Scenario 2

1
Scenario 2
Scenario 2 Fast transport of vesicles
2
Scenario 2

3
Scenario 2

• Fast axonal flow
• Organelles such as mitochondria and precursors of
  synaptic vesicles
• Transported from near centrosome in cell body to
  axon ending
• Movements observable with light microscope, using
  phase contrast or interference contrast

4
Scenario 2

Vesicles in axon
5
Scenario 2

• Discovery of the motor protein kinesin
• Transport inhibited by AMPPNP (non-hydrolyzable
  analogue of ATP
• In AMPPNP, organelles are arrested attached to
  microtubules cf rigor
• Microtubules purified from brain in presence
  AMPPNP have ATP-ase activity

6
Scenario 1
EGTA binds Ca 2 Leaving free Mg 2

7
Scenario 2

• Discovery of the motor protein kinesin
• Microtubules purified from brain in presence
  AMPPNP have ATP-ase activity
• Assay purified motor protein by observing mt
  gliding in vitro

8
Assay for motor activity
Scenario 2

Microtubules migrating on kinesin bound to surface
9
Scenario 2

ATP binding, ATPase activity, mt binding all are
properties of the motor domains
10
Scenario 2
A single motor domain of kinesin
11
Scenario 2
O ATP D ADP

Animation of kinesin walking
12
Scenario 2

13
Introduction
The sub-cellular biological world is full of
phenomena that challenge physical intuition
single-molecule machines, self-assembling
architectures and spontaneous information
processing. These phenomena derive from the
physical character of biological macromolecules,
which have passed through the evolutionary
design process and acquired the character of a
technology. Deborah Fygenson

14
Scenario 2

Single kinesin moving a bead
15
Scenario 2
Visualisation of single kinesin-GFP molecules
moving processively
16
Scenario 2

17
Scenario 2

• Motor characteristics of kinesin I
• Dilution of kinesin shows a single molecule
  moves mt as fast as any greater number
• Can study force exerted by single molecules
• Kinesin moves in 8 nm steps ( 1 heterodimer)
  towards mt plus end i.e. Anterograde transport
• Movement through many cycles without detachment
  is named processive

18
Scenario 2

19
Scenario 2

Conserved 350-residue motor domain defines a
diverse kinesin superfamily Vesicle
transport Membrane movements Mitotic
associated with spindle and chromosomes
20
Scenario 2

Cytoplasmic dynein is a minus-end directed motor
21
Scenario 2

Other proteins are required for attaching cargo
to motor In absence of cargo, tail folds and
blocks ATP-ase
22
Scenario 2

Amyloid Precursor Protein APP (relationship to
Alzheimers disease) may be a cargo receptor
for kinesin
23
Scenario 2

• Linkage of kinesins to cargo vesicles
• Drosophila gene Sunday Driver has phenotype
• similar to kinesin mutants, homologues in worm
• mouse and human.
• Amyloid Precursor Protein (APP), important in
  Alzheimers disease, may be a kinesin cargo
  receptor

24
Scenario 2

25
Introduction
Microtubules Microfilaments Intermediate
filaments

Cytoskeleton

Kinesin Dynein Myosin
Motor proteins