Neuronal Cytoskeleton14

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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

• Linear array

• Asymmetric

• a

-tubulin at one end

• b

-tubulin at other end

• Same polarity

• Plus end = fast growing

• Minus end = slow growing

+ -

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 Assembly-Promoting and MT-Stabilizing

Property

HMW-τ

LMW-τ

• MAP-1A

-1B

-2A, B

• -2C, D

• -4

DCX

LLS1

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

CLIP-170

APC

EB1

Kinesins

Dyneins

Gephyrin

OP18/ stathmin

Microtubule end-binding Protein

Attachment of Microtubules to endosomes

Attachment of Microtubules to cell cortex

Attachment of Microtubules to cell cortex

Microtubule-Activated ATPases

• Move organelles from “minus” to “plus” ends.

• Move organelles from “plus” to “minus” ends.

Proteins Anchoring MTs to Membrane Receptors

Binds glycine receptors

Microtubule-destabilizing Proteins

Highly abundant – favours MT destabilization

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

– 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 P i

» 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

• Motor proteins

– Kinesin

Cytoskeleton

Cytoskeleton

• Motor proteins

– Kinesin

• 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

ATP Cycle

Attached

τ on

Detached

τ off

δ = WD = step size;

V = ATPase activity v = in vitro sliding velocity

Attachement

Power stroke

Back stroke

δ = working distance

Detachment

Cytoskeleton

• Motor proteins

– Kinesin

• One member of a superfamily of related proteins

– 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

• 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

Mass (kDal) and Distribution

Class and Protein

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

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

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.

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