1. The Axonal Cytoskeleton and Molecular Motors
Lecture by Dr. Dirk Lang
Dept. of Human Biology
UCT Medical School
Room
Phone:
08/2007

2. The Cytoskeleton Microfilaments Intermediate Filaments 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
EXPRESSION
All cells
Different families in various cell types

3. Cytoskeleton and Axon Growth

4. Formation of Nerve Connections:
„Newborn“ neurones form axons that are guided towards their target cells
(Wolpert)

5. Biochemical Guidance Cues:
Short range
Contact-mediated interaction
Promote or inhibit axon growth
Long range
Diffusible
Establish gradients
Attract or repel growing axons

6. The Neurone During Axon Growth:
The tip of a growing axon is called the
growth cone
(Kandel/Schwartz)

7. The moving and sensing tip of a growing axon
The Growth Cone:
The moving and sensing tip of a growing axon
(Wolpert)

8. Growth Cone – Molecular Equipment:
Cytoskeletal elements
for structural maintenance, elongation and navigation
(Immunostaining of tubulin (red) and actin (green))
(Gilbert)

10. Dynamics of actin polymerisation

12. Molecular “Switches”:
Central elements in the response of growth cones to guidance cues:
Small GTPases Rho
(mediates repulsion)
and Rac (mediates attraction)

13. Rho and Rac

14. Retinal axons growing on a laminin substrate…
Stimulation of Rac

15. Retinal axons with soluble Nogo-A protein added to culture medium…
Stimulation of Rho

16. Axonal Transport and Molecular Motors

17. Neurons have proximal-distal (=basal-apical) polarity
(Kandel/Schwartz)

18. Axonal Transport: Anterograde: Towards synapse Retrograde:
Towards cell body
(Kandel/Schwartz)

19. How are molecules transported in axons?
Membrane-bound vesicles and proteins are transported many micrometers along very well defined routes and delivered to specific addresses
Rapid transport (3mm/s)
250mm/day
Slow transport
1mm/day
Anterograde transport - towards synapse
Retrograde transport - towards cell body

20. Axonal transport does not require an intact cell
Extruded axoplasm assays - Cytosol
is squeezed from the axon with a
roller onto a glass coverslip.
Addition of ATP shows movement
by videomicroscopy
Vesicle movement in this system
is about 1-2um/s similar to fast axonal
transport.

21. Intracellular transport requires microtubules
Stain with an anti-gamma tubulin antibody (red). Gamma-tubulin at initiates synthesis at one end (-) (green)

22. Microtubules in Neurones

23. Microtubules provide tracks for movement of vesicles along the axon

24. Movement occurs in individual filaments and is cargo-specific
Antrograde transport
Retrograde transport

25. Microtubule Motor Proteins

26. Molecular structure of dyneins and kinesins
10nm
Light
chains
dynein
kinesin
Heavy
chains
25nm
microtubule
Minus end
Plus end
Dyneins - composed of 2-3 heavy chains with a total Mr of 1,000kD
- interact with microtubules indirectly through microtubule binding proteins

27. Kinesin Dimer of a heavy chain complexed to a light chain Mr= 380kD
Three domains:
Large globular head
Binds microtubules and ATP
2) Stalk
3) Small globular head
Binds to vesicles
To date 12 different family
Members have been identified

28. Structure of Kinesin

29. How does Kinesin catalyze transport?

30. Beads coated with kinesin binds to microtubules and move along
Dynein promotes movement in the opposite direction

31. How does Kinesin catalyze transport?

32. How to build directionality and specificity?
Multiple motor proteins can bind to a given cargo
Each kinesin/dynein transports a specific cargo

33. Interaction between cargo and motor protein is indirect

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

35. Movement of pigmented granules in a cell

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

37. Muscle Contraction;
The Sarcomere

38. Light microscopic structure of myofibrils

39. The Contractile Apparatus:
A highly ordered array of myofilaments
Each muscle fibre contains numerous myofibrils
Myofibrils are made up from subunits called sarcomeres
Sarcomeres are the actual contractile units, composed of myofilaments
Muscle fibre (LM):
Myofibrils (EM):
Myofibril
(Wheater’s)
Sarcomere

40. Myofilaments in the Sarcomere:
(Wheater’s)

41. The Sliding Filament Theory:
Sliding action of actin- and myosin myofilaments
Myosin
Actin
(Wheater’s)

42. Composition of Myofilaments:
A set of proteins make up thin and thick filaments
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
Thin filament:
Thick filament:
(Stevens/Lowe)

43. 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)