1. Motor Proteins

2. 2 Motor Systems Actin-based motility: motor proteins are myosins
Tubulin-based motility: motor proteins are kinesins and dyneins

3. Roles of the actin-based system
Cell Crawling - lymphocytes
Growth cone extension
‘Muscle-like’ contraction of ovarian follicles, mammary gland ducts, etc.
Muscle contraction
Distribution of vesicles, intermediate filaments and organelles within the cytoplasm

4. Actin-based motility: the Myosins
The head is both actin-binding and ATP binding; the purple light chain has a regulatory role. Myosin II is muscle myosin.

5. There are lots of myosins
18 different myosin families have been identified (I –XVIII)
There are a total of 40 myosin genes in the human genome – i.e. there are multiple copies of some of the genes

6. The Myosin Activity Cycle
Myosin head energized, with bound ADP and Pi,, not attached to actin
ATP hydrolyzed
Myosin head attaches to actin
HEAD DETACHED
Head detaches from thin filament
HEAD ATTACHED
Head rotates - Powerstroke transmits force to thin filament – head is deenergized
ATP replaces ADP and Pi on myosin head

7. Preparation of actin cables pointing in same direction
Study of the movement of myosin against actin filaments – an in vitro motility assay
Preparation of actin cables pointing in same direction
Used cells from a giant alga that uses these cables for moving chloroplasts around.
Open algal cell and add yellow fluorescent beads coated with myosin, then add ATP and take time lapse photography

8. Cartoon of myosin coated bead actin preparation

9. A series of exposures taken at intervals of 1s.
This picture shows that the myosin coated beads moved along the actin cables
Red dots are chloroplasts which fluoresce red

10. Speed of movement
Myosin coated beads moved unidirectionally and movement was dependent on ATP
The speed of beads coated with muscle myosin is 5 µm/s which is the same speed as the contraction of sarcomeres in muscle
Different myosins move at different speeds. Smooth muscle myosin moves at 1 µm/s.

11. Studies of movement due to a single myosin molecule
Use a setup in which focused laser beams create “optical traps”. These optical traps can hold small objects. The force is controlled by adjusting the intensity of the laser beam.

12. Actin filament is held in optical trap via one or two attached beads
Myosin concentrations are kept low so that only one myosin contacts the actin filament

13. ATP is also kept low so that only one ATP binds to each myosin head

14. displacement
time
Results show that the myosin pulls on the actin filament in a stepwise, or ratchet-like fashion

15. A single ATP molecule is hydrolyzed resulting in a power stroke and displacement of around 10 nm.

16. The force generated can also be determined and it is around 3-7 picoNewtons (pN)

17. Is the force and displacement what you would expect from the energy supplied by 1 molecule of ATP?
DG= -12 kcal/mole for ATP hydrolysis = 16 x cal per molecule ATP

18. 1 pN x 10 nm displacement = 2.5 x 10-21 cal
3-7 pN of force generated ---> 7.5 to 17.5 x calories.
So the force and displacement for each step with actin/myosin motor is equivalent to the energy yield from the hydrolysis of 1 ATP

19. The neuron’s growth cone extends by building microfilaments at the + ends in response to growth cues from the environment…

20. Distribution of a vesicle along the actin network is polarized

21. Putting it together: Actin and Intermediate filaments
Myosin V is the link between actin, which serves as a rigid skeletal element, and the intermediate filament, which is being delivered to another part of the cell.

22. Tubulin-based motility

23. Roles of the tubulin-based system
Axoplasmic transport
Positioning organelles within the cell
Mitotic spindle
Cilia and flagella

24. Axoplasmic Transport
Anterograde – from cell body toward synapse – ie toward + end of the tubule - driven by kinesins
Retrograde – from synapse toward cell body – driven by dyneins
‘Fast’ – works for cargo carried in vesicles – mm/day.
‘Slow’ – for individual protein molecules – net rate is less than 10 mm/day, but apparently this reflects a stop and go aspect of the process

25. Functions of axoplasmic transport
Delivers proteins, mitochondria, vesicles to synapses
Removes recycled proteins and organelles to cell body for destruction by lysosomes
Carries intracellular chemical messages from synapse to cell body
Delivers neuron-specific viruses (herpes and pox viruses) from peripheral sensory nerve endings to cell bodies in the CNS

26. Actin and tubulin based systems can cooperate

27. Consequences of interfering with axoplasmic transport
Interruption of axoplasmic transport causes a traffic jam on the proximal side of the interruption and Wallerian degeneration of distal parts of neuron.
Anticancer drugs targeted against microtubules have neuronal toxicity.

