Rat sensory growth cones in vitro Flashcards
What is the anatomy of the growth cone?
3 domains:
- Central
- Transitional
- Peripheral
- Lamellapodium
- Filopodium
What is the difference between lamellapodia and filopodia?
Made of different kinds of F-actin:
- Lamella - actin bundles are CROSSLINKED into a NET
- Filopodia - actin bundles are POLARISED to form larger bundles
What happens to actin in a resting growth cone?
Actin is added at the ENDS of the filopodia and ‘treadmills’ back towards the central domain (actin is severed in the central domain)
Tubulin (from the central domain) is dragged sporadically into the filopodia
What is microtubule capture?
Sporadic movement of tubuliin into the filopodia
When does microtubule capture happen more dramatically?
When the growth cone comes into contact with an ATTRACTIVE CUE
What happens to the growth cone when it comes into contact with an attractive cue?
It REORGANISES itself to establish a new direction:
1) F-actin treadmilling SLOWS
2) F-actin ACCUMULATION
What does F-actin accumulation cause?
Filopodium to be STABILISED
Microtubule capture
What part of the growth cone is/isn’t attached to the substrate?
Is attached by:
The central domain (palm)
Not attached:
Filopodia
Is attachment of the growth cone to the substrate enough to drive the growth cone forwards?
NO - need stimulus of cue to rearrange cytoskeleton
What 2 things lead to reorientation of microtubules and filopodia extension in the growth cone?
1) MOLECULAR clutch
2) Actomyosin-based actin-tubilin link
What does the molecular clutch do?
Slows rearward actin treadmillling
Forward movement of filopodium
Microtubules extend up the back of the filopodium
What does the actomyosin-based actin-tubulin link do?
Captures microtubules in the wake of the extending filopodium - to reorganise them
What is the evidence for the molecular clutch?
What does this show?
1) N-cadherin and cytophobic agent in a dish and put neurons over the top
N-cadherin-GFP in the neuron
Where neuron sticks to Ncad in the dish - bright colour
2) Labelled actin in the cell - allows to track what happens to actin over the point of contact with the substrate
Shows:
- Actin treadmills back but SLOWS DOWN/STATIONARY where there is Ncad
What is Ncad?
An adhesion molecule (cadherin)
Does the clutch require F-actin to be joined directly to the substrate?
Not necessarily
Describe uncrosslinked F-actin
In the lamella:
Little strength
No net movement
Treadmilling occurs
What happens when encounter an attractive cue in the lamellopodia?
- Drives a signal
- Activates intracellular CROSS-LINKING protiens via SECONDARY MESSENGERS
- Cross-linking stops the treadmilling of the fibres - pushes the membrane out –> membrane moves FORWARDS
What is the crosslinking protein in the growth cone?
Likely to be a myosin
What are the 2 alternative ideas for how the clutch works?
1) Physical cross-linking of the membrane receptor to the actin cytoskeleton
2) Secondary messengers and cross-linking agents
What is sempahorin?
A NEGATIVE cue that causes growth cone collapse (destabilisation of the F-actin)
What are Rho GTPases?
What are they important in?
MOLECULAR SWITCHES that respond to EXTRACELLULAR signals
Important in regulating the actin cytoskeleton
What is the ON state of a Rho GTPase?
What is the OFF state?
ON - bound to GTP
OFF - bound to GDP
What molecule turns GTPases ON?
GEF (Guanine nucleotide exchange factor)
Donates a phosphate –> converts bound GDP to GTP
What molecule turns GTPases OFF?
GTPase activating protein!! (GAP)
(GTPase function is to convert GTP–>GDP, so if activated, GTP is turned to GDP, GTPase bound to GDP is OFF)
(activates the cleavage of bound GTP –> GDP)
What effects do RhoGTPases have on the actin cytoskeleton?
Depends on which Rho GTPase is activated:
- RhoA
- Rac
- Cdc42
Why are Rho GTPases called RHO??
Because one of their members is called Rho
But have many different other members that aren’t called Rho
What happens when CA RhoA?
Stress fibre formation
What happens when CA Rac?
Lamellipodia formation
What happens when CA Cdc42?
Filopodia formation
What happens when DN RhoA, Rac or Cdc42?
How?
SUPRESS formation of their structures
By COMPETING with the normal Rho-GTPases, turning them OFF
What does modulation of GTPases do?
Control axon growth
What RhoGTPase(s) are POSITIVE regulators of axon growth when they are ON?
Rac and Cdc42
What RhoGTPase(s) are NEGATIVE regulators of axon growth when they are ON?
RhoA
What is a positive regulator of axon growth?
Negative regulator?
Positive - STIMULATES axons growth
Negative - INHIBITS axon growth
What does DN RhoA cause?
Neurite retraction
What does DN Cdc42 cause?
BLOCKS formation of BOTH dendrites and axons
What does DN Rac cause?
BLOCKS formation of AXONAL growth
What is unusual about CA Rac/Cdc42?
What does this suggest?
Also BLOCKS formation of AXON growth (even though DN block axon growth)
Suggests structures whose formation cdc42 and rac normally promote may need to be disassembled as well as assembled for ordinary growth!!!
Where is assembly and dissembly for normal growth seen?
1) Actin treadmilling (addition of F-actin at one end, disassembly at the other)
2) During guidance - f-actin is broken down in the direction the growth cone is no longer travelling in and used for growth in the new direction
How does DN/CA rac/cdc42 block growth?
