Transcript Neuronal Cytoskeleton14

Neuronal Cytoskeleton:
Structure and Function
Cytoskeleton
• Eukaryotic cell Skeletal System
– Three well defined filamentous structures
• Microtubules
• Microfilaments
• Intermediate filaments
Cytoskeleton
• Eukaryotic cell Skeletal System
– Micotubules
• Rigid tubes
• Tubulin
– Microfilaments
• Solid / thinner
• Actin
– Intermediate filaments
• Tough ropelike fibres
• Many related proteins
Cytoskeleton
• Functions of Cytoskeleton
– 1 Dynamic scaffold
– 2 Internal framework
– 3 Network of highways
– 4 Force generating apparatus – cell movement
– 5 m-RNA anchoring
– 6 Cell division
Cytoskeleton
• Microtubules – Structure and Composition
– Components of a diverse array of structures
• Mitotic spindle
• Core of flagella and cilia
– Tubes of globular proteins
• Longitudinal rows
• Protofilaments
• Cross-section – 13 rows of protofilaments – circular
– Dimeric building blocks – a-tubulin and b-tubulin
Cytoskeleton
• Microtubules – Structure and Composition
– a-tubulin and b-tubulin
• Similar 3-D structure
• Form dimers
• Fit together – non-covalent bonds
Cytoskeleton
• Microtubules – Structure and Composition
– a-tubulin and b-tubulin
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Linear array
Asymmetric
a-tubulin at one end
b-tubulin at other end
Same polarity
Plus end = fast growing
Minus end = slow growing
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Components of Neuronal Cytoskeleton
(cont’d)
• Microtubules
- Formed by 13 longitudinal strands arranged
in helical configuration.
- Each strand is composed of aligned globular
heterodimers consisting of α- and β-tubulin
subunits.
- This leads to polarized assembly with one
end having mainly exposed α subunits and the
other end having mainly exposed β subunits.
Cytoskeleton
• Microtubules – Structure and Composition
– a-tubulin
• Bound GTP
– Not hydrolysed
– Non-exchangable
– b-tubulin
• Bound GDP
• Exchanged for GTP prior to assembly
The Cytoskeleton in Neuronal
Morphogenesis
• Neurite (out)growth of growth cones.
- Occurs through the protrusion of filopodia
and lamellipodia and the subsequent invasion
of the expanded bases of filopodia and
lamellipodia by MTs.
- The bundling of the invading MTs constitutes
the consolidation of the growth of the neurite.
The Cytoskeleton in Neuronal
Morphogenesis (cont’d)
• Axonal Maturation –
-When axons reach their targets, the
cytoskeleton of the GC is remodeled and
converted into the cytoskeleton of the
presynaptic terminal.
- Motility and extension cease.
- Synapsin accumulates and cross-links
synaptic vesicles to microfilaments.
Model showing the Origin of Axonal
Microtubules
The structure and action of growth cones
Components of Neuronal Cytoskeleton
(cont’d) Microtubules (cont’d)
Protein
• HMW-τ
• LMW-τ
• MAP-1A
• -1B
• -2A, B
• -2C, D
• -4
• DCX
• LLS1
Assembly-Promoting and MT-Stabilizing
Property
• Present in PNS.
• Abundant in axons of CNS; Contributes to MT
stabilization
• Abundant in dendrites and mature neurons
• Present in both axons and dendrites; contributes
to neural migration and initial neurite outgrowth.
• Present in both soma and dendrites (spines too).
• Present in axon, dendrites, and glial cells.
• Present in glial cells and in immature neurons.
• Contributes to neuronal migration.
• Contributes to neuronal migration.
Components of Neuronal Cytoskeleton
(cont’d) Microtubules (cont’d)
Protein
Microtubule end-binding Protein
• CLIP-170 • Attachment of Microtubules to endosomes
• APC
• Attachment of Microtubules to cell cortex
• EB1
• Attachment of Microtubules to cell cortex
Microtubule-Activated ATPases
• Kinesins
• Move organelles from “minus” to “plus” ends.
• Dyneins
• Move organelles from “plus” to “minus” ends.
