"Picture yourself hiking through the woods
or walking across a lawn," says Elizabeth Haswell, PhD, assistant
professor of biology in Arts & Sciences at Washington University in St. Louis. "Now ask yourself: Do the bushes know that
someone is brushing past them? Does the grass know that it is being crushed
underfoot? Of course, plants don't think thoughts, but they do respond to being
touched in a number of ways."
"It's
clear," Haswell says, "that plants can respond to physical stimuli,
such as gravity or touch. Roots grow down, a 'sensitive plant' folds its
leaves, and a vine twines around a trellis. But we're just beginning to find
out how they do it," she says.
In the
1980s, work with bacterial cells showed that they have mechanosensitive
channels, tiny pores in the cells membrane that open when the cell bloats with
water and the membrane is stretched, letting charged atoms and other molecules
rush out of the cell. Water follows the ions, the cell contracts, the membrane
relaxes, and the pores close.
Genes
encoding seven such channels have been found in the bacterium Escherichia
coli and 10 in Arabidopsis
thaliana, a small flowering plant related to mustard and cabbage.
Both E. coli and Arabidopsis serve as model organisms in Haswell's
lab.
She
suspects that there are many more channels yet to be discovered and that they
will prove to have a wide variety of functions.
Recently,
Haswell and colleagues at the California Institute of Technology, who are
co-principal investigators on an National Institutes of Health (NIH) grant to
analyze mechanosensitive channels, wrote a review article about the work so far
in order to "get their thoughts together" as they prepared to write
the grant renewal. The review appeared in the Oct. 11 issue ofStructure.
Swelling
bacteria might seem unrelated to folding leaflets, but Haswell is willing to
bet they're all related and that mechanosensitive ion channels are at the
bottom of them all. After all, plant movements -- both fast and slow -- are
ultimately all hydraulically powered; where ions go the water will follow.
Giant E. coli cells
The big
problem with studying ion channels has always been their small size, which
poses formidable technical challenges.
Early
work in the field, done to understand the ion channels whose coordinated
opening and closing creates a nerve impulse, was done in exceptionally large
cells: the giant nerve cells of the European squid, which had projections big
enough to be seen with the unaided eye.
Experiments
with these channels eventually led to the development of a sensitive electrical
recording technique known as the patch clamp that allowed researchers to
examine the properties of a single ion channel. Patch clamp recording uses as
an electrode a glass micropipette that has an open tip. The tip is small enough
that it encloses a "patch" of cell membrane that often contains just
one or a few ion channels.
Patch
clamp work showed that there were many different types of ion channels and that
they were involved not just in the transmission of nerve impulses but also with
many other biological processes that involve rapid changes in cells.
Mechanosensitive
channels were discovered when scientists started looking for ion channels in
bacteria, which wasn't until the 1980s because ion channels were associated
with nerves and bacteria weren't thought to have a nervous system.
In E.
coli, the ion channels are embedded in the plasma membrane, which
is inside a cell wall, but even if the wall could be stripped away, the cells
are far too small to be individually patched. So the work is done with
specially prepared giant bacterial cells called spherophlasts.
These
are made by culturing E. coli in
a broth containing an antibiotic that prevents daughter cells from separating
completely when a cell divides. As the cells multiply, "snakes" of
many cells that share a single plasma membrane form in the culture. "If
you then digest away the cell wall, they swell up to form a large sphere,"
Haswell says.
Not
that spheroplasts are that big. "We're doing most of our studies in Xenopus oocytes (frog eggs), whose diameters
are 150 times bigger than those of spheroplasts," she says.
Three
mechanosensitive channel activities
To find
ion channels in bacteria, scientists did electrophysiological surveys of
spheroplasts. They stuck a pipette onto the spheroplast and applied suction to
the membrane as they looked for tiny currents flowing across the membrane.
"What
they found was really amazing," Haswell says. "There were three
different activities that are gated (triggered to open) only by deformation of
the membrane." (They were called "activities" because nobody
knew their molecular or genetic basis yet.)
The
three activities were named mechanosensitive channels of large (MscL), small
(MscS) and mini (MscM) conductance. They were distinguished from one another by
how much tension you had to introduce in order to get them to open and by their
conductance.
One of
the labs working with spheroplasts was led by Ching Kung, PhD, at the
University of Wisconsin-Madison. The MscL protein was identified and its gene
was cloned in 1994 by Sergei Sukharev, PhD, then a member of Kung's lab. His
tour-de-force experiment, Haswell says, involved reconstituting fractions of
the bacterial plasma membrane into synthetic membranes (liposomes) to see
whether they would confer large-channel conductance.
In
1999, the gene encoding MscS was identified in the lab of Ian Booth, PhD, at
the University of Aberdeen.
Comparatively, little work has been done on the mini channel, which is finicky
and often doesn't show up, Haswell says, though a protein contributing to MscM
activity was recently identified by Booth's group.
Once
both genes were known, researchers did knockout experiments to see what
happened to bacteria that didn't have the genes needed to make the channels.
What they found, says Haswell, was that if both the MscL and MscS genes were
missing, the cells could not survive "osmotic downshock," the
bacterial equivalent of water torture.
"The
standard assay," Haswell says, "is to grow the bacteria for a couple
of generations in a very salty broth, so that they have a chance to balance
their internal osmolyte concentration with the external one." (Osmolytes
are molecules that affect osmosis, or the movement of water into and out of the
cell.) "They do this," she says, "by taking up osmolytes from the
environment and by making their own."
