Neuronal growth cone is where new membrane is added

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• Neuronal growth cone is where new membrane is
  added
• Evidence
• 1. Dye particles - staining neurite shaft remain
  in fixed location relative to cell body -
  although axon elongates
• 2. Cut and remove cell body in culture growth
  cone grows and pathfinding persists.
• Where is new membrane added?
• at tips of filopodia ?
• at base of growth cone?
• does it happen asymmetrically?
• what controls this?
• Filopodia are in constant motion
  extending/retracting
• some adhere, generate force and pull the growth
  cone forward
• there is also a pushing force from new
  materials arriving at growth cone eg membrane
  vesicles.What causes this?
• Cajal 1900 - tied a thread around nerve and
  showed that materials build up on either side of
  the blockade
• established anterograde and retrograde axonal
  transport to and from the cell body.

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• Different materials are transported at different
  rates
• - use neuronal cytoskeleton to do this - composed
  of
• 1. Microfilaments - single filaments 7nm diameter
  bundles are called - F-actin (filamentous)
• 2. Microtubules - 25nm diameter
• 3. Neurofilaments - 8-10nm diam - mostly
  structural
• Polymers of variable length do not extend full
  length of axon.
• Microfilaments are polymers of g-actin (globular)
  42kD protien 375 amino acids
• this self-assembles into a twisted helix of
  staggered parallel rows of monomers - equivalent
  to thin filaments of skeletal muscle (see Figure)
• F-actin is polarized - monomer addition faster at
  one end than the other
• label with protein called meromyosin
• one end looks barbed - () end - always anchored
  to inner membrane at this end - assemble monomers
  faster here
• other end looks pointed (-) end - disassemble
  monomers here

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• Microtubules - polymers of tubulin.
• Tubulin forms in dimers composed of ? and ?
  tubulin -each 54kD.
• Dimers form protofilaments with ? and ?
  repeating along length of each protofilament.
• In EM cross section - 13 protofilaments are
  grouped to form a hollow cored cylinder - the
  microtubule (see fig).
• Extremely dynamic - change length by dimer
  addition / subtraction - so cytoplasm contains
  large soluble pool of tubulin.
• MTs also are polarised
• fast growing ends are ALL oriented distally in
  axons (towards growth cone, away from cell body)
  
• in dendrites 50/50 /- ends oriented
  distally
• In general the cytoskeleton is organized
  spatially in axons
• Microfilaments mostly found immediately
  underneath membrane
• neurofilaments and microtubules more common in
  central part of axon.

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• Microtubules extend into palm of growth cone
• microfilaments predominate in periphery and
  extend as bundles to form the core of filopodia.
• Often closely associated
• MT-MF in or near base of filopodia
• also contacts between fast growing barbed end of
  microfilaments and inner face of plasma membrane
• Push and contact integral membrane-spanning
  proteins.
• Rapid freeze-fracture technique shows 3-D picture
  of cytoskeleton - extensively crosslinkages
• MT-MT MF-NF NF-NF
• MT-organelles MF-MT NF-organelles
• MF-membrane NF-membrane

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• Almost all protein synthesis is in cell body
• anterograde transport supplies
• neurotransmitters new membrane new cytoskeleton
  
• retrograde transport functions
• recycle lysosomal products
• signaling eg NGF-receptor complex moves to
  nucleus
• so signals damage if interrupted
• Different substances and organelles are
  transported at different rates and in different
  directions eg
• slow anterograde transport
• components of NF and MT 0.1 - 2 mm/day
• actin next slowest 2 - 4
  mm/day
• Fast anterograde/retrograde transport moves
  membrane bound organelles eg. Mitochondria/vesicle
  s at 100-400mm/day.
• Mechanism of fast transport known.
• Microscopy allows visualization of dynamics of
  single MT (25nm diameter)

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• Squeeze out squid axoplasm
• directly watch single MTs acting as rails for
  organelle transport
• MTs supports BI-directional transport
• 2 organelles can pass each other- so separate
  tracks on a single MT.
• Organelles may possess multiple MT binding sites
  - also exist variety of MT-associated proteins.
• Kinesin protein present in axoplasm
• mix latex microbeads MT kinesin - kinesin
  binds to beads and they are moved in an
  anterograde direction.
• dynein - responsible for retrograde transport
• adsorb dynein to glass coverslip add ATP MTs
  directly observe MT gliding 1 - 2 ?m/s
  (see Figure)
• ends forward (all ends point distally in
  axon) so coverslip is moving in opposite
  direction towards - end of MTs - retrogradely
• Kinesin and dynein are major part of the cross
  bridges between MT- organelles in freeze fracture
  images
• conformational change produces the power stroke
  to drive organelles walking along a tubule.

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• (See handout figures)
• Cell surface receptors mediate substrate
  attachment - via proteins called integrins
• these bind directly through the plasma membrane
  to the extracellular matrix.
• create a direct link between internal signaling
  molecules and extracellular proteins eg. laminin
  / fibronectin
• filopodia contact and bind integrins
• some proteins eg. talin directly couples
  integrins to the microfilaments
• binding may immobilize the actin
  microfilaments, due to a rigid mechanical
  coupling between cytoskeleton and extracellular
  matrix.
• myosin and associated stable sub-membrane
  elements now move in direction of barbed ends on
  immobilised actin filaments.

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• (Material not covered in lecture)
• How is the cytoskeleton transported?
• Used to think that it was assembled in cell body
  and transported as part of slow component - as
  moving intact/preformed matrix BUT
• 1. Pools of unassembled actin and tubulin are
  present in growth cone
• 2. ends (preferred assembly of MTs are all
  distal in axon
• 3. Microapply MT inhibitor colchicine - most
  effective at blocking growth at growth cone.
• MTs not continuous distal ends occur along the
  length of the axon.
• Recent controversy - actin and tubulin
  transported as monomers - exchanged with intact
  cytoskeleton in passing
• ie. Cytoskeleton is dynamically changing, but
  stationary as a matrix.
• Evidence from mouse sensory neurone work (drg)
  indicates polymers of bot actin and tubulin are
  both tranlocating and exchanging subunits - more
  recently this has been questioned in frog work.

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• Microinject photoactivateable tubulin in to 2
  cell stage frog embryo - incorporates into all
  MTs in neurites.
• Activate with local flash creates a fluorescent
  mark - observe movement. Find polymer
  translocation - transport MT whole down axon -
  different technique and different neuronal type
  might explain different conclusion??
• Also used caged fluorescein labelled tubulin -
  injected into both mouse and frog tissue.
• Mouse drg neurones see stationary areas of
  photoactivated tubulin - no translocation of MTs
  and no movement of polystyrene beads attached to
  plasma membrane of neurite shaft
• Frog nerves - translocation of whole
  photoactivated zone - MTs move as a block
  correlated with beads moving anterogradely on
  plasma membrane surface at same rate.
• But this would suggest that polymers of the
  cytoskeleton and elements of the cell membrane
  were translocated by different means in 2
  different nerve types. Seems unlikely /
  unsatisfactory.
• Resolved with use of nM vinblastine which arrests
  assembly of MTs but does NOT allow existing MT to
  disassemble
• See progressive increase MTs in growing axon and
  progressive decrease in MTs in cell body, so must
  be transported down axon.

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• But remain very short - much shorter than normal
  - implies usually elongate by local assembly in
  axon. Ie. Need both transport and local assembly
• ALSO - MTs in axons growing in nM vinblastine,
  uniformly oriented with ends pointing distally.
  
• Implies that in absence of MT assembly, the
  transport properties / mechanisms can impose the
  characteristic polarity on MTs.
• Actin dynamics and forward movement