Saturday, May 21, 2011

New technique sheds light on the mysterious process of cell division

 Using a new technique in which models of primitive cells are constructed from the bottom up, scientists have demonstrated that the structure of a cell's membrane and cytoplasm may be as important to cell division as the specialized machinery -- such as enzymes, DNA or RNA -- which are found within living cells.

Christine Keating, an associate professor of chemistry at Penn State University, and Meghan Andes-Koback, a graduate student in the Penn State Department of Chemistry, generated simple, non-living model "cells" with which they established that asymmetric division -- the process by which a cell splits to become two distinct daughter cells -- is possible even in the absence of complex cellular components, such as genes. The study, which will be published in the Journal of the American Chemical Society, may provide important clues to how life originated from non-life and how modern cells came to exhibit complex behaviors.

Keating explained that how biological cells split into asymmetrical daughter cells with very different compositions and different "fates" is something of a mystery. Cellular differentiation -- the process by which an unspecialized cell, such as a stem cell, becomes a specialized cell -- requires that different biological components reorganize themselves into each of the resulting daughter cells. For this apparently complex task to be accomplished, some important mechanism must guide both the reorganization of cellular parts and the maintenance of polarity -- the property of a cell to exhibit distinct front and back "sides" with specific placement and distribution of cellular machinery. "Many genes have been implicated in the maintenance of cell polarity and the facilitation of division into nonidentical daughter cells. It's thanks to changes in the expression of these genes that a skin cell becomes a skin cell and a heart cell becomes a heart cell," Keating said. "But our research took a different approach. We asked: In addition to the genetic factors that guide asymmetrical cell division and polarity maintenance, what structural, biophysical factors might be at work, and how might these factors have predated the evolution of the complex genetic systems known to exist in modern cells?"

The team began with the hypothesis that because new daughter cells arise by division of existing mother cells, certain inherited material -- such as the cell membrane -- could serve as a sort of informational "landmark." This landmark could set in motion and guide a cascade of chemical events related to ordered cell division and polarity maintenance. To test this hypothesis, Keating and Andes-Koback built model cells from the bottom up, allowing water, lipids, and polymers to assemble into mimics of the most basic constituents of real, living cells -- such as a membrane and cytoplasm. They then altered the osmotic pressure outside of the "cells" by adding sugar, which forced them to divide in a way that is reminiscent of how living, biological cells split under natural conditions.

"We observed that even model cells can divide in a structured way, which implies a kind of intrinsic order," Andes-Koback said. She explained that, like a biological cell, the model mother cell was designed to exhibit asymmetry in both its membrane and its cellular interior. The membrane asymmetry was modeled using two distinct lipid domains, while the cellular interior was modeled using two distinct polymers called polyethylene glycol (PEG) and dextran. These polymers form distinct domains, or compartments, on the inside of the model cells, with the dextran-rich compartment containing a higher concentration of a particular protein. The team observed that when the asymmetric mother cell divided, one daughter inherited one lipid domain surrounding the PEG-rich interior, and the other daughter inherited the other membrane domain surrounding the dextran-rich interior, which contained the larger portion of the protein. "Most importantly, we also found that when we varied the relative size of the two lipid domains, one daughter cell got both types of membrane and the other daughter got only one type," Andes-Koback said. "This was possible since the interior aqueous phases controlled the fission plane, and it is important because it provides a way to achieve a patch of distinct membrane to serve as a landmark for polarity in subsequent 'generations.'"

The team members note that the new modeling technique seems to suggests that simple chemical and physical interactions within cells -- such as self-assembly, phase separation, and partitioning -- can result in seemingly complex behaviors -- like asymmetric division -- even when no additional cellular machinery is present. "Since there were no nucleic acids nor enzymes present, we clearly didn't have genes governing how our model cells would behave," Keating said. "So our study supports the hypothesis that structural and organizational 'cues' work in concert with genetic signals to achieve and maintain polarity through successive cell-division cycles."

Keating added that a working model of cellular dynamics requires a good understanding, not just of the role of genes, but also of the role of the structural organization of cells. "Once we have a firm grasp of what guides a cell's behavior, we might one day be able to design better disease treatments based on targeting errors in intracellular organization," she said.