Proteins Anchoring MTs to Membrane Receptors
• Gephyrin
• Binds glycine receptors
Microtubule-destabilizing Proteins
• OP18/
• Highly abundant – favours MT destabilization
stathmin
Cytoskeleton
• Microtubule Associate Proteins (MAPs)
– Mostly in brain
– Exception – MAP4 – many cell types (non-neuronal)
• Domain attaches to microtubule
• Domain extends out – filament
• Various roles
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Cross-bridges connecting microtubules
Increase microtubule stability
Alter microtubule rigidity
Alter microtubule rate of assembly
Activity – phosphatases and phosphokinases
Cytoskeleton
• Microtubules – structural roles
– Determine cell shape
• Axons of nerve cells
– Internal organization
– Axonal transportation
• Materials moved from cell body – along axon
– Anteriograde
• From axon to cell body – endocytosis
– Retrograde
– Axons have microfilaments, intermediate filaments
and microtubules
• Interconnected
Interactions Among Cytoskeletal Components
AL, axolinin (squid giant axon MAP); RB, actin MF-assoc domain; RA MT-assoc domain
PL, plasma membrane
Cytoskeleton
• Microtubules – structural roles
– Passive
– Tracks for many motor proteins
– Motor proteins use ATP
• Move cellular cargo
– Vesicles, Mitochondria, Lysosomes, Chromosomes
– Motor proteins – Three families
• Myosins
• Kinesins
• Dyneins
– Kinesins and Dyneins – move on microtubules
Cytoskeleton
• Motor proteins
– Move unidirectionally
– Stepwise
– Series of conformational changes
• A mechanical cycle
• Coupled to chemical cycle – Energy
– Steps –
» ATP binding to motor
» Hydrolysis of ATP
» Release of ADP and Pi
» Binding of new ATP
Cytoskeleton
• Motor proteins
– Kinesin
• Tetramer
– 2 identical heavy and 2 identical light chains
• Functional domains
– Pair of globular heads
» Bind microtubule
» ATP-hydrolysing
– Neck / stem and tail
– Tail binds cargo
• Move toward plus end of microtubule
– Plus end directed
Cytoskeleton
• Motor proteins
– Kinesin
Cytoskeleton
• Motor proteins
– Kinesin
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Velocity proportional to [ATP]
Move one heterodimer at a time (step)
One head – always attached
Heads are coordinated
– Each at different stages of chemical and mechanical cycles
– When one head binds
» Conformational change in adjacent neck region
» Swings other head forward
• Kinesin – ‘walks’ along microtubule
Common Properties of Kinesin
1. Structure
N-terminal globular head:
motor domain, nucleotide binding and hydrolysis, specific
binding sites for the corresponding filaments.
C-terminal: structural and functional role: myosins
2. Mechanical properties, function
cyclic function and work:
motor  binding to a filament  force  dissociation 
relaxation.
1 cycle requires 1 ATP hydrolysis.
They can either move (isotonic conditions) or produce
force (isometric conditions)
The ATP Hydrolysis Cycle
δ = working distance
ATP Cycle
Attached
τon
Power stroke
Detached
τoff
Attachement
δ = WD = step size;
V = ATPase activity
v = in vitro sliding
velocity
Detachment
Back stroke
Cytoskeleton
• Motor proteins
– Kinesin
• One member of a superfamily of related proteins
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Kinesin related proteins – KRPs
Kinesin-like proteins – KLPs
> 50
Heads similar
Tails heterogenous – binding different cargoes
• Motor proteins
Cytoskeleton
– Kinesin-mediated organelle transport
• Kinesins aligned with plus ends away from nucleus
• Tend to move organelles in anterograde direction
• Motor proteins
Cytoskeleton
– Cytoplasmic Dynein
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Movement of cilia and flagella
And ubiquitous motor protein in eukaryotic cells
Huge - > 1.5 Mda
2 identical heavy chains
Many intermediate and light chains
Heavy chain
– Large globular head
– Force generating engine
– Minus end directed
Cytoskeleton
• Motor proteins
– Cytoplasmic Dynein – Two roles
• Force generation – spindle – mitosis
• Minus-end directed motor for Golgi Complex and vesicles
• Requires a sub-unit complex dynactin
1.1-MDal protein (10-11 pps), which include p150-Glued
and the filament-forming actin-related protein (ARP1).
Dynactin and actin bind via the p150-Glued subunit.
So, dynactin increases the run length of the dynein-driven
movements, acting as a processivity factor for the dyneindriven motor on the MT.
Components of Neuronal Cytoskeleton
• Microfilaments
- Composed of polymerization of actin (α and
β monomers.
- Must bind ATP to polymerize.
Dynamics occur through the incorporation and release of tubulin
heterodimers at the ends of polymer
Microfilament dynamics are also associated with the
Exchange of actin monomers at the polymer ends.
Note the replacement of subunits.
Myosin
The headgroup of mysosin walks toward the
head group of the actin filament (microfilament)
Components of Neuronal Cytoskeleton
(cont’d)
• Intermediate Filaments
– About 12 different isoforms, based on sequence
homologies.
– Expression is developmentally dependent.
- Neural stem cells express nestin (Class VI).
- Before differentiation, neuroblasts and neurons
express vimentin (Class III).
- See next slide for Table
Polymerization of Intermediate Filaments
Central rods of the α-helix
are hydrophobic interX 
coiled-coil dimer:
Dimer tetramer
(antiparallel structure).
