Essential Cell Biology 5th edition

Microtubules
589
Figure 17–22 Kinesin causes microtubule gliding in vitro.
In an in vitro motility assay, purified kinesin is mixed with
microtubules in the presence of ATP. When a drop of the
mixture is placed on a glass slide and examined by videoenhanced
microscopy, individual microtubules (artificially
colored) can be seen gliding over the slide. They are driven
by kinesin molecules, which attach to the glass slide by
their tails. Images were recorded at 1-second intervals. The
microtubules moved at about 1–2 μm/sec. (Courtesy of Nick
Carter and Rob Cross.)
time = 0 sec time = 1 sec time = 2 sec
1 µm
microtubules with what they hoped were the motor
proteins still attached. Vale and his team then added
ATP to release the attached proteins, and they found a
110-kilodalton polypeptide that could stimulate the
gliding of microtubules along a glass cover slip (Figure
17–22). They dubbed the molecule kinesin (from the
Greek kinein, “to ECB5 move”). e17.21/17.22
Similar in vitro motility assays have been instrumental
in the study of other motor proteins—such as myosins,
which move along actin filaments, as we discuss later.
Subsequent studies showed that kinesin moves along
microtubules from the minus end to the plus end; they
also identified many other motor proteins of the kinesin
family.
Lights, camera, action
Combining such assays with ever more refined microscopic
techniques, researchers can now monitor the
movement of individual motor proteins along single
microtubules, even in living cells.
Observation of kinesin molecules, labeled with a fluorescent
marker protein, revealed that this motor protein
marches along microtubules processively—that is, each
molecule takes multiple “steps” along the filament (100
or so) before falling off. The length of each step is 8 nm,
which corresponds to the spacing of individual tubulin
dimers along the microtubule. Combining these observations
with assays of ATP hydrolysis, researchers have
confirmed that one molecule of ATP is hydrolyzed per
step. Kinesin can move in a processive manner because
it has two heads. This enables it to walk toward the plus
end of the microtubule in a “hand-over-hand” fashion,
each head repetitively binding and releasing the filament
as it swings past the bound head in front (Figure
17–23). Such studies now allow us to follow the footsteps
of these fascinating and industrious proteins—step
by molecular step.
(A)
1
2
(B)
3
minus
end
kinesin
tail
microtubule
1 µm
1
plus
end
2
3
16 nm
4
kinesin
heads
5
Figure 17–23 A single molecule of
kinesin moves along a microtubule.
(A) Three frames, separated by intervals
of 1 second, record the movement of an
individual, fluorescently labeled kinesin
molecule (red ) along a microtubule
(green); the labeled kinesin moves at
a speed of 0.3 μm/sec. (B) A series of
molecular models of the two heads of
a kinesin molecule, showing how they
are thought to walk processively (left to
right) along a microtubule in a series of
8-nm steps in which one head swings
past the other (Movie 17.6). (A and B,
courtesy of Ron Vale.)