Keating also explained that experimentation on non-living model cells that contain no DNA could help point to clues explaining the mysterious process of abiogenesis -- the formation of life from non-living matter, an event that happened at least once during our Earth's history. "Scientists have simulated early-Earth conditions in laboratories and have demonstrated that many amino acids -- the biochemical constituents of proteins -- can form through natural chemical reactions," Keating said. "We hope our research helps to fill in another part of the puzzle: how chemical and spatial organization may have contributed to the success of early life forms."

The work was funded by the Chemistry and Molecular and Cellular Biosciences divisions of the National Science Foundation and by the National Institutes of Health.

Story Source:

The above story is reprinted (with editorial adaptations) from materials provided by Penn State.

Journal Reference:

Meghan Andes-Koback, Christine D. Keating. Complete Budding and Asymmetric Division of Primitive Model Cells to Produce Daughter Vesicles with Different Interior and Membrane Compositions. Journal of the American Chemical Society, 2011; 110518124742024 DOI: 10.1021/ja202406v

'Critical baby step' taken for spying life on a molecular scale

The ability to image single biological molecules in a living cell is something that has long eluded researchers; however, a novel technique, using the structure of diamond, may well be able to do this and potentially provide a tool for diagnosing, and eventually developing a treatment for, hard-to-cure diseases such as cancer.

In a study published May 19 in the Institute of Physics and the German Physical Society's New Journal of Physics, researchers have developed a technique, exploiting a specific defect in the lattice structure of diamond, to externally detect the spins of individual molecules.

Magnetic Resonance Imaging (MRI) has already taken advantage of a molecule's spin to give clear snapshots of organs and tissue within the human body, however to get a more detailed insight into the workings of disease, the imaging scale must be brought down to individual biomolecules, and captured whilst the cells are still alive.

Co-lead author Professor Phillip Hemmer, Electrical & Computer Engineering, Texas A&M University, said, "Many conditions, such as cancer and aging, have their roots at the molecular scale. Therefore if we could somehow develop a tool that would allow us to do magnetic resonance imaging of individual biomolecules in a living cell then we would have a powerful new tool for diagnosing and eventually developing cures for such stubborn diseases."

To do this, the researchers, from Professor Joerg Wrachtrup's group at the University of Stuttgart and Texas A&M University, used a constructed defect in the structure of diamond called a nitrogen vacancy (NV) -- a position within the lattice structure where one of the carbon atoms is replaced with a nitrogen atom.

Instead of bonding to four other carbon atoms, the nitrogen atom only bonds to three carbon atoms leaving a spare pair of electrons, acting as one of the strongest magnets on an atomic scale.

The most important characteristic of a diamond NV is that it has an optical readout -- it emits bright red light when excited by a laser, which is dependent on which way the magnet is pointing.

The researchers found that if an external spin is placed close to the NV it will cause the magnet to point in a different direction, therefore changing the amount of light emitted by it.

This change of light can be used to gauge which way the external molecule is spinning and therefore create a one-dimensional image of the external spin. If combined with additional knowledge of the surface, or a second NV nearby, a more detailed image with additional dimensions could be had.

To test this theory, nitrogen was implanted into a sample of diamond in order to produce the necessary NVs. External molecules were brought to the surface of the diamond, using several chemical interactions, for their spins to be analyzed.

Spins that exist within the diamond structure itself have already been modelled, so to test that the spins were indeed external, the researchers chemically cleaned the diamond surface and performed the analysis again to prove that the spins had been washed away.

Professor Hemmer continued, "Currently, biological interactions are deduced mostly by looking at large ensembles. In this case you are looking only at statistical averages and details of the interaction which are not always clear. Often the data is taken after killing the cell and spreading its contents onto a gene chip, so it is like looking at snapshots in time when you really want to see the whole movie."

"Clearly there is much work to be done before we can, if ever, reach our long-term goal of spying on the inner workings of life on the molecular scale. But we have to learn to walk before we can run, and this breakthrough represents one of the first critical baby steps."

Story Source:

The above story is reprinted (with editorial adaptations) from materials provided by Institute of Physics, via EurekAlert!, a service of AAAS.

