Elizabeth Haswell, PhD, assistant professor of biology in Arts & Sciences at Washington University in St. Louis, in a growth chamber with her “lab rats,” Arabidopsis plants she uses to understand how plants respond to touch, gravity and other mechanical forces. If wild-type Arabidopsis plants are touched frequently. their growth is stunted. Image: David Kilper/WUSTL |
“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 to 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 laboratory.
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 of Structure.
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 activites
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
Representations of the pore section of the MscS channel in E. coli in its nonconducting (top) and open (bottom) configurations are based on X-ray crystallization studies of the protein’s structure. The transition between closed and open states is often described as similar to the narrowing and expanding of the pupil of the eye. The “closed” state can still appear to have an opening because amino acids around the opening act as a “hydrophobic plug” that prevents ions from moving through it. Image: E. Haswell/WUSTL |
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 coauthors 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.