28. Role of microtubules in positioning organelles within the cytoplasm
1. The endoplasmic reticulum is stretched towards the periphery by its connections to the microtubules.
2. Lysosomes are pushed toward the periphery by microtubules.
3. Three different kinesins are implicated in the movement of mitochondria along microtubule paths to the part of the cell where they are needed.
4. Dynein positions the Golgi; without microtubules, the Golgi breaks up into a lot of little vesicles that disperse in the cytoplasm.
5. Axoplasmic transport of vesicles to the axon terminals and relay of trophic substances (and herpes and chickenpox viruses) to the soma relies on the connections formed with kinesins and dyneins.

29. Microtubules are associated with motor proteins: Dynein and Kinesin (kinesin is thought to have evolved from myosin)

30. Microtubules are oriented: kinesin takes its cargo to the + end and dynein transports to the - end

31. Cartoons of microtubule transport

32. Specific proteins mediate attachment of cargo to dyneins
This cartoon is included to remind you that there must be a mechanism that designates particular vesicles for anterograde or retrograde transport

33. The mitotic spindle

34. Some confusing terms
Organizing center for microtubules in an interphase cell = centrosome (usually); 1 centrosome consists of 2 centrioles; each centriole consists of a pinwheel array of triplet microtubules. 1 basal body (cilia and flagella) = 1 centriole
Centromere = attachment point for spindle microtubule on chromosome = kinetochore

35. The sequence of mitotic processes
Nuclear membrane disassembled, chromosomes condense
Interphase microtubules dissassemble
Centrosome is duplicated – this initiates formation of mitotic spindle
At prometaphase, new microtubules form with their – ends attached to the centrosomes – the extending MTs randomly contact the kinetochores of the chromosomes and attach to them
During anaphase, sister chromosomes separate (anaphase A) and the spindle poles move further apart (anaphase B)
During telophase, daughter cells separate (cytokinesis- actin-myosin is involved in this) and the nuclear envelopes reform.

36. Some questions How do chromosomes line up at the metaphase plate?
They are pushed there by net growth of microtubules, with polymerization occurring at the + end
How can microtubules draw sister chromosomes apart in anaphase A?
During anaphase A, spindle microtubules shrink by depolymerization near the + end - not the – end. No ATP is necessary for this process
How do the spindle poles move further apart from each other in anaphase B?
Dyneins push on microtubules in the overlap zone, while kinesins pull each end of the spindle toward the plasma membrane. ATP is required for this process

37. Polymerization pushes chromatids to the metaphase plate during prometaphase
Depolymerization pulls chromosomes toward the spindle poles during anaphase A

38. The role of kinesins (green) and dyneins (pink) in anaphase B separation of spindle poles – at the same time, growth of microtubules at the + ends causes the spindle to elongate

39. Cilia and Flagella

40. Cilia and Flagella Structure – basically the same structure
Differences: Cilia are shorter and numerous, whereas flagella are long and exist alone or as pairs.
The basal body that organizes the cilia or flagellum is identical in structure to the centrioles (right, below) that are present as a pair in the centrosome (left, below):

41. Functions of Cilia and Flagella
Respiratory airway (mucociliary escalator)
Oviduct (egg and sperm transport)
Flagella
Spermatozoa
Renal tubule

42. Basic facts about cilia and flagella
All eucaryotic cilia and flagella contain 9 outer bundles of doublet microtubules with a central singlet pair of microtubules – this entire structure is called an axoneme
Bending of the axoneme is the result of sliding of adjacent doublets relative to one another
Dynein arms generate the sliding forces – dyneins are attached to the b tubule of each doublet and their heads apply force to the adjacent a tubule

43. Cilia and Flagella: motility results from microtubule sliding within the axoneme

44. Cilia and Flagella: an axoneme (a cylinder of tubules: 9+2) connected to a basal body and covered by membrane

45. The axoneme
The machinery inside cilia and flagella is constructed of a ring of 9 microtubule doublets (A=13, B=11) linked by nexin and powered by 2 dynein arms that have ATPase activity. The spokes link the ring to the inner 2 microtubules
Inner arm dyneins are responsible for axoneme bending – outer arm dyneins just contribute to sliding but do not produce bends

46. Bacterial flagella and eucaryotic flagella are not homologous