Journal Reference:

Grotz et al. Sensing external spins with NV diamond. New Journal of Physics, 2011; [link]

Nuclear magnetic resonance with no magnets

Nuclear magnetic resonance (NMR), a scientific technique associated with outsized, very low-temperature, superconducting magnets, is one of the principal tools in the chemist's arsenal, used to study everything from alcohols to proteins to such frontiers as quantum computing. In hospitals the machinery of NMR's cousin, magnetic resonance imaging (MRI), is as loud as it is big, but nevertheless a mainstay of diagnosis for a wide range of medical conditions.

It sounds like magic, but now two groups of scientists at Berkeley Lab and UC Berkeley, one expert in chemistry and the other in atomic physics, long working together as a multidisciplinary team, have shown that chemical analysis with NMR is practical without using any magnets at all.

Dmitry Budker of Berkeley Lab's Nuclear Science Division, a professor of physics at UC Berkeley, is a protean experimenter who leads a group with interests ranging as far afield as tests of the fundamental theorems of quantum mechanics, biomagnetism in plants, and violations of basic symmetry relations in atomic nuclei. Alex Pines, of the Lab's Materials Sciences Division and UCB's Department of Chemistry, is a modern master of NMR and MRI. He guides the work of a talented, ever-changing cadre of postdocs and grad students known as the "Pinenuts" -- not only in doing basic research in NMR but in increasing its practical applications. Together the groups have extended the reach of NMR by eliminating the use of magnetic fields at different stages of NMR measurements, and have finally done away with external magnetic fields entirely.

Spinning the information

NMR and MRI depend on the fact that many atomic nuclei possess spin (not classical rotation but a quantum number) and -- like miniature planet Earths with north and south magnetic poles -- have their own dipolar magnetic fields. In conventional NMR these nuclei are lined up by a strong external magnetic field, then knocked off axis by a burst of radio waves. The rate at which each kind of nucleus then "wobbles" (precesses) is unique and identifies the element; for example a hydrogen-1 nucleus, a lone proton, precesses four times faster than a carbon-13 nucleus having six protons and seven neutrons.

Being able to detect these signals depends first of all on being able to detect net spin; if the sample were to have as many spin-up nuclei as spin-down nuclei it would have zero polarization, and signals would cancel. But since the spin-up orientation requires slightly less energy, a population of atomic nuclei usually has a slight excess of spin ups, if only by a few score in a million.

"Conventional wisdom holds that trying to do NMR in weak or zero magnetic fields is a bad idea," says Budker, "because the polarization is tiny, and the ability to detect signals is proportional to the strength of the applied field."

The lines in a typical NMR spectrum reveal more than just different elements. Electrons near precessing nuclei alter their precession frequencies and cause a "chemical shift" -- moving the signal or splitting it into separate lines in the NMR spectrum. This is the principal goal of conventional NMR, because chemical shifts point to particular chemical species; for example, even when two hydrocarbons contain the same number of hydrogen, carbon, or other atoms, their signatures differ markedly according to how the atoms are arranged. But without a strong magnetic field, chemical shifts are insignificant.

"Low- or zero-field NMR starts with three strikes against it: small polarization, low detection efficiency, and no chemical-shift signature," Budker says.

"So why do it?" asks Micah Ledbetter of Budker's group. It's a rhetorical question. "The main thing is getting rid of the big, expensive magnets needed for conventional NMR. If you can do that, you can make NMR portable and reduce the costs, including the operating costs. The hope is to be able to do chemical analyses in the field -- underwater, down drill holes, up in balloons -- and maybe even medical diagnoses, far from well-equipped medical centers."

"As it happens," Budker says, "there are already methods for overcoming small polarization and low detection efficiency, the first two objections to low- or zero-field NMR. By bringing these separate methods together, we can tackle the third objection -- no chemical shift -- as well. Zero-field NMR may not be such a bad idea after all."

Net spin orientation can be increased in various ways, collectively known as hyperpolarization. One way to hyperpolarize a sample of hydrogen gas is to change the proportions of parahydrogen and orthohydrogen in it. Like most gases, at normal temperature and pressure each hydrogen molecule consists of two atoms bound together. If the spins of the proton nuclei point in the same direction, it's orthohydrogen. If the spins point in opposite directions, it's parahydrogen.