Tetramers are connected
Longitudinally
(protomers).
8 protofilaments 
1 filament
Intermediate Filament Proteins
Class and Protein
Mass (kDal) and Distribution
I. Acidic cytokeratins
II. Basic Cytokeratins
III. Vimentin
Desmin
GFAP
Periferin
IV. NF-L
NF-M
NF-H
α-interferon (NF-/66)
V. Lamins
VI. Nestin
• (40-64); Epithelial cells
• (52-68); Epithelial cells
• (55); Mesenchymal cells,
immature neurons, glial cells
• (53); Myocytes
• (51); Astroglial cells
• (57); PNS neurons
• (68); Neurons
• (145); Neurons
• (200); Neurons
• (66); CNS neurons
• (66-72); All cells
• (240); CNS neural stem cells
The Cytoskeleton in Neuronal Morphogenesis
(cont’d): Axonal Maturation (cont’d)
• Myelination –
Characterized by the radial growth of the axon (increased
diameter), which is because of increased neurofilament
expression and its phosphorylation.
Next slide: Stimulation of axonal neurofilament
phosphorylation by myelinating Schwann cells.
- Note the interaction between Schwann cell
membrane and axonal membrane molecules triggering
either the activation of a neurofilament kinase (k) or
the inhibition of a phosphatase (P)  enhanced
phosphorylaiton of the ‘tail’ domains of the NF-H and
NF-M.
Regulation of Myelination
• Lateral projections of the NF polymers and
high degree of phos  electrostatic repulsion
 wide interfilament spacing and incr axonal
calibre.
• In nonmyleinated axon segments, the activity
of the phosphatase > kinase activity  NF less
phosphorylated  narrower interfilament
spacing and decreased axonal diam.
Neuronal Polarity
Dendrites
Axons
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Uniform calibre
Few branches
Lack polysomes
Little, if any, protein synthesis
Fast growth
Neurofilament abundant
Uniform polarity of
microtubles
Narrow spacing between
microtubules
Abundance of tau protein
Presence of αγ spectrin
Highly phosphorylated NF-M
and NF-H
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Tapered morphology
Highly branched
Presence of polysomes
Some protein synthesis
Slow growth
Abundance of microtubles
Mixed polarity of microtubules
Wide spacing between
microtubules
• Presence of MAP2A, B
• Presence of αβ spectrin
• Nonphosphorylated NF-M and
NF-H
Cytoskeleton in Neuronal Plasticity
• Dendritic spines as postsynaptic structures.
• Actin – provides the main structural basis for
cytoskeletal organization within dendritic spines
(lack MTs and IFs).
• Actin rearranges in synaptic plasticity (neuronal
connectivity).
• LTP of synapses in hipp DG assoc with
phosphorylation of cofilin, which  incr in f-actin
within spines  growth and strengthening of
synapses.
• Cytoskeletal modifications also alter neuronal
physiology through modulating nt receptors and
ion channels, which are anchored to the
membrane cytoskeleton.
Neurons are Highly Polarized Cells whose
Organelles and Proteins are Differentially
Distributed
• The soma is the main site of macromolecule
synthesis.
• The dendrites contain free ribosomes and
synthesize some of their proteins.
- mRNA trafficking and local protein synthesis
in dendrites.
• The axon, to a large extent, lacks protein
synthesis machinery.
Axonal Transport Allows Bidirectional
Communication between the Soma and the
Axon Terminals
• Fast anterograde axonal transport is
responsible for the movement of membranous
organelles from the soma towards the axon
terminal, and allows for renewal of axon
proteins.
- Recall the role of kinesin and ATP.
Retrograde axonal transport returns old
membrane constituents, trophic factors,
exogenous materials to the soma.
• Dynein.
• Mechanism that regulates the direction of
vesicle movement.
• Functions of retrograde transport.
Slow Anterograde Axonal Transport Moves
Cytoskeletal Proteins and Cytosoluble
Proteins
• The different cytoskeletal elements are assembled
and connected by bridges in soma.
• Cytoskeletal proteins are transported in a soluble
form or as isolated fibrils and assembled during
their progression.
• The transport of microtubles and neurofilaments
is bidirectional, intermittent, asynchronous, and
occurs at the fast rate of known motors.
Axonal and Dendritic Intraneuronal
Transport
• Slow Component A: Moves proteins at a rate of
0.2-1 mm day-1; Consists mostly of pps assoc with
NFs and MTs.
• Slow Component B: Comprises > 100 pps moving
at 2-8 mm day-1. Transport of MTs and actin
filaments including their assoc proteins.
• Intermediate Component: Mitochondria
conveyed along MTs at 50-100 mm day-1.
• Fast Component: Complex group of membraneassoc proteins moving at 200-400 mm day-1 and
corresponds to most membrane organelles along
MTs.