are the pioneer axons?
-first axons to form a pathway -often used by later axons to navigate -pioneer axons can use local cues on their journey -critical for development, but studies show that nothing that different about them, can ablate it and another will take up that role
What are the guidepost cells?
-local cues used by growing axons are termed guidepost cells
What is the role of guidepost neurons in the developing grasshopper?
-cell body on apendages and extend axons to the CNS -pioneer neurons always grow close to Tr1 and Cx1 cells -if ablate Cx1= they don’t grow in the right direction -so the Cx1 cell must provide something that is essential to the axons navigate = guide post cell
What are the Mauthner cells in salamander?
-pair of large cells in the hindbrain of fish amd amphibians -responsible for the escape response
What was the experiment with Mauthner cells?
-showed that axon navigation relies on cues from the eternal environment -removed part of the hindbrain and flipped it 180 degrees =within a segment the axons did the right thing -once it got to the boundary that made it turn around and grew in the direction that was needed
What do growing axons do?
-reconise various cues on the surface of other axons and cells -respons to diffusible molecules -use intermediate targets -in vivo= axons grow in a complex environment, cannot be reproduced properly inexperiments
What is the growth cone?
-specialised tip of a growing axon -have sensory, motor, integrative and adaptive abilities -can continue to grow and navigate correctly when removed from the cell body –growing axon always has a specialised tip, always larger than the othe rparts of the axon
What was the experiment proving that the growth cone can navigate and grow without the cell body?
embryonic frog brain:
- can cut the optic nerve, the axon will contunue to grow and find target
- proves that doesn’t need the cell body for information
- only lasts some time as it runs out of supplies from the nucleus it usually gets
How long have people known about growth cones?
-a long time -Cajal 1880s in chick neural tube -Speidel 1930s in a frog tail -Harrison early 1900s, neural explants
How can growth cones be visualized?
-can be visualized in living neurons in culture or in vivo using GFP or Dil
What are the processes of a growth cone called?
-have filopodia (string feet) and lamellipodia (flat feet) -these are at the tips of the growth cone explores and makes decisions as to where to go
What are the variations in growth cones?
-can vary in speed of advance and in morpholgy= pioneers vs followers -pioneer axons often have more alaborate growth cones
What does this picture show?
- example of change in morphology in growth axons pioneer vs follower axon growth cone
- it is the axons doing the most exploring that have the most elaborate growth cones
- the number of filopodia and lamepodia varies as an axon extends
How do axons grow as the growth cones advance?
-new material must be incorporated into the growing axon -new material is assembled distally in the growing axon, near the growth cone -addition of material is Ca2+ dependent, probably by Ca2+ mediated fusion of internal vesicles to the growth cone surface -addition of cytoskeletal components (actin filaments and microtubules) also occurs at the tips of the growth cones of growing axons
What is the growth like the closer to the tip of the growth cone you are?
the closer to the tip you are the more relative growth occurs
What was the experiment that proved the axons grow distally?
proves that it is material added to the growing tip (distally) is pushing the whole thing
Where does growth occur in axons?
- the growth happens at the tips of the growth cone - filamentous actin added at tips of filopodia -actin filaments become incorporated in the axon cortex
What is the structure of the growth cone?
several domains c-domain= cortex domain -f-actin= filamentous actin bundles -some microtubules are the dynamic ones= on the outside and in middle stable micritubule
What is the molecular structure of the growth cone?
actin monomers at filopodia tips -when go back on the filopodia= there are receptors and ligands= detect what is in the environment -the tubulin at the end is unstable= dynamic
What is the cytoskeleton of the growth cone?
-microtubules extend down the axon and into the central domain of the growth cone, a small number of dynamic microtubules also extend into the peripheral domain -actin fibres are prominent in the peripheral domain; actin filaments in the filopodia are assembled by the addition of G-actin monomers
What does the growth cone actually look like? (picture)
what it actually looks like the growth cone -g actin at edges -filamentous actin more inside -the microtubules that are unstable project to the outside bit
What are the 3 key cytoskeletal molecules in growth cone?
-actin, myosin, microtubules
What leads to axon advance in a particular direction?
- retention and thickening of selected filopodia
- this is key!
- what happens when growth cone decides to turn?: get filopodia withdrawal on one side and dilation on the other side and get microtubule invasion into the direction you want to go
What is the role of actin in the growth cone?
-Cytochalasin(actin depolymerizing drug( inhibits filopodia formation, and growth cone advance is slowed -actin filaments are essential for growth cone navigation
What was the experiment involving cytochalasin?
