recap Flashcards
What are the ways axon guidance cues can be present in the environment?
- patterning
- molecules present on the cell surfaces guiding axons
- secreted molecules steering axonal growth
causes the ‘growing tip’ to feed its way through the environment seen in cajals growth cones
How does the axons become polarised to have a growing tip?
- Highly polarised microtubules with +ve end towards the growth cones
- Due to the localisation of different microtubules-associated proteins determining how the microtubules are cross linked
- e.g. axons express Tau
What is the difference between polarisation in the dendrites compared to the axons?
- Dendrites microtubules are less ordered with mixed orientations
- Dendrites express Map2 rather than Tau
What is meant by a neurite?
Any projection from the cell body
How can we observe where axons are forming?
- Due to axon microtubules being polarised and positive at the growing end, GFP-labeled plus end directed kinesins mark where axons are forming
- Kinesins move along microtubules towards + end
How is microtubule stabilisation relevant to axon formation?
Stabilized microtubules (MTs) are crucial for axon initiation.
Stable MTs: Acetylated.
Unstable MTs: Tyrosinated (early-stage neurites).
Experiment Insight:
Local stabilization via taxol selects one neurite for axon formation.
If the selected axon is removed, another neurite is chosen.
Therefore Axons compete to stabilize MTs using feedback loops.
How does taxol work?
- Taxol binding pushes microtubules in to the stable state where it is associated with GTP in order to polymerise/depolymerise
- Used as a cancer treatment as it prevents depolymerisation of the mitotic spindle which is skewing cell division
How do environmental cues influence neurite selection?
Polarity Formation:
- Established as neurons arise from dividing radial glial cells.
- RGCs generate neurons and guides migration while adapting to environmental signals.
Environmental Cues:
Extracellular Molecules: Growth factors, guidance cues (e.g., netrins, semaphorins), extracellular matrix, signals from neighboring cells and glia.
What is the anatomy of a growth cone?
- Lamella and filopodia are made up of different types of F-actin
- In lamella the actin bundles are cross linked in to net
- In filopodia the actin bundles are polarised to form larger bundles
- Neither are stuck down but instead are highly motile
- Flattened in vitro study makes it easy to image
What is meant by ‘Focal treadmilling’?
Focal treadmilling is when actin filaments undergo continuous turnover.
Polymerization at the Barbed End (+): Actin filaments grow as new actin monomers are added to the barbed end.
Depolymerization at the Pointed End (−): Actin filaments shrink as monomers are removed from the pointed end.
- Exploration of filopodia via microtubules
How has focal treadmilling been shown experimentally?
- Attractive bead = F-actin treadmilling slows and F-actin accumulates
- Accumulation stabilises filopodium and drags microtubules into the back of the filopodium as ‘microtubule capture’
- Immobile bead = growth cone reorganises microtubules to a new growth direction
What happens to the treadmilling when an attractive cue is encountered?
- Molecular clutch is engaged and rearward actin treadmilling slows resulting in forward movement
- Actin-tubulin link captures microtubules
What evidence is there for a molecular clutch?
- Growth cones on stimulating substrates (e.g., N-cadherin) show more forward movement with arrested F-actin flow.
- N-cadherin promotes neural growth and arrests actin flow where growth cones contact it.
- Mapping F-actin flow shows actin spends more time over N-cadherin spots, consistent with a molecular clutch arresting actin movement upon contact with guidance cues.
When has growth cone collapse been observed?
- When sensory and retinal axons come in to contact, the growth cone collapses back away from it and into another direction
- repelling cue
How do Rho GTPases regulate the actin cytoskeleton?
Their activity is regulated by GAPs and GEFs
GAPs put Rho GTPases unto ‘OFF’ state by activating cleavage of bound GTP to GDP
GEFs put Rho GTPase into ‘ON’ state by donating a phosphate to converte bound GDP to GTP
GAP turns off, GEF turns on (contradicts normal)
What are some Rho GTPases that are associated with axons?
- RhoA is associated with stress fibres which are the structures associated with cell attachment
- Rac is associated lamellipodia
- Cdc42 affects filopodia
What is meant by a constitutively activated mutant and a dominant negative mutant?
- Constitutively activated mutants of Rho-GTPases are in ‘always on’ state
- Dominant negative is the notion where something cant be turned on
How are GTPases relevant in axon growth?
- Activated RhoA (RhoA-GTP): Inhibits axon growth (causes neurite retraction).
- Activated Rac & Cdc42 (Rac-GTP, Cdc42-GTP): Stimulate axon growth.
Experimental Evidence:
CA RhoA:* Causes neurite collapse; DN RhoA blocks collapse.
DN Cdc42: Blocks both axon and dendrite formation.
DN Rac: Blocks only axon growth.
CA Rac/Cdc42:* Unexpectedly blocks axon growth.
What is the importance of the dynamic between the assembly and disassembly of actin structures?
Blocking/activating Rac prevents axon growth meaning there must be a requirement for assembly and disassembly
Must be DYNAMIC (bit of both for axon growth)
What is the difference between an instructive cue or a permissive cue?
An instructive cue actively directs cells to follow a specific developmental path or outcome.
A permissive cue creates conditions that allow a process to occur but does not dictate the specific outcome.
What evidence suggests that Rac/Rho are instructive rather than permissive?
- Semaphorins bind to plexins on growth cones (e.g. plexin B), activating RhoA and inhibiting Rac. -
- RhoA activation causes growth cone collapse, while Rac inhibition prevents growth.
- The balance between RhoA and Rac determines the growth cone’s response (collapse or extension), showing that Rac and Rho actively instruct axon guidance, rather than just allowing it.
What does it mean if the growth cone has more plexins?
- More sensitivity of motor axons to the semaphorins
How else can guidance signals regulate Rho GTPases?
- via their GEFs or GAPs
- The GEF and GAP family is a lot larger- more scope for specificity
- Ephrins, inhibitory cues, signal via a GEF called Ephexin, simultaneously regulates RhoA, Rac and cdc42
How does Ca2+ signalling regulate axon formation and direction?
- Increase Ca2+ activity as filopodia extend
- Transient, localised Ca2+ levels can turn a growth cone
Explain how a ‘calcium cage’ works in axon experiments
- Ca2+ is in a ‘chemical cage’
- Flash a light
- Chemical cage breaks down from light activating electrons
- Ca2+ released
- Allows you to see what localised Ca2+ release looks like and its effects in the axon
How does Ca2+ regulate Rho GTPases?
- Ryanodine activates calcium in the growth cone, can make it turn towards you
- After ryanodine induction, you can see significant increase in Rac and significant decrease in RhoA
- This balance again is essential
What proteins are involved in modulating the actin cytoskeleton?
- Coffilin - Binds to actin filaments and accelerates their disassembly
- Arp2/3 - nucleates new actin filaments to promote brancing
- WASP/WAVE - activate Arp2/3 in reponse to signalling
- Profilin - promotes addition of actin to growing filament ends
- Thymosin - prevents actin monomers from polymerising into filaments
