L1: The growth cone

The organelle that allows neurons to send processes from one part of the nervous system to another

Textbook chapters for this

Development of the Nervous system

• Chpt 5, 6, and 8

Learning objectives

1. What growth cones do

2. cytoskeletal dynamics underlie navigation

3. Guidance cures-how these regulate the cytoskeleton

Questions being explored in this lecture

• Once different neuronal cell types are specified, each neuron sends out a primary neutrite (the axon) with which to reach the brain regions and connect other neurons

But

1. How do neurons know which direction

2. and along which path to extend?

What was the clash of view of Golgi and Cajal

• Golgi→ Reticularists

  • Invented a way of visualising NS with golgi black stain

  • labeled only a few cells so could see them more clearly

  • though the nervous system is a reitculum→ a physical continuum extends between nerve cells forming a ‘nrve net’

• Cajal→ Neuronists→ each neuron is an independent cell

What did Cajal’s work on fixed embryonic neural tissue show

• described the specialised structures at distal tips of axons→ growth cones

• He imaged these as

  • battering rams with which axons might force their way through the embryonic tissue

  • cone was a like a club or fingerlike protrusion

    • force its way through tissue to make connections with other nerve cells

Ross Harrison’s work and observations

Experiment:

• Tissue culture→ drop of lymph fluid and hunf upside down

• observe live growth cones in real time using cultured pieces of embryonic neural tube

Observations:

1. cones are dynamic structures

2. showed motile contractile forces and pulling→ on laminin bead

3. hand-like appearance→ equipped with finger-like filopodal extnesions

   • these are continuously sent out and rapidly retracted

   • (as if being used to sample the environment)

4. Membranous lamellipodia extend between filopodia

Work and observations of William Harris of growth cones

Experiment

• retinal ganglion cells sent out axons (labeled with neural tracer HRP)

• always tipped with a growth cone→ from the retina to the optic tectum

• But then the axon was separated from the cell body

Obersvation:

• Time lapsed→ growth cone still navigate for several hours

  • along correct pathway into optic tectum

• THEREFORE: shows growth cone has everything it needs for navigation

Therefore in understanding the growth cones we can understand

• how nerve cells can send their axons from one part to another

→ next things to investigate→

• How does it turn left and right? What mechansism?

  • does it push or pull?

• how does it know when to turn left and right?

Compartments of the growth cone: 3 Morpholigically distinct domains

1. Central domain (contains organelles)→where axons terminates

2. Transition zone (abuts the central)→ shows characteristic membrane ruffling activities

3. Peripheral domain (distal extent of the transition zone)→ consist of filo and lamellipodia

How are these three domains separated

• Different distributions of cytoskeleton (actin and tubulin mainly)

1. Central domain→ bundles of essentially parallel microtubules from the axon invade the central domain → where they fan out

2. Transition zone→ distal tips (‘plus’ ends) of MT above reach into transition zone

3. Peripheral domain→ individual MT extend their plus ends into the peripheral domain→ BUT→ primarily contains filamentous actin and few MTs

How do growth cones show polarity

• Actin and microtubules are key elements are are both polarised

How do the actin and microtubules work for growth

1. Actin filament

   • energy needed for polymerisation is released during depolymerisation

   • Treadmilling→ when the rate of polymerisation is equal to that od depolymerisation

2. Microtubule

   • GTP cap favours growth but when lost→ rapid depolymerisation ensures

   • Alternation between slow growth and rapid disassembly is called dynamic instability

Due to the assymetric components of actin and MT that make the pushing and pulling

What are the general mechanics of growth cone propulsion

• Combination of pushing a pulling

Three sets of experiments to show the pushing and pulling

1. Cultured microtubules fluorescently labelled

   • if a small spot of fluorescence is photo bleached with laser near distal end→ the bleached spot remains stationary but the axonal extension continues

   • can see where new radiolabelled components are added

   • Suggests→ axons extend by inserting new microtubule building components at their distal tips→ PUSHING growth cones forward