By the mathematics of quantum mechanics, adding up the spin states of the two protons and two electrons in a hydrogen molecule equals three ways for orthohydrogen to reach spin one; parahydrogen can only be spin zero, however. Thus orthohydrogen molecules normally account for three-quarters of hydrogen gas and parahydrogen only one-quarter.

Parahydrogen can be enhanced to 50 percent or even 100 percent using very low temperatures, although the right catalyst must be added or the conversion could take days if not weeks. Then, by chemically reacting spin-zero parahydrogen molecules with an initial chemical, net polarization of the product of the hydrogenation may end up highly polarized. This hyperpolarization can be extended not only to the parts of the molecule directly reacting with the hydrogen, but even to the far corners of large molecules. The Pinenuts, who devised many of the techniques, are masters of parahydrogen production and its hyperpolarization chemistry.

"With a high proportion of parahydrogen you get a terrific degree of polarization," says Ledbetter. "The catch is, it's spin zero. It doesn't have a magnetic moment, so it doesn't give you a signal! But all is not lost…."

And now for the magic

In low magnetic fields, increasing detection efficiency requires a very different approach, using detectors called magnetometers. In early low-field experiments, magnetometers called SQUID were used (superconducting quantum interference devices). Although exquisitely sensitive, SQUID, like the big magnets used in high-field NMR, must be cryogenically cooled to low temperatures.

Optical-atomic magnetometers are based on a different principle -- one that, curiously, is something like NMR in reverse, except that optical-atomic magnetometers measure whole atoms, not just nuclei. Here, an external magnetic field is measured by measuring the spin of the atoms inside the magnetometer's own vapor cell, typically a thin gas of an alkali metal such as potassium or rubidium. Their spin is influenced by polarizing the atoms with laser light; if there's even a weak external field, they begin to precess. A second laser beam probes how much they're precessing and thus just how strong the external field is.

Budker's group has brought optical-atomic magnetometry to a high pitch by such techniques as extending the "relaxation time," the time before the polarized vapor loses its polarization. In previous collaborations, the Pines and Budker groups have used magnetometers with NMR and MRI to image the flow of water using only the Earth's magnetic field or no field at all, to detect hyperpolarized xenon gas (but without analyzing chemical states), and in other applications. The next frontier is chemical analysis.

"No matter how sensitive your detector or how polarized your samples, you can't detect chemical shifts in a zero field," Budker says. "But there has always been another signal in NMR that can be used for chemical analysis -- it's just that it is usually so weak compared to chemical shifts, it has been the poor relative in the NMR family. It's called J-coupling."

Discovered in 1950 by the NMR pioneer Erwin Hahn and his graduate student, Donald Maxwell, J-coupling provides an interaction pathway between two protons (or other nuclei with spin), which is mediated by their associated electrons. The signature frequencies of these interactions, appearing in the NMR spectrum, can be used to determine the angle between chemical bonds and distances between the nuclei.

"You can even tell how many bonds separate the two spins," Ledbetter says. "J-coupling reveals all that information."

The resulting signals are highly specific and indicate just what chemical species is being observed. Moreover, as Hahn saw right away, while the signal can be modified by external magnetic fields, it does not vanish in their absence.

With Ledbetter in the lead, the Budker/Pines collaboration built a magnetometer specifically designed to detect J-coupling at zero magnetic field. Thomas Theis, a graduate student in the Pines group, supplied the parahydrogen and the chemical expertise to take advantage of parahydrogen-induced polarization. Beginning with styrene, a simple hydrocarbon, they measured J-coupling on a series of hydrocarbon derivatives including hexane and hexene, phenylpropene, and dimethyl maleate, important constituents of plastics, petroleum products, even perfumes.

"The first step is to introduce the parahydrogen," Budker says. "The top of the set-up is a test tube containing the sample solution, with a tube down to the bottom through which the parahydrogen is bubbled." In the case of styrene, the parahydrogen was taken up to produce ethylbenzene, a specific arrangement of eight carbon atoms and 10 hydrogen atoms.

