Scientists Create Cell Assembly Line: New Technology Synthesizes Cellular Structures from Simple Starting Materials

Scientists Create Cell Assembly Line: New Technology Synthesizes Cellular Structures from Simple Starting Materials

  12:29:00 AM    Science Lover

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Borrowing a page from modern manufacturing, scientists from the Florida campus of The Scripps Research Institute have built a microscopic assembly line that mass produces synthetic cell-like compartments.

Artist's rendering of cell structure. 
(Credit: iStockphoto/Sebastian Kaulitzki)

 

The new computer-controlled system represents a technological leap forward in the race to create the complex membrane structures of biological cells from simple chemical starting materials.

"Biology is full of synthetic targets that have inspired chemists for more than a century," said Brian Paegel, Scripps Research assistant professor and lead author of a new study published in the Journal of the American Chemical Society. "The lipid membrane assemblies of cells and their organelles pose a daunting challenge to the chemist who wants to synthesize these structures with the same rational approaches used in the preparation of small molecules."

While most cellular components such as genes or proteins are easily prepared in the laboratory, little has been done to develop a method of synthesizing cell membranes in a uniform, automated way. Current approaches are capricious in nature, yielding complex mixtures of products and inefficient cargo loading into the resultant cell-like structures.

The new technology transforms the previously difficult synthesis of cell membranes into a controlled process, customizable over a range of cell sizes, and highly efficient in terms of cargo encapsulation.

The membrane that surrounds all cells, organelles and vesicles -- small subcellular compartments -- consists of a phospholipid bilayer that serves as a barrier, separating an internal space from the external medium.

The new process creates a laboratory version of this bilayer that is formed into small, cell-sized compartments.

How It Works

"The assembly-line process is simple and, from a chemistry standpoint, mechanistically clear," said Sandro Matosevic, research associate and co-author of the study.

A microfluidic circuit generates water droplets in lipid-containing oil. The lipid-coated droplets travel down one branch of a Y-shaped circuit and merge with a second water stream at the Y-junction. The combined flows of droplets in oil and water travel in parallel streams toward a triangular guidepost.

Then, the triangular guide diverts the lipid-coated droplets into the parallel water stream as a wing dam might divert a line of small boats into another part of a river. As the droplets cross the oil-water interface, a second layer of lipids deposits on the droplet, forming a bilayer.

The end result is a continuous stream of uniformly shaped cell-like compartments.

The newly created vesicles range from 20 to 70 micrometers in diameter -- from about the size of a skin cell to that of a human hair. The entire circuit fits on a glass chip roughly the size of a poker chip.

The researchers also tested the synthetic bilayers for their ability to house a prototypical membrane protein. The proteins correctly inserted into the synthetic membrane, proving that they resemble membranes found in biological cells.

"Membranes and compartmentalization are ubiquitous themes in biology," noted Paegel. "We are constructing these synthetic systems to understand why compartmentalized chemistry is a hallmark of life, and how it might be leveraged in therapeutic delivery."

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Male Fertility Is in the Bones: First Evidence That Skeleton Plays a Role in Reproduction

Male Fertility Is in the Bones: First Evidence That Skeleton Plays a Role in Reproduction

  11:19:00 AM    Science Lover

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Researchers at Columbia University Medical Center have discovered that the skeleton acts as a regulator of fertility in male mice through a hormone released by bone, known as osteocalcin.

Researchers have found an altogether unexpected 
connection between a hormone produced in bone and male 
fertility. The study shows that the skeletal hormone known as 
osteocalcin boosts testosterone production to support the 
survival of the germ cells that go on to become mature sperm. 
(Credit: iStockphoto/Max Delson Martins Santos)

 

The research, led by Gerard Karsenty, M.D., Ph.D., chair of the Department of Genetics and Development at Columbia University Medical Center, is slated to appear online on February 17 in Cell, ahead of the journal's print edition, scheduled for March 4.

Until now, interactions between bone and the reproductive system have focused only on the influence of gonads on the build-up of bone mass.

"Since communication between two organs in the body is rarely one-way, the fact that the gonads regulate bone really begs the question: Does bone regulate the gonads?" said Dr. Karsenty.

Dr. Karsenty and his team found their first clue to an answer in the reproductive success of their lab mice. Previously, the researchers had observed that males whose skeletons did not secrete a hormone called osteocalcin were poor breeders.

The investigators then did several experiments that show that osteocalcin enhances the production of testosterone, a sex steroid hormone controlling male fertility. As they added osteocalcin to cells that, when in our body produce testosterone, its synthesis increased. Similarly, when they injected osteocalcin into male mice, circulating levels of testosterone also went up.

