Motor Proteins

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Motor Proteins
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2 Motor Systems

• Actin-based motility motor proteins are myosins
• Tubulin-based motility motor proteins are
  kinesins and dyneins

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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

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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.

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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

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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
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Study of the movement of myosin against actin
filaments

• 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

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Cartoon of myosin coated bead actin preparation
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• 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

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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

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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.

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• 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

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

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displacement
time

• Results show that the myosin pulls on the actin
  filament in a stepwise, or ratchet-like fashion

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

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

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• 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
  10-21 cal per molecule ATP

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• 1 pN x 10 nm displacement 2.5 x 10-21 cal
• 3-7 pN of force generated ---gt 7.5 to 17.5 x
  10-21 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

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The neurons growth cone extends by building
microfilaments at the ends in response to
growth cues from the environment
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Distribution of a vesicle along the actin network
is polarized
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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.

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Tubulin-based motility
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Roles of the tubulin-based system

• Axoplasmic transport
• Positioning organelles within the cell
• Mitotic spindle
• Cilia and flagella

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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
  50-400 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

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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

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Actin and tubulin based systems can cooperate
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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.

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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.

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Microtubules are associated with motor proteins
Dynein and Kinesin (kinesin is thought to have
evolved from myosin)
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Microtubules are oriented kinesin takes its
cargo to the end and dynein transports to the -
end
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Cartoons of microtubule transport
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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
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The mitotic spindle
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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) and the nuclear envelopes reform.

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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?
• Kinesins push on microtubules in the overlap
  zone, while dyneins pull each end of the spindle
  toward the plasma membrane. ATP is required for
  this process

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Polymerization pushes chromatids to the metaphase
plate during prometaphase
Depolymerization pulls chromosomes toward the
spindle poles during anaphase A
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The role of kinesins (pink) and dyneins (green)
in anaphase B separation of spindle poles at
the same time, growth of microtubules at the
ends causes the spindle to elongate
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Cilia and Flagella
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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
  centromeres (right, below) that are present as a
  pair in the centrosome (left, below)

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Functions of Cilia and Flagella

• Cilia
• Respiratory airway (mucociliary escalator)
• Oviduct (egg and sperm transport)
• Flagella
• Spermatozoa
• Renal tubule

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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

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Cilia and Flagella motility results from
microtubule sliding within the axoneme
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Cilia and Flagella an axoneme (a cylinder of
tubules 92) connected to a basal body and
covered by membrane
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The axoneme

• The machinery inside cilia and flagella is
  constructed of a ring of 9 microtubule doublets
  (A13, B11) 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
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Bacterial flagella and eucaryotic flagella are
not homologous