Essential Cell Biology 5th edition

588
HOW WE KNOW
PURSUING MICROTUBULE-ASSOCIATED MOTOR PROTEINS
The movement of organelles throughout the cell cytoplasm
has been a subject of observation, measurement,
and speculation since the middle of the nineteenth
century. But it was not until the mid-1980s that biologists
identified the molecules that drive this movement
of organelles and vesicles from one part of the cell to
another.
Why the lag between observation and understanding?
The problem was in the proteins—or, more precisely, in
the difficulty of studying them in isolation outside the cell.
To investigate the activity of an enzyme, for example,
biochemists first purify the protein: they break open cells
or tissues and separate the enzyme from other molecular
components (see Panels 4–4 and 4–5, pp. 166–167).
They can then study the enzyme in a test tube (in vitro),
controlling its exposure to substrates, inhibitors, ATP,
and so on. Unfortunately, this approach did not seem to
work for studies of the motile machinery that underlies
intracellular transport. It is not possible to break open a
cell and pull out an intact, fully active transport system,
free of extraneous material, that continues to carry mitochondria
and vesicles from place to place.
That problem was solved by technical advances in
two separate fields. First, improvements in microscopy
allowed biologists to see that an operational transport
system (with extraneous material still attached) could be
squeezed from the right kind of living cell. At the same
time, biochemists realized that they could assemble a
working transport system from scratch—using purified
cytoskeletal filaments, motors, and cargo—outside the
cell. One such breakthrough started with a squid.
Teeming cytoplasm
Neuroscientists interested in the electrical properties of
nerve cell membranes have long studied the giant axon
from squid (see How We Know, pp. 412−413). Because
of its large size, researchers found that they could
squeeze the cytoplasm from the axon like toothpaste,
and then study how ions move back and forth through
various channels in the empty, tubelike plasma membrane
(see Figure 12–33). These investigators discarded
the extruded cytoplasmic jelly, as it appeared to be inert
(and thus uninteresting) when examined under a standard
light microscope.
Then along came video-enhanced microscopy. This
type of microscopy, developed by Shinya Inoué, Robert
Allen, and others, allows one to detect structures that
are smaller than the resolving power of standard light
microscopes, which is only about 0.2 μm, or 200 nm
(see Panel 1−1, pp. 12–13). The resulting images are
captured by a video camera and then enhanced by computer
processing to reduce the background and heighten
contrast. When researchers in the early 1980s applied
this new technique to preparations of squid axon cytoplasm
(axoplasm), they observed, for the first time, the
motion of vesicles and other organelles along cytoskeletal
filaments.
Under the video-enhanced microscope, extruded axoplasm
is seen to be teeming with tiny particles—from
vesicles 30–50 nm in diameter to mitochondria some
5000 nm long—all moving to and fro along cytoskeletal
filaments at speeds of up to 5 μm per second. If the axoplasm
is spread thinly enough, individual filaments can
be seen.
The movement continues for hours, allowing researchers
to manipulate the preparation and study the effects.
Ray Lasek and Scott Brady discovered, for example, that
the organelle movement requires ATP. Substitution of
ATP analogs, such as AMP-PNP, which resemble ATP
but cannot be hydrolyzed (and thus provide no energy),
inhibit the translocation.
Snaking tubes
More work was needed to identify the individual components
that comprise the transport system in squid
axoplasm. What kind of filaments support this movement?
What are the molecular motors that shuttle the
vesicles and organelles along these filaments? Identifying
the filaments was relatively easy: antibodies to tubulin
revealed that they are microtubules. But what about the
motor proteins? To find these, Ron Vale, Thomas Reese,
and Michael Sheetz set up a system in which they could
fish for proteins that power organelle movement.
Their strategy was simple yet elegant: add together
microtubules and organelles and then look for molecules
that induce motion. They used purified microtubules
from squid brain, added organelles isolated from squid
axons, and showed that organelle movement could be
triggered by the addition of an extract from squid axoplasm.
In this preparation, the researchers could either
watch the organelles travel along the microtubules or
watch the microtubules glide snakelike over the surface
of a glass cover slip that had been coated with an axoplasm
extract (see Question 17–18). Their challenge was
to isolate the protein responsible for movement in this
reconstituted system.
To do that, Vale and his colleagues took advantage
of the earlier work with the ATP analog AMP-PNP.
Although this analog inhibits the movement of vesicles
along microtubules, it still allows organelles to attach to
the microtubule filaments. So the researchers incubated
the axoplasm extract with microtubules and organelles
in the presence of AMP-PNP; they then pulled out the