What's Behind Cellular Morphing?
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What's Behind Cellular Morphing?
Research on Cytoskeleton and Signal Transduction Join to Reveal
How Cells Move
Cell biology does not usually remind one
of stargazing. But as Le Ma, in a darkened room, mixes two pinhead-size
drops of clear liquid onto a coverslip, a view quite like the night
sky opens up under the microscope, complete with stars and comets.
Comets? Yes, luminous tails appear against a black background dotted
with bright points, and successive pictures taken over the course
of some 10 minutes actually show those tails shooting along in curved
trajectories (see image).
|Image by Le Ma
tails" shown in this series of video images are not celestial
bodies. About one-thousandth of an inch long, they are
made of actin, a protein component of every cell's inner
skeleton. In this test tube experiment, the comets move
out of, or into, the visual field as millions of actin
proteins get assembled in a series of biochemical reactions.
These reactions are started by the tiny sphere made of
cell membranes that sits atop each comet tail. The growing
tail propels the vesicle along like a rocket engine. The
images are stills from a video, taken at two-minute intervals.
This observationenjoyed in various forms
by biologists for seven yearsexemplifies how seemingly bizarre
findings in one area of research can literally propel another one
Microbiologists curious about how food-poisoning
bacteria crisscross infected host cells sparked a line of intense
study that is now producing a molecular understanding of a fundamental
question in cell biology, namely, how cells move in response to
changes occurring outside their membrane.
Shaping the Cell
Scientists have long been fascinated by
the question of what controls a cell's inner skeleton. This medley
of individual proteins can polymerize into weight-bearing threads
at one moment and dissolve into a soft gel the next. One such protein,
actin, forms a filament network running just underneath the cell's
outer membrane. The actin cytoskeleton is like a scaffold that is
constantly being built and taken apart in ways tightly controlled
in space and time, depending on whether the cell changes its shape,
moves, or divides.
Scientists in this burgeoning fieldthe
past year alone saw more than 900 published papers mentioning the
actin cytoskeletonhave spent the last four years trying to
understand the precise molecular channels of communication that
link a specific outside stimulus to the assembly of actin filaments.
Though they have identified myriad molecular players in these signal
transduction pathways and glimpsed partial connections between sets
of two or three of them, complete chains of command remained elusiveuntil
Recently four reports have begun to fill this
gap, two of them by Harvard scientists. In the April 16 Cell,
Marc Kirschner, the Carl W. Walter professor of cell biology, graduate
student Le Ma, and others describe how they staged in the test tube
a series of signaling events that lead from specialized membrane
lipids all the way to polymerization of actin filaments. Rong Li,
HMS assistant professor of cell biology, and her colleagues report
in the April 26 Current Biology a similar link in live yeast cells.
Across Harvard, many scientists study this question
in cell types ranging from the epithelial cells involved in breast
cancer, to blood and immune cells (see sidebar, p.7), to neurons
5, 1999). The breadth of the field is unsurprising, Kirschner
says. "We could spend the next 20 minutes listing biological problems
that occur through regulated changes in the actin cytoskeleton,
from development to angiogenesis and metastasis, to phagocytosis
The problem is also a knotty one. Central dogmas
in biologythe DNA, RNA, and protein sequencesare essentially
linear. Not encoded in the DNA, however, is the assembly of proteins
into intricate 3-D architectures, which differ among hundreds of
cell types and organisms, and change with time. "This is one of
the great, least understood problems in biology," Kirschner says.
Kirschner's current research grew out of work by others, including
Tim Mitchison, HMS professor of cell biology, who was then at the
University of California, San Francisco. Studying how pathogenic
bacteria called Listeria monocytogenes managed to travel through
cellsan unusual feat for bacteria, which generally live in
the spaces between, not inside, host cellsthe scientists found
in 1992 that listeria propelled itself sitting atop a tail of actin
filaments. Stationary themselves, the filaments continuously grew
just underneath the bug.
Mitchison suspected that the pathogen might hijack
a normal, cellular mechanism for making actin filaments. He found
that listeria's propulsive mechanism also worked in extracts of
frog eggs, an experimental system much simpler to study than infected
This work was most significant, Kirschner says,
for providing a way to study the initial steps of the de novo assembly
of filaments from individual actin proteins, a mysterious process
called actin nucleation. Previous work in the field had focused
on ways of manipulating existing filaments, such as severing or
bundling them, mostly because the nucleation of a new filament is
a fleeting event hard to capture in an experiment.
In 1997, the Mitchison lab described the function
of what has become a key player in all signaling chains controlling
actin polymerization. Called the Arp2/3 complex, this set of seven
proteins is thought somehow to enable three individual actin proteins
to overcome energetic barriers and form a germinal filament, onto
which additional actin proteins can settle easily.
The key question in the field then became: how
does this complex connect back up to the membrane? This is where
the current set of papers comes in.
|Stephen Moskowitz, Advanced
|This diagram depicts
how a signal arriving at the outside of a cell (far left) gets
passed on inside through a series of proteinprotein interactions,
which ultimately translate the original signal into changes
in the cell's inner skeleton (far right). If staged in vitro,
this series gives rise to moving "comet tails" that form as
individual actin proteins (red) assemble into cross-linked threads.
In a cell, these actin threads push into the membrane, causing
it to extend the tiny spikes that typically appear as the cell
stirs in response to the signal. See story for molecular details.
As often happens in signal transduction research, Kirschner's
study ran into the Arp2/3 complex by coming from the opposite direction.