2. Growth cone filopodia cultured neurons

   • shows tugging on other axons

   • if individual filopodia lifted off substrate with fine glass needle

   • Shows→ growth cone snaps into new direction

   • Suggests→ filopodia exert a tensile force

3. Actin depolymerising agent cytochalasin-B applied at concentrations where selectively disrupts the formation of filopodia at the growth cone

   • affects most senstiive part to the drug)

   • Shows→ slowing down and stabilising of axonal growth

     • Pathfinding ability is also lost

Overall what do these experiments show as to how filopodia work

1. probe their environment for directional cues

2. Pull the growth cone forward

3. Microtubule polymerisation in the central domain→ may help push it forward

Overall: the extension and retration cycles of filopodia are achieeved by a combination of three independent processes

1. Rapid actin assembly from G-actin monomers at the leading edge at the tips of filopodia

2. Myosin-powered retrograde flow of filamentous actin networks from leading edge to the transitional zone

3. Proximal recyling of filamentous actin inthe transitional zone

The rate of growth cone advance is determined by

1. Balance of actin assembly at the leading edge (pushing)

2. Rate of retrograde translocation of actin filaments towards the transition zone (pulling)

3. If anything is stopping it→ substrate

Therefore: growth cone advance cold be achieved by

1. an increase in rate of actin assembly at the leading edge

or

2. descreasing the rate by which myosin motors drive F-actin retrograde translocation (flow)

Dynamic microtubules are important in growth cone guidance→ what do they do

1. Constantly extend into the peripheral domain (guided by F-actin)→ into Filopodia

experimental evidence shows:

2. Local stabilisation of dynamic microtubules leads to→ growth cone turning toward the side of stabilisation

3. Local destabilisaition→ opposite effect

Demonstrates→ local sttabilisaition of dynamic MT is critical for growth cone navigation

How do these processes cause the growth cones to advance→ actin filaments in peripheral domain flow retrogradely

How do they flow retrogradely:

1. Assembly at the leading edge (‘plus’ ends)

2. translocation proximally by myosin motors

   • (actin is pulled back by myosins into transition zone)

3. enzyme mediated disassembly/recycling in the transition zone

   • F-actin is enzymatically disassembled (at ‘minus’ ends) and recyled

→ therefore→ a retrograde flow of F-actin maintained

Image→ can see the retrograde flow

How do the actin filaments interact with dynamic MTs

• interact, guiding dynamic MTs into the peripheral domain

• then shunting them out

• transient invasions of dynamic MTs precede the advance of stable MTs into the peripheral domain

Imaging MT dynamics

Dynamic MTs from the central domain transiently invade the peripheral domain

• Central domain→ stable MTs in parallel bunds

• Peripheral domain→ probed by single dynamic MTs

Imaging the interactions between F-actin and MTs (what do the F-actin bundles do)

Filopodial F-actin bundles

1. Guide dynamic MTs into peripheral domain

2. Clear dynamic MTs from the peripheral domain

MTs can be linked to F-actin (by MAPS)→ then tugged back into the central domain

→ can also be affected by the substrate it is on

How do growth cones achieve directed growth?

1. Adhesion of filopodium to a substrate via cell surface receptors and cell adhesion moelcules→ transduced to actin cytoskeleton

2. this decreases the myosin powered retrograde flow of F-actin

3. Thus decrease reate at which dynamic MTs can be shunted out of the filopodia

4. therefore reduced F-actin retrograde flow favours the establishemnt of MTs within the filopodium

5. Stabilisation of a dynamic microtubule within a filopodium promotes invasion of other microtubles

6. This stabilises the filopodium

7. thereby determines the direction of growth cone advance

Testing the role of filopodia in the turning

Procedure

• removing filopodia by using cytochalasin

• find a concetration where the filopodia just disappears

Results:

• Without filopodia→ aberrant pathway finding

• Remove the drug→ wash away→ get direction back

A single filopodium can direct growth

• If prevent filopodia from form by cytochalasin→ growth cones fail to navigate properly