CA - blocks disassembly
DN - blocks assembly
Does evidence indicate that RhoGTPases are involved in guiding axons (instructive) or just allow axons to grow (permissive)
INSTRUCTIVE
What is the relationship between Rac and RhoA in growth cone turning?
BALANCE between the two determined growth cone turning
How do factors that COLLAPSE the growth cone affect Rac and RhoA?
ACTIVATE RhoA
DOWNREGULATE Rac
How do factors that EXTEND the growth cone affect Rac and RhoA?
DOWNREGULATE RhoA
ACTIVATE Rac
Which 2 ways do guidance factor receptors regulate Rho GTPases?
1) Bind DIRECTLY and modulate the GTPase
2) Bind to GEFs and GAPs, which regulate the GTPase
What is plexin B?
Component of the semaphorin receptor
Regulates Rac by directly binding to it
How are semaphorins used in flies?
They are expressed by some muscles but not others - to guide innervation by motor neurons
What muscles do motor axons AVOID in the flies?
Avoid muscles that make the semaphorins
What muscles do motor axons INNERVATE in the flies?
Muscles that don’t make the semaphorins
What happens to the motor neurons when increase plexin on motor neuron axons?
Become more sensitive to sema in the environment
Fail to innervate muscle
What happens to the motor neurons when DECREASE rac?
More sensitive to sema (repulsive cue)
Fail to innervate muscle
What happens to the motor neurons when INCREASE rac?
LESS sensitive to sema
Innervate muscle
What happens to the motor neurons when DECREASE RhoA?
Less sensitive to sema
Innervate muscle
What does it mean if something is INSTRUCTIVE in guiding axons?
They GUIDE axons:
Cues can directly switch them on/off
(downstream of attractive/repulsive cue)
What does it mean if something is PERMISSIVE in guiding axons?
They allow axon growth in general:
- Upstream of the attractive/repulsive cues
What is the model for sema signalling?
- Sema binds Plexin receptor
- Receptor gets crosslinked
- Receptor sequesters Rac in the environment
- Less free Rac in the environment
- More RhoA than free Rac
- Growth cone COLLAPSE
When there is more Rac than RhoA what happens to the GC?
Extension
When there is more RhoA than Rac what happens to the GC?
Collapse
What determines the outcome of the growth cone?
BALANCE between free RhoA and Rac
How many members of the Rho GTPase family?
What does this mean?
6 in fly
20 in humans
Very small - hard to confer specificity to pathways
Why would signalling through GEFs and GAPs allow more regulation and specificity of pathways than just using Rho GTPases?
Much larger family
More RESTRICTED expression (GTPases have broad expression)
What are ephrins?
INHIBITORY cues
How do ephrins ultimately regulate RhoA, Rac and cdc42?
Signal via GEF called Ephexin, which regulates RhoA, Rac and Cdc42
How does Epherin control RhoA, Rac and cdc42?
TURNS OFF Rac and cdc42
TURNS ON RhoA
What secondary messenger may guidance cues act through?
How?
Calcium
Affects the Rac/RhoA balance
How does calcium effect growth cones?
Localised calcium flux TURNS growth cones
Growth cones turn towards flux of calcium
What happens to intracellular calcium as the growth cone navigates in vitro/vivo?
Transient fluxes in calcium - happens constantly in response to cues in the environment
What visualises calcium in a neuron?
Fluo-3 dye
What happens when a growth cone is treated with ryanodine?
Why?
Growth cone turns towards ryanodine:
- Ryanodine causes release of calcium from stores
- Calcium flux occurs
- Ryanodine activates Rac/cdc42
- Ryanodine supressed RhoA
How guidance cues modulate the actin cytoskeleton? (5 ways)
1) Filament disassembly
2) Branching
3) Termination of branch extension
4) Filament assembly
5) Actomyosin contractility
What are the known point of input into the actin cytoskeleton for guidance cues?
Through RhoGEFs and RhoGAPs which control Rho, RacA and cdc42
What is filament disassembly activated by?
Cofilin
What is branching initiated by?
Arp2/3 in response to activation by WAVE/WASP proteins
What is termination of branch extension caused by?
Capping proteins
What is filament assembly regulated by?
Profilin and Thymosin
What determines actomyosin contractility?
Cross-linking between branched structures
What is the evidence that MT may be involved earlier than actin in movement of the growth cone?
When put guidance cue on growth cone - IMMEDIATELY tubulin accumulates underneath
What is the role of MT in growth cone guidance?
Bring in important elements (eg. signal transduction elements, factors required for membrane protrusion) into the are of contact with the cue
Bring these elements into the filopodia
Elements important in response of the filopodia to cues
What ‘goodies’ are brought into the filopodia by the microtubules?
Monomeric actin
Collapsin Response Mediator Protein (CRMP)
Transduction elements
What does CRMP do?
Promotes MT polymerisation and drives WASP complex proteins into the filopodia
Promotes actin crosslinking
What controls the growth cone behaviour?
Intimate interaction of pathways that control actin cytoskeleton and pathways that control the MT
What is Rac signalling important in?
How?
Establishment of polarity
Is involved in the PI3K responses
How does cdc42 act in a feedback loop?
- Ras activates PI3K
- PI3K activates PIP3 levels
- PIP3 activates Cdc42
- Cdc42 has effect on the Par3 complex
How else does sem3A effect polarity?
Via cGMP/cAMP
What does the cGMP/cAMP ratio affect?
Responses to guidance cues
What are axon guidance cues a continuation of?
How?
The polarity process
Attraction enhances axonal polarity, repulsion inhibits it