He and Ma began by working with Lewis Cantley, HMS professor of
cell biology at Beth Israel Deaconess Medical Center. Over the past
15 years, Cantley and others had found that certain lipids in the
cell's outer membrane were not simply structural components of that
membrane but helped convey signals to the cytoskeleton, among other
On this project, the researchers initially dropped
bits of real cell membrane into frog egg extracts. Presto! Comet
tails of actin appeared behind the vesicles, telling the researchers
that some component of the membrane started the process. To find
out which one, the scientists embedded into artificial lipid spheres
PIP2, a twice-phosphorylated lipid known to somehow modulate the
cytoskeleton. Again, comet tails corkscrewed these spheres about
under the microscope, the scientists reported last year.
Kirschner, Ma, and others went on to isolate
biochemically the components that lay between PIP2 and the actin
tails. With them, they then reconstructed the chain of events in
a completely purified system. These other players are a protein
called cdc42, and N-WASP, a variant of WASP, the protein mutated
in an immune disorder marked by cytoskeletal defects (see sidebar).
Both had already been implicated in actin remodeling, but never
in the context of a complete pathway. (For example, Tomas Kirchhausen,
HMS associate professor of cell biology at the Center for Blood
Research, and others at Harvard reported in 1996 that cdc42 and
How can one transfer Kirschner's in vitro data
back into a real live context? Consider this simplified example:
The ligand platelet-derived growth factor, emanating from a blood
clot plugging a wound, docks onto the receptor on a nearby fibroblast,
telling it to repair the damage.
In response, several enzymes tack phosphate groups
onto the water-loving end of inositide lipids floating in the fibroblast's
membrane, creating PIP2 (see diagram, p.5). These phosphate groups
fit like plugs into sockets called PH domains. The N-WASP protein
has such a domain and, with it, anchors itself to the membrane.
Once activated by yet other proteins called exchange factors, cdc42
binds N-WASP, which sticks to the membrane by holding on to cdc42
and PIP2. This allows N-WASP, a protein that is usually clamped
shut, to spring open and bind the Arp2/3 complex.
WASP does two things, Kirschner says. Its binding
sites for many different proteins make it a hub that integrates
several lines of upstream information. But it then also works with
the Arp2/3 complex in somehow lining up those first few actin monomers
needed to make a filament.
Once that happens, actin filaments grow, align
themselves perpendicularly to the membrane, and push into it. In
this way, they gradually generate one of the cell's finger-like
spikes, a process much like sticking a finger out from inside a
balloon, Cantley says. The comet tails generated in vitro by lab-made
lipids may simulate that.
And on the hypothetical fibroblast, the spikes
represent the first stirrings towards moving into the wound.
Many questions remain, however. The Kirschner
paper only presents a pathway that can nucleate actin in vitro.
It does not capture the myriad variations that will be found in
different cell types in vivo, or even prove that this ever occurs
in a living cell.
Rong Li's study is the only one of the current
set to have tested this pathway in vivo. Her lab found that the
yeast version of WASP indeed activates the Arp2/3 complex to build
the actin structures that help yeast grow. But she already has data
suggesting that yet other proteins work with Arp2/3, too. "The story
does not end there," she says. Stay tuned.
Seen Through Lens of Cytoskeleton, Immune Disorders' Symptoms
Start Making Sense
Raif Geha has treated patients with WiskottAldrich
Syndrome (WAS) for 25 years, but just recently has begun
to understand their panoply of problems much more clearly.
The Prince Turki Bin Abdul Aziz Al-Saud professor of pediatrics
at Children's Hospital, he says that soon after the underlying
gene defect was first described five years ago by Stanford
scientists, basic cell biological research converged with
clinical observations to explain this rare immune system
Symptoms of WAS include bleeding, immune deficiency,
eczema, inflammatory bowel disease, and lymphomas. Considered
independent at first, they now appear linked through a flaw
in controlling a cell's inner skeleton. The affected protein,
WASP, turned out to play a central role in coordinating
incoming signals into the proper cytoskeletal change.
WAS can sometimes be cured by bone marrow transplants,
but better treatments are still needed. Researchers at Harvard
and elsewhere are studying gene therapy approaches.
T cells, monocytes, and mast cells all share aspects
of a central pathway of signal transduction (described in
the main story), says Geha. Yet cell-specific variations
are important. For instance, Geha studies SLP-76, a protein
active at the beginning of this pathway. It connects signals
coming in through the cell-specific receptor, such as the
T cell receptor, and going to an exchange factor called
Vav, which activates cdc42. From then on, immune cells use
the common pathway. Geha's lab also has discovered a protein
that functions at the end of the pathway. Called WIP, it
is thought to cooperate with WASP and actin monomers in
making filaments and turning them into the little spikes
extended by active cells.
In WAS patients this pathway is disrupted, suggesting
a reason why their platelets cannot stop bleeding quickly
and why T cells cannot mount adequate immune responses.
The cytoskeleton is one part of the explanation, says Geha;
the other lies in the fact that the signals that ultimately
reshape the cytoskeleton also branch off to the nucleus,
where they control growth.
Geha suspects that the cytoskeleton might also play
a role in the eczema and inflamed intestine often seen in
WAS. T cells' hampered motility might trap those that somehow
enter the skin and the intestine, though evidence for this
The pathway keeps pointing out unexpected connections.
In this month's Journal of Clinical Investigation, Geha's
group reports that mice lacking SLP-76 cannot release granules
from their mast cells. Known to require cytoskeletal changes,
this process underlies common seasonal allergies.
"When we started work on these proteins, we had no
idea they were going to act in the same pathway," recalls
Geha. But, he adds, in this bench-to-bedside research, "projects
eventually come together, and not always by design."