-low doses of cytochalasin in a grasshopper don’t completely block actin polymerization -high dose of cytochalasin then stop actin polymerization completely -lower not completely, but growth cones cannot navigate completely when actin polymerization partially blocked -also done in frogs
What are the characteristics of actin filaments in the filopodia?
-occur in bundles -plus ends (fast growing) point towards periphery -new actin monomers added at the tip -actin filaments disassembled into monomers at the minus ends can be recycled -flow rearwards -but do not change length!
How is actin polymerized in the growth cone?
normally they are dynamic but do not change length
What is the situation like in a stationary growth cone?
- retrograde F-actin flow, driven mostly by myosin activity counteracts actin polymerisation
- extension of distal zone depends on balance of F-actin polymerisation and retrograde flow
- even when not receiving any positive or negative signal, non growing= still get recycling of actin, without moving
- extension of distal zone depends on the balance, if on the front balanced the retrograde flow= then balanced, but if growing= the the front outdoes the retrograde on
- proven by putting a bead in and it moves! even though the growth cone doesn’t
What is the evidence for role of myosin in the retrograde flow?
-forward flow is slowed when myosin blocked -filopodia lengthen because they are no longer being pulled rearward -less retrograde flow of actin when myosin is blocked!
How does the growth cone advance (engaging the clutch),
- crucial for the growth cone to adhere to substrate -through cadherins can interact with the extracellular matrix= this blocks the retrograde flow and the actin monomers can be added without the retrograde flow so the cone will advance -actin polymerization pushes filopodium forwards
What is the advancing growth cone like (part 2)?
normally have polymerization balanced by retrograde flow= when not moving -it also enables the microtubules to advance down this peripheral zone (this happens once the growth cone interacts with the substrate and the retrograde flow is blocked)
What is the second theory about axonal growth?
second theory= filopodium is growing and it creates space
- so passive process the microtubule -the other theory that it is more active, that is showed here
- myosin pulls on actin cables and hence pulls the main body of the growth cone forward if the filopodium is attached to the substrate
What is the summary of steps involved in axonal growth?
- A.actin filaments polymerize near the leading edge
- B.actin filaments are dragged back towards the central domain by myosin= no net progress
- C.adhesive interactions with the substrate inhibits the retrograde flow of actin filaments resulting in forwards movement
- D.polymerization driven forward progression of axon filaments
- E.lack of retrograde flow allows microtubules polymerization proceed into the peripheral domain
What are the characteristics of microtubules?
-microtubules are polymers of tubulin -run straight and parallel along an axon until they enter the base of the growth cone -growth cones contain unassembled tubulin, and microtubule stabilizing proteins (e.g. Tau) -most microtubules are in the central domain, but some unbundled, dynamic microtubules invade the periphery
Characteristics of microtubules part 2:
-plus end of microtubules (where polymerization takes place) occurs in the growing tip of the growth cones -a C-terminal tyrosine is added to alpha-tubulin by the enzyme tubulin tyrosine ligase -tyrosinated form of tubulin is sensitive to depolymerisation agents and is very dynamic (within the growth cone) -microtubules in which tubulin is acetylated (in the axons) are stable
What are the interactions between actin and microtubules?
-actin cables linked to microtubules via cross linking molecules -actin cables in filopodia provide channels for dynamic microtubules to invade -retrograde flow of actin breaks the microtubules, new plus ends that can be elongated and minus ends that can be depolymerized -new growing dynamic microtubules polymerize and extend into the periphery of the growth cone -microtubules in the peripheral domain can be stabilised and promote directional axon growth -these interactions are crucial for growth cone navigation
What decides the direction of growth cone advance?
- in culture growth cones move straight ahead= similar numbers of filopodia on each side
- depolymerize actin filaments on one side, growth cone steers to the other side
- local stabilization of microtubules on one side turns the axon to that side
- local destabilization of microtubules on one side= turns away from that side!
What are MAPs?
-microtubule associated proteins -show differential localizations -e.g. MAP2 mainly found in dendrites, Tau in axons -play a variety of roles -e.g. orbit/clasp promotes microtubule invasion of the peripheral zone and cross linking to actin (stabilization, SCG10, is a destabilizing protein) -gain or loss of function of MAPs alter the rate of axon growth
What are the actin associated proteins?
-cofilin depolymerizes F-actin into G-actin monomers (similar role to SCG10) -Arps promote branching of actin filaments -Ena-Vasp are anti-capping agents and promote growth of actin at the leading adge -loss of Ena-Vasp= loss of filopodia (lamellopodia still present), but can still navigat in vivo