Conversely, when osteocalcin is not present, testosterone levels drop, which causes a decline in sperm count, the researchers found. When osteocalcin-deficient male mice were bred with normal female mice, the pairs only produced half the number of litters as did pairs with normal males, along with a decrease in the number of pups per litter.

Though the findings have not yet been confirmed in humans, Dr. Karsenty expects to find similar characteristics in humans, based on other similarities between mouse and human hormones.

If osteocalcin also promotes testosterone production in men, low osteocalcin levels may be the reason why some infertile men have unexplained low levels of testosterone.

Skeleton Regulates Male Fertility, But Not Female

Remarkably, although the new findings stemmed from an observation about estrogen and bone mass, the researchers could not find any evidence that the skeleton influences female reproduction.

Estrogen is considered one of the most powerful hormones that control bone; when ovaries stop producing estrogen in women after menopause, bone mass rapidly declines and can lead to osteoporosis.

Sex hormones, namely estrogen in women and testosterone in men, have been known to affect skeletal growth, but until now, studies of the interaction between bone and the reproductive system have focused only on how sex hormones affect the skeleton.

"We do not know why the skeleton regulates male fertility, and not female. However, if you want to propagate the species, it's probably easier to do this by facilitating the reproductive ability of males," said Dr. Karsenty. "This is the only rationale I can think of to explain why osteocalcin regulates reproduction in male and not in female mice."

Other Novel Functions of Osteocalcin Reported Earlier

The unexpected connection between the skeleton and male fertility is one of a string of surprising findings in the past few years regarding the skeleton. In previous papers, Dr. Karsenty has found that osteocalcin helps control insulin secretion, glucose metabolism and body weight.

"What this work shows is that we know so little physiology, that by asking apparently naïve questions, we can make important discoveries," Dr. Karsenty says. "It also shows that bone exerts an important array of functions all affected during the aging process. As such, these findings suggest that bone is not just a victim of the aging process, but that it may be an active determinant of aging as well."

Next Steps and Potential Drug Development

Next, the researchers plan to determine the signaling pathways used by osteocalcin to enhance testosterone production.

And as for potential drug development, since the researchers have also identified a receptor of osteocalcin, more flexibility in designing a drug that mimics the effect of osteocalcin is expected.

Whether it's for glucose metabolism or fertility, says Dr. Karsenty, knowing the receptor will make it easier for chemists to develop a compound that will bind to it.

"This study expands the physiological repertoire of osteocalcin, and provides the first evidence that the skeleton is a regulator of reproduction," said Dr. Karsenty.

Authors of the Cell study are: Franck Oury, Ph.D. (CUMC); Grzegorz Sumara, Ph.D. (CUMC); Olga Sumara, Ph.D. (CUMC); Mathieu Ferron, Ph.D. (CUMC); Haixin Chang, Ph.D. (Cornell University); Charles E. Smith, Ph.D. (McGill University); Louis Hermo, Ph.D. (McGill University); Susan Suarez, Ph.D. (Cornell University); Bryan L Roth, Ph.D.(UNC-Chapel Hill); Patricia Ducy, Ph.D. (CUMC); and Gerard Karsenty, M.D., Ph.D. (CUMC).

This study was supported in part by the National Institute of Child Health Research (NICHR) and the National Institutes of Health (NIH).

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Neuron Connections Seen in 3-D

Neuron Connections Seen in 3-D

  3:44:00 PM    Science Lover

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A team of researchers from the Max Planck Institute of Biochemistry, in Germany, led by the Spanish physicist Rubén Fernández-Busnadiego, has managed to obtain 3D images of the vesicles and filaments involved in communication between neurons. The method is based on a novel technique in electron microscopy, which cools cells so quickly that their biological structures can be frozen while fully active.

This three-dimensional visualization of synapses shows the tomography mail synaptic vesicles (yellow), cell membrane (purple), connectors between vesicles (red), filaments that anchor the vesicles to the cell membrane (blue), microtubule (dark green), material synaptic space (light green) and postsynaptic density (orange). (Credit: Fernández-Busnadiego et al.)

"We used electron cryotomography, a new technique in microscopy based on ultra-fast freezing of cells, in order to study and obtain three-dimensional images of synapsis, the cellular structure in which the communication between neurons takes place in the brains of mammals" Rubén Fernández-Busnadiego, lead author of the study which features on the front cover of this month's Journal of Cell Biology and a physicist at the Max Planck Institute of Biochemistry, in Germany, said.