Therefore: filopodia are essential for steering

Experiments showing how Dynamic MTs determine growth cone turning

Micropiteppte experiments

• control→ random growth

• Taxol→ stabilises MTs→ so go twards the direction of the substance

• Nocodazole→ depolymerises→ MTs are more stable on the opposite side to the substance

Overview of actin filaments and dynamic microtubules

• Actin filaments in filopodia serve as tracks

• Therese direct dynamic microtubules→ which themselves determine growth cone turning

Growth cone turning is regulated through changing the balance between:

1. Actin polymerisation vs retrograde flow and depolymerization

   • like non muscle myosin II motors→ seeding and serving proteins

2. Microtubule growth dynamics

   • capping proteins that stabilize vs severing proteins

3. Interactions between actin and microtubule cytoskeletons

   • like linker proteins

Any and all proteins involved in these processes are potential points for regulating growth cone guidance

How do substrates and guidance cues direct growth cone navigation?→ Electron microscopy grid experiments Letourneau

Procedure:

• coating islands in one substrate compared to others

• generate artificial landscapes of differential adhesivness

• have different adhesievness

• test for growth

Results:

• Most growth from intermediate stickiness

• showed distinct preferences of growth cones to extend over some substrates but noth otherrs

• What this shows→ need to be sticky enough for traction but not glued down

Results found for Concanavalin-A (most adhesive)

• Most adhesive substrates

BUT

• in fact poor supporters of axonal growth

  • seem to glue axons down to the extent of immobolising them

• We now know that→ such adhesive landscapes also exist in the developing embryo

How do these interactions work in vivo?

Combinations of receptors confer substrate choices→ Form a ‘molecular clutch’

• Components remain incompletely characterised

  • Extracellular matrix proteins (ECM)→ (substrate)

    • e.g Lamin and fibronectin and various collagens

    • → promote axon outgrowth

  • Intracellular→ integrin receptors (alpha and beta subunits)

    • particular neuron expresses at a given developmetnal stage

    • differen ECM molecule preference

      • types of substances it interacts with depends on the types of receptors it can interact with

These interactions can change in development

• e.g chick retinal ganglion cell axons:

  • alpha-6 integrin subunit→ prefer lamin as a substrate over fibronectin

• BUT→ with maturation of alpha-6 expression

Integrins can also interact with the neuronal guidance cues

• Netrins and Semaphorins

How do interactions with extracellular substrates change the dynamics of F-actin and microtubules inside the growth cone

• Integrin receptor complexes form a direct link betweenreceptor

• -substrate interactions act like a clutch

  • slowing down the F-actin back flow

1. Linkage of filopodial F-actin to a substrate (via receptor complex)

2. lead to attenuation of F-actin retrograde flow

3. Shifts the balance to favour net extension of F-actin

4. Permits dynamic MTs to extend towards the sit of growth cone-target interaction

Examples of CAMs

• Some have been found to interact directly with the cytoskeleton

• e.g Neural Cell Adhesion Moelcule (NCAM) isofroms 140 and 180 are associated with alpha-actinin and tubulins

The numerous proteins nucleate, stbilise fragment, cap and crosslink actin filaments and microtubules

• the activities and localisation of these proteins can be regulated through receptors on the growth cone surface

Other guidance cues come in the form of

• Cadherins

• many cell adhesion molecules (CAMs) largely of the immunoglobulin superfamily

  • e.g N-CAM

  • some mediate homophilic

  • some mediate heterophilic interactions

Overall Ascepts of growth gruidance

1. Differential adhesiveness

2. Guidance cues that function as signals

What is axon guidance controlled by

• Long range cues→ secreted (soluble)

• Short range cues→ membrane bound (physical attachment) immobilised

What do these cues do

• trigger an intracelular signalling cascade that modify the cytoskeletal dynamics

