The Axonal Cytoskeleton and Molecular Motors

08/2007

Lecture by Dr. Dirk Lang

Dept. of Human BiologyUCT Medical School

Room 6.10.1Phone: 406-6419E-Mail: DIRK.LANG@UCT.AC.ZA

The Cytoskeleton

Microfilaments IntermediateFilaments

Microtubules

SIZE 7 – 9 nM 10 nM 24 nM

STRUCTURE Thin filaments (+/-)

Rope-like filaments Hollow tubules(+/-)

STRENGTH Fragile Strong, flexible Rigid

SUBUNITS Globular actin Extended α-helix α- and β-tubulin

ENERGY ATP Phosphorylation GTP

EVOLUTION Highly conserved Variable Highly conserved

EXPRESSION All cells Different families in various cell types

All cells

Cytoskeleton and Axon Growth

(Wolpert)

„Newborn“ neurones

form axons that are guided towards

their target cells

Formation of Nerve Connections:

• Short range Contact-mediated interaction Promote or inhibit axon growth

• Long range Diffusible Establish gradients Attract or repel growing axons

Biochemical Guidance Cues:

The Neurone During Axon Growth:

(Kandel/Schwartz)

The tip of a growing axon is called the

growth cone

The Growth Cone:

(Wolpert)

The moving and sensing tip of a growing axon

(Gilbert)

Growth Cone – Molecular Equipment:

Cytoskeletal elementsfor structural maintenance, elongation and navigation

(Immunostaining of tubulin (red) and actin (green))

Dynamics of actin polymerisation

Molecular “Switches”:Central elements in the response of growth cones to guidance cues:

Small GTPases Rho(mediates repulsion)and Rac (mediates attraction)

Rho and Rac

Retinal axons growing on a laminin substrate…

Stimulation of Rac

Retinal axons with soluble Nogo-A protein added to culture medium…

Stimulation of Rho

Axonal Transport and Molecular

Motors

(Distal)

(Kandel/Schwartz)

(Proximal)

Neurons have proximal-distal (=basal-apical)

polarity

(Kandel/Schwartz)

Axonal Transport:

Towards synapse

Anterograde:

Retrograde:

Towards cell body

How are molecules transported in axons?

Anterograde transport - towards synapse

Retrograde transport - towards cell body

Rapid transport (3m/s)250mm/day

Slow transport1mm/day

Membrane-bound vesicles and proteins are transported many micrometers along very well defined routes and delivered to specific addresses

Axonal transport does not require an intact cell

Extruded axoplasm assays - Cytosolis squeezed from the axon with aroller onto a glass coverslip.

Addition of ATP shows movementby videomicroscopy

Vesicle movement in this systemis about 1-2um/s similar to fast axonaltransport.

Stain with an anti-gamma tubulin antibody (red). Gamma-tubulin at initiates synthesis at one end (-) (green)

Intracellular transport requires microtubules

Microtubules in Neurones

Microtubules provide tracks for movement of vesicles along the

axon

Movement occurs in individual filaments and is

cargo-specific

Retrograde transport

Antrograde transport

Microtubule Motor Proteins

10nm

dynein kinesin

Light chains

Heavy chains

Minus end Plus end

dynein kinesin

25nmmicrotubule

Molecular structure of dyneins and kinesins

Dyneins - composed of 2-3 heavy chains with a total Mr of 1,000kD

- interact with microtubules indirectly through microtubule- binding proteins

Kinesin

Dimer of a heavy chain complexed to a light chain

Mr= 380kD

Three domains:1) Large globular headBinds microtubules and ATP2) Stalk3) Small globular headBinds to vesicles

To date 12 different familyMembers have been identified

Structure of Kinesin

How does Kinesin catalyze transport?

Beads coated with kinesin binds to microtubules and move along

Dynein promotes movement in the opposite direction

How does Kinesin catalyze transport?

How to build directionality and specificity?

• Multiple motor proteins can bind to a given cargo

• Each kinesin/dynein transports a specific cargo

Interaction between cargo and motor protein is indirect

Intracellular transport, positioning of organelles and growth of ER requires motor proteins and microtubules

Movement of pigmented granules in a cell

In addition to kinesin and dynein, myosin can also function as a motor protein on actin filaments

Muscle Contraction;The Sarcomere

Light microscopic structure of myofibrils

The Contractile Apparatus:A highly ordered array of myofilaments

(Wheater’s)

Muscle fibre (LM):

• Each muscle fibre contains numerous myofibrils• Myofibrils are made up from subunits called sarcomeres• Sarcomeres are the actual contractile units, composed of myofilaments

Myofibrils (EM):

Sarcomere

Myofibril

Myofilaments in the Sarcomere:

(Wheater’s)

The Sliding Filament Theory:

(Wheater’s)

Sliding action of actin- and myosin myofilaments

ActinMyosin

Composition of Myofilaments: A set of proteins make up thin and thick filaments

Thin filament:

• Thin filaments are formed by polymerisation of G-actin into F-actin• Thick filaments are assembled from myosin heavy and light chains• Actin filaments anchored to Z-line, myosin filaments to M-line

Thick filament:

(Stevens/Lowe)

Muscle Contraction Requires Energy: Sliding of myofilaments powered by ATP hydrolysis

• Hydrolysis of myosin-bound ATP causes myosin to bind to actin• Myosin undergoes conformational change, moves along actin• Replacement of ADP by fresh ATP causes detachment of myosin

(Stevens/Lowe)