"Then,"
she says, "you take these bacteria that are chockfull of osmolytes and
throw them into fresh water. If they don't have the MscS and MscL proteins that
allow them to dump ions to avoid the uncontrolled influx of water, they don't survive."
It's a bit like dumping saltwater fish into a freshwater aquarium.
Why are
there three mechanosenstivie channel activities? The currently accepted model,
Haswell says is that the channels with the smaller conductances are the first
line of defense. They open early in response to osmotic shock so that the
channel of large conductance, through which molecules the cell needs can
escape, doesn't open unless it is absolutely necessary. The graduated response
thus gives the cell its best chance for survival.
Crystallizing
the proteins
The
next step in this scientific odyssey, figuring out the proteins' structures,
also was very difficult. Protein structures are traditionally discovered by
purifying a protein, crystallizing it out of a water solution, and then
bombarding the crystal with X-rays. The positions of the atoms in the protein
can be deduced from the X-ray diffraction pattern.
In a
sense crystallizing a protein isn't all that different from growing rock candy
from a sugar solution, but, as always, the devil is in the details. Protein
crystals are much harder to grow than sugar crystals and, once grown, they are
extremely fragile. They even can even be damaged by the X-ray probes used to
examine them.
And to
make things worse MscL and MscS span the plasma membrane, which means that
their ends, which are exposed to the periplasm outside the cell and the
cytoplasm inside the cell, are water-loving and their middle sections, which
are stuck in the greasy membrane, are repelled by water. Because of this double
nature it is impossible to precipitate membrane proteins from water solutions.
Instead
the technique is to surround the protein with what have been characterized as
"highly contrived detergents," that protect them -- but just barely
-- from the water. Finding the magical balance can take as long as a scientific
career.
The
first mechanosensitive channel to be crystallized was MscL -- not the protein
in E. coli but
the analogous molecule (a homolog) from the bacterium that causes tuberculosis.
This work was done in the lab of one of Haswell's co-authors, Douglas C. Rees a
Howard Hughes investigator at the California Institute of Technology.
MscS
from E. coli was
crystallized in the Rees laboratory several years later, in 2002, and an MscS
protein with a mutation that left it stuck in the presumed open state was
crystallized in the Booth laboratory in 2008. "So now we have two crystal
structures for MscS and two (from different bacterial strains) for MscL,"
Haswell says.
Of
plants and mutants
Up to
this point, mechanosensitive channels might not seem all that interesting
because the lives of bacteria are not of supreme interest to us unless they are
making us ill.
However,
says, Haswell, in the early 2000s, scientists began to compare the genes for
the bacterial channels to the genomes of other organisms and they discovered
that there are homologous sequences not just in other bacteria but also in some
multicellular organisms, including plants.
"This
is where I got involved," she says. "I was interested in gravity and
touch response in plants. I saw these papers and thought these homologs were
great candidates for proteins that might mediate those responses."
"There
are 10 MscS-homologs in Arabidopsis and no MscL homologs," she says.
"What's more, different homologs are found not just in the cell membrane
but also in chloroplast and mitochondrial membranes. "
The
chloroplast is the light-capturing organelle in a plant cell and the
mitochondria is its power station; both are thought to be once-independent
organisms that were engulfed and enslaved by cells which found them useful.
Their membranes are vestiges of their free-living past.
The
number of homologs and their locations in plant cells suggests these channels
do much more than prevent the cells from taking on board too much water.
So what
exactly were they doing? To find out Haswell got online and ordered Arabidopsis seeds from the Salk collection in La
Jolla, Calif., each
of which had a mutation in one of the 10 channel genes.
From
these mutants she's learned that two of the ten channels control chloroplast
size and proper division as well as leaf shape. Plants with mutations in these
two MscS channel homologs have giant chloroplasts that haven't divided
properly. The monster chloroplasts garnered her lab the cover of the August
issue of The Plant Cell.
"We
showed that bacteria lacking MscS and MscL don't divide properly
either,"Haswell says, "so the link between these channels and
division is evolutionarily conserved."
The big
idea
But
Haswell and her co-authors think they are only scratching the surface. "We
are basing our understanding of this class of channels on MscS itself, which is
a very reduced form of the channel," she says. "It's relatively
tiny."
"But
we know that some of the members of this family have long extensions that stick
out from the membrane either outside or inside the cell. We suspect this means
that the channels not only discharge ions, but that they also signal to the
whole cell in other ways. They may be integrated into common signaling
pathways, such as the cellular osmotic stress response pathway.
We
think we may be missing a lot of complexity by focusing too exclusively on the
first members of this family of proteins to be found and characterized,"
she says. "We think there's a common channel core that makes these
proteins respond to membrane tension but that all kinds of functionally
relevant regulation may be layered on top of that."
"For
example," she says, "there's a channel in E.
coli that's closely
related to MscS that has a huge extension outside the cell that makes it
sensitive to potassium. So it's a mechanosensitive channel but it only gates in
the presence of potassium. What that's important for, we don't yet know, but it
tells us there are other functions out there we haven't studied."
What
about the sensitive plant?
So are
these channels at the bottom of the really fast plant movements like the
sensitive plant's famous touch shyness? (To see a movie of this and other
"nastic" (fast) movements, go to the Plants in Motion site maintained
by Haswell's colleague Roger P. Hangartner of Indiana University).
Haswell
is circumspect. "It's possible," she says. "In the case of Mimosa
pudica there's
probably an electrical impulse that triggers a loss of water and turgor in
cells at the base of each leaflet, so these channel proteins are great
candidates.
