Motor Proteins
2 Motor Systems
• Actin-based motility: motor proteins are
myosins
• Tubulin-based motility: motor proteins are
kinesins and dyneins
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
Actin-based motility: the Myosins
The head is
both actinbinding and
ATP binding;
the purple light
chain has a
regulatory role.
Myosin II is
muscle myosin.
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
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
ATP replaces
ADP and Pi on
myosin head
Head rotates - Powerstroke
transmits force to thin
filament – head is
deenergized
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
Cartoon of myosin coated bead
actin preparation
• 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
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.
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.
• 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
• ATP is also kept low so that only one ATP
binds to each myosin head
time
• Results show that the myosin pulls on the
actin filament in a stepwise, or ratchet-like
fashion
• A single ATP molecule is hydrolyzed
resulting in a power stroke and
displacement of around 10 nm.
• The force generated can also be
determined and it is around 3-7
picoNewtons (pN)
• 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
• 1 pN x 10 nm displacement = 2.5 x 10-21
cal
• 3-7 pN of force generated ---> 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
The neuron’s growth cone extends by building microfilaments at
the + ends in response to growth cues from the environment…
Distribution of a vesicle along the
actin network is polarized
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.
Tubulin-based motility
Roles of the tubulin-based system
•
•
•
•
Axoplasmic transport
Positioning organelles within the cell
Mitotic spindle
Cilia and flagella
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 – 50400 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
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
Actin and tubulin based systems can
cooperate
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.
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.
Microtubules are associated with motor proteins:
Dynein and Kinesin
(kinesin is thought to have evolved from myosin)
Microtubules are oriented: kinesin takes its cargo
to the + end and dynein transports to the - end
Cartoons of microtubule transport
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
The mitotic spindle
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
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 (cytokinesisactin-myosin is involved in this) and the nuclear envelopes
reform.
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
Polymerization
pushes
chromatids to the
metaphase plate
during
prometaphase
Depolymerization pulls
chromosomes toward
the spindle poles during
anaphase A
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
Cilia and Flagella
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):
Functions of Cilia and Flagella
• Cilia:
– Respiratory airway (mucociliary escalator)
– Oviduct (egg and sperm transport)
• Flagella
– Spermatozoa
– Renal tubule
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
Cilia and Flagella: motility results from microtubule
sliding within the axoneme
Cilia and Flagella: an axoneme (a cylinder of tubules: 9+2) connected to a basal
body and covered by membrane
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
The axoneme
Inner arm dyneins are responsible for axoneme bending – outer
arm dyneins just contribute to sliding but do not produce bends
Bacterial flagella and eucaryotic flagella are not homologous