29-Oct-2010
Work
could help unravel complexities of the cell and lead to new
antibiotics and disease treatments
LA JOLLA,
CA – October 29, 2010
Identifying
and observing the molecules that form ribosomes—the cellular
factories that build the proteins essential for life—has for
decades been a key goal for biologists but one that had seemed
nearly unattainable. But the new Scripps Research study, which
appears in the October 29, 2010 issue of the journal Science,
yielded pictures of the chemical intermediate steps in ribosome
creation.
"For me
it was a dream experiment," said project leader James Williamson,
Ph.D., professor, member of the Skaggs Institute for Chemical
Biology, and dean of graduate and postgraduate studies at Scripps
Research, who credits collaborators at the Scripps Research
National Resource for Automated Molecular Microscopy (NRAMM)
facility for making it possible. "We have great colleagues at
Scripps to collaborate with who are willing to try some crazy
experiments, and when they work it's just beautiful."
Past studies
of the intermediate molecules that combine to form ribosomes
and other cellular components have been severely limited by
imaging technologies. Electron microscopy has for many years
made it possible to create pictures of such tiny molecules,
but this typically requires purification of the molecules. To
purify, you must first identify, meaning researchers had to
infer what the intermediates were ahead of time rather than
being able to watch the real process.
"My lab
has been working on ribosome assembly intensively for about
15 years," said Williamson. "The basic steps were mapped out
30 years ago. What nobody really understood was how it happens
inside cells."
Creating
a New View
The NRAMM
group, led by Scripps Research Associate Professors Clinton
Potter and Bridget Carragher and working with Scripps Research
Kellogg School of Science and Technology graduate students Anke
Mulder and Craig Yoshioka, developed a new technique, described
in the Science paper and dubbed discovery single-particle profiling,
which dodges the purification problem by allowing successful
imaging of unpurified samples. An automated data capture and
processing system of the team's design enables them to decipher
an otherwise impossibly complex hodgepodge of data that results.
For this
project, second author Andrea Beck, a research assistant in
the Williamson laboratory, purified ribosome components from
cells of the common research bacterium Escherichia Coli. She
then chemically broke these apart to create a solution of the
components that form ribosomes. The components were mixed together
and then were rapidly stained and imaged using electron microscopy.
"We went in with 'dirty' samples that contained horribly complex
mixtures of all different particles," said Williamson.
Mulder,
who is first author on the paper, collected and analyzed the
particles that were formed during the ribosome assembly reaction.
Using the team's advanced algorithms, they were able to process
more than a million data points from the electron microscope
to ultimately produce molecular pictures.
The Pieces
Fit
The team
produced images that the scientists were able to match like
puzzle pieces to parts of ribosomes, offering strong confirmation
that they had indeed imaged and identified actual chemical intermediates
in the path to ribosome production. "We always saw the same
thing no matter how we processed the data, and this led us to
believe this was real," said Williamson.
Further
confirmation came as the researchers imaged components from
different timeframes. After breaking down ribosome components,
the scientists prepared samples at various stages allowing enough
time for the molecular mix to begin combining as they do during
ribosome creation in cells.
Imaging
this time series, the team was able to show higher concentrations
of larger, more complex molecules and fewer smaller molecules
as time elapsed. These results fit with the limited information
that was already available about the timing of formation steps,
providing further confirmation of the team's success.
Interestingly,
this work also confirmed that there are more than one possible
paths in ribosome formation, a phenomenon known as parallel
assembly that been suggested by prior research but never definitively
confirmed.
Long-Term
Potential
Williamson
says that with the information now at hand, they will be able
to move forward with studies of which additional molecules might
be present in cells and essential for ribosome formation. Such
data could offer exciting medical potential.
All bacteria
contain and are dependent on ribosomes. Identification of molecules
required for ribosome assembly could offer new targets for antibiotic
drugs aimed at killing bacteria. "If we can figure out how to
inhibit assembly, that would be a very important therapeutic
avenue," said Williamson.
There are
also indications that some diseases such as Diamond Blackfan
Anemia might be caused, at least in some cases, by errors in
ribosome production. Better understanding of that production
could also reveal ways such errors might be repaired to cure
or prevent disease.
At the more
basic level, this successful project has also proven techniques
that Scripps Research scientists and other researchers can apply
to allow similar imaging and understanding of other complex
but critical cellular processes.
###
In addition
to Williamson, Mulder, Beck, Yoshioka, Potter, and Carragher,
authors of the paper, entitled "Visualizing Ribosome Biogenesis:
Parallel Assembly Pathways for the 30S Subunit," were Anne Bunner
and Ronald Milligan from Scripps Research.
This research
was supported by the National Institutes of Health and a fellowship
from the National Science Foundation.
About
The Scripps Research Institute
The Scripps
Research Institute is one of the world's largest independent,
non-profit biomedical research organizations, at the forefront
of basic biomedical science that seeks to comprehend the most
fundamental processes of life. Scripps Research is internationally
recognized for its discoveries in immunology, molecular and
cellular biology, chemistry, neurosciences, autoimmune, cardiovascular,
and infectious diseases, and synthetic vaccine development.
An institution that evolved from the Scripps Metabolic Clinic
founded by philanthropist Ellen Browning Scripps in 1924, Scripps
Research currently employs approximately 3,000 scientists, postdoctoral
fellows, scientific and other technicians, doctoral degree graduate
students, and administrative and technical support personnel.
Headquartered in La Jolla, California, the institute also includes
Scripps Florida, whose researchers focus on basic biomedical
science, drug discovery, and technology development. Scripps
Florida is located in Jupiter, Florida. For more information,
see http://www.scripps.edu/
Contact:
Mika Ono
mikaono@scripps.edu
858-784-2052
Scripps Research Institute