• causing

  • advance→ attractive

  • collapse/retraction→ repulsive cues

How do guidance cues and their receptors regulate growth cone dynamics

1. Receptors to the Slit, Netrin, Ephrin and Semaphorin families of guidance cues signal through intracellular pathways

2. Part of these are the Rho family of small GTPases

3. act as integrators of multiple signalling pathways

4. Attractive response → activation of the small Rho family GTPases Rac and CDC42 →Promotes actin polymerisation

5. Repulsive cues→ activate RhoA→ decrease actin polymerisation

Summary of how cues work

1. Myosin motors→ changing rate of F-actin flow

2. F-actin polymerisation

3. F-actin depolymerisation

4. F-actin- MT coupling

5. MT polymerisation

6. MT stability (end capping)

Some cues can be attractive or repulsive→ depending on the context example

Netrin signalling through the netrin receptor DCC

1. Generally→ attraction

2. Addition of Netrin co-receptor Unc-5→ repulsion

What are the growth cone responses to these cues mediated by

Intracellular [cGMP] : [cAMP]

• High [cAMP]/[cGMP]→ attraction

• Low [cAMP]/[cGMP]→ repulsion

How makes netrin repulsive vs attractive at certain points:

• due to activity of kinases (PKA)

• if PKA is inhibited→ changes the ratio of cGMP to cAMP

How does the cAMP/cGMP ratio regulate the response? Attraction

It regulates Calcium release from internal calcium stores

Attraction:

1. cue triggers influx of extracellular calcium through L-type calcium channels

2. Ca2+ signal is amplified by calcium induced calcium release (CICR) from ER

   • via ryanodine receptors (RyR) or InsP3 receptors

3. Generates high amplitude local clacium signals

4. activate Ca2+/ calmodulin-dependent kinase CamKII

5. CamKII can phosphoryalte microtuble motors

6. initiating directed vesciel exocytosis at the side of elevated calcium

7. provides plasma membrane necessary for growth cone tunring toward an attractive cue

8. as well as directed deliverly of signalling and adhesion complex components

9. in paralle, cytoskeletal dynamics are changed to favour stabilisation and polymerisation of microtubules and filamentous actin

How does the cAMP/cGMP ratio regulate the response? Repulsion

1. repulsive cue triggers low amplitude calcium influx

2. not amplified by internal stores

3. low ampltitude signals appear to act via Ca2+/calmodulin-dependent phosphatase, Calcineurin

4. This has a higher affinity to Ca2+ than CamKII→ so can be activated at lower [Ca2+]

5. Calcineruin activation triggers clathrin mediated endocytosis

6. leads to growth cone retraction

   • partly by removal of adhesion complex components such as integrins

What is decicevness facilitated by

• mutual inhibition between cGMP and cAMP signalling

  • cGMP→ inhibits

  • cAMP→ promotes calcium influx

Because growth cones can navigate even if cut off from cell body this show

• growth cones can navigate autonomously

What is the machinery that is necessary for pathfinding and how is it regulated?

1. Is transciption required?

   • Cut off cell body

   • result→ still navigate

   • shows→ Do not need transciption

2. Is protein synthesis required?

   • add protein synthesis inhibitors (cycloheximide, anisomycin)

   • result→ unbiased no direction

   • shows→ protein synthesis is required

Local translation is required for growth cone steering

for example:

• attractive or repulsive turning reponses of growth cones to netrin-1 and Semaphorin3A (respectively)

• require local translation witin the growth cone

• among transcipts regulated by local tranlsation in respoponce to guidance in response to guidance cues are:

  • beta-actin (attraction)

  • cofilin

  • RhoA→ repulsion→ including actin depolymerisation

in summary→ different mRNA activated

• difference receptors causes different pools of mRNAs to be activated

• how does mRNAs

  • stya, get there and get pooled

• mRNA increase the seniticity of the direction due to the cue sensed by the receptors seen above

What else is also important in growth cone guidance and adaptation and re-sentiisation

• local endocytosis of receptors and targeting of components

Summary of growth cone guidance