Immediately below the test tube sits the magnetometer's alkali vapor cell, a device smaller than a fingernail, microfabricated by Svenja Knappe and John Kitching of the National Institute of Standards and Technology. The vapor cell, which sits on top of a heater, contains rubidium and nitrogen gas through which pump and probe laser beams cross at right angles. The mechanism is surrounded by cylinders of "mu metal," a nickel-iron alloy that acts as a shield against external magnetic fields, including Earth's.

Ledbetter's measurements produced signatures in the spectra which unmistakably identified chemical species and exactly where the polarized protons had been taken up. When styrene was hydrogenated to form ethylbenzene, for example, two atoms from a parahydrogen molecule bound to different atoms of carbon-13 (a scarce but naturally occurring isotope whose nucleus has spin, unlike more abundant carbon-12).

J-coupling signatures are completely different for otherwise identical molecules in which carbon-13 atoms reside in different locations. All of this is seen directly in the results. Says Budker, "When Micah goes into the laboratory, J-coupling is king."

Of the present football-sized magnetometer and its lasers, Ledbetter says, "We're already working on a much smaller version of the magnetometer that will be easy to carry into the field."

Although experiments to date have been performed on molecules that are easily hydrogenated, hyperpolarization with parahydrogen can also be extended to other kinds of molecules. Budker says, "We're just beginning to develop zero-field NMR, and it's still too early to say how well we're going to be able to compete with high-field NMR. But we've already shown that we can get clear, highly specific spectra, with a device that has ready potential for doing low-cost, portable chemical analysis."

Story Source:

The above story is reprinted (with editorial adaptations) from materials provided by DOE/Lawrence Berkeley National Laboratory.

Journal Reference:

T. Theis, P. Ganssle, G. Kervern, S. Knappe, J. Kitching, M. P. Ledbetter, D. Budker, A. Pines. Parahydrogen-enhanced zero-field nuclear magnetic resonance. Nature Physics, 2011; DOI: 10.1038/nphys1986

Rainbows without pigments offer new defense against fraud

Scientists from the University of Sheffield have developed pigment-free, intensely coloured polymer materials, which could provide new, anti-counterfeit devices on passports or banknotes due to their difficulty to copy.

The polymers do not use pigments but instead exhibit intense colour due to their structure, similar to the way nature creates colour for beetle shells and butterfly wings.

These colours were created by highly ordered polymer layers, which the researchers produced using block copoylmers (an alloy of two different polymers). By mixing block copolymers together, the researchers were able to create any colour in the rainbow from two non-coloured solutions.

This type of polymer then automatically organises itself into a layered structure, causing optical effects similar to opals. The colour also changes depending on the viewing angle. This system has huge advantage in terms of cost, processing and colour selection compared to existing systems.

The complexity of the chemistry involved in making the polymer means they are very difficult for fraudsters to copy, making them ideally suited for use on passports or banknotes.

The academics used Diamond Light Source, the UK's national synchrotron science facility in Oxfordshire, to probe the ordered, layered structures using high power X-rays. This helped them understand how the colours were formed, and how to improve the appearance.

Dr Andrew Parnell, from the University of Sheffield's Department of Physics and Astronomy, said: "Our aim was to mimic the wonderful and funky coloured patterns found in nature, such as Peacock feathers. We now have a painter's palette of colours that we can choose from using just two polymers to do this. We think that these materials have huge potential to be used commercially."

Professor Nick Terrill, Principal Beamline Scientist for I22, the Diamond laboratory used for the experiment, explained: "Small Angle X-ray Scattering is a simple technique that in this case has provided valuable confirmatory information. By using Diamond's X-rays to confirm the structure of the polymer, the group was able to identify the appropriate blends for the colours required, meaning they can now tailor the polymer composition accordingly."

The institutions involved in the research include the University of Sheffield, the University of Hull and Diamond Light Source in Oxfordshire.

Story Source:

The above story is reprinted (with editorial adaptations) from materials provided by University of Sheffield.

Journal Reference:

Andrew J. Parnell, Andrew Pryke, Oleksandr O. Mykhaylyk, Jonathan R. Howse, Ali. M. Adawi, Nicholas J. Terrill, J. Patrick A. Fairclough. Continuously tuneable optical filters from self-assembled block copolymer blends. Soft Matter, 2011; 7 (8): 3721 DOI: 10.1039/C0SM01320J