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Nanoelectronic Transistor Combined With Biological Machine Could Lead To Better Electronics

Nanoelectronic Transistor Combined With Biological Machine Could Lead To Better Electronics

  9:44:00 PM    Science Lover

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If artificial devices could be combined with biological machines, laptops and other electronic devices could get a boost in operating efficiency.

An artist's representation of a nanobioelectronic device incorporating alamethycin biological pore. In the core of the device is a silicon nanowire (grey), covered with a lipid bilayer (blue). The bilayer incorporates bundles of alamethicin molecules (purple) that form pore channels in the membrane. Transport of protons though these pore channels changes the current through the nanowire. (Credit: Image by Scott Dougherty, LLNL)

Lawrence Livermore National Laboratory researchers have devised a versatile hybrid platform that uses lipid-coated nanowires to build prototype bionanoelectronic devices.

Mingling biological components in electronic circuits could enhance biosensing and diagnostic tools, advance neural prosthetics such as cochlear implants, and could even increase the efficiency of future computers.

While modern communication devices rely on electric fields and currents to carry the flow of information, biological systems are much more complex. They use an arsenal of membrane receptors, channels and pumps to control signal transduction that is unmatched by even the most powerful computers. For example, conversion of sound waves into nerve impulses is a very complicated process, yet the human ear has no trouble performing it.

“Electronic circuits that use these complex biological components could become much more efficient,” said Aleksandr Noy, the LLNL lead scientist on the project.

While earlier research has attempted to integrate biological systems with microelectronics, none have gotten to the point of seamless material-level incorporation.

“But with the creation of even smaller nanomaterials that are comparable to the size of biological molecules, we can integrate the systems at an even more localized level,” Noy said.

To create the bionanoelectronic platform the LLNL team turned to lipid membranes, which are ubiquitous in biological cells. These membranes form a stable, self-healing,and virtually impenetrable barrier to ions and small molecules.

“That's not to mention that these lipid membranes also can house an unlimited number of protein machines that perform a large number of critical recognition, transport and signal transduction functions in the cell,” said Nipun Misra, a UC Berkeley graduate student and a co-author on the paper.

Julio Martinez, a UC Davis graduate student and another co-author added: “Besides some preliminary work, using lipid membranes in nanoelectronic devices remains virtually untapped.”

The researchers incorporated lipid bilayer membranes into silicon nanowire transistors by covering the nanowire with a continuous lipid bilayer shell that forms a barrier between the nanowire surface and solution species.

“This 'shielded wire' configuration allows us to use membrane pores as the only pathway for the ions to reach the nanowire,” Noy said. “This is how we can use the nanowire device to monitor specific transport and also to control the membrane protein.”

The team showed that by changing the gate voltage of the device, they can open and close the membrane pore electronically.

The research appears Aug. 10 in the online version of the Proceedings of the National Academy of Sciences.

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Spontaneous Assembly: A New Look At How Proteins Assemble And Organize Themselves Into Complex Patterns

Spontaneous Assembly: A New Look At How Proteins Assemble And Organize Themselves Into Complex Patterns

  9:28:00 PM    Science Lover

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Self-assembling and self-organizing systems are the Holy Grails of nanotechnology, but nature has been producing such systems for millions of years. A team of scientists has taken a unique look at how thousands of bacterial membrane proteins are able to assemble into clusters that direct cell movement to select chemicals in their environment. Their results provide valuable insight into how complex periodic patterns in biological systems can be generated and repaired.

PALM is an an ultrahigh-precision visible light microscopy
technique that enables scientists to photo-actively fluoresce
and image individual proteins. This PALM composite of an
E.coli bacterial cell shows the organization of proteins in
the chemotaxis signaling network.
(Credit: Image courtesy of DOE/Lawrence Berkeley National Laboratory)

Researchers with Berkeley Lab, the University of California (UC) Berkeley, the Howard Hughes Medical Institute, and Princeton University, used an ultrahigh-precision visible light microscopy technique called PALM - for Photo-Activated Localization Microscopy - to show that the chemotaxis network of signaling proteins in E.coli bacteria is able to spontaneously form from clusters of proteins without being actively distributed or attached to specific locations in cells. This simple organizational mechanism - dubbed “stochastic self-assembly” - is related to the self-organizing patterns first described in 1952 by the British computer scientist Alan Turing.

“It is not widely appreciated that complex periodic patterns can spontaneously emerge from simple mechanisms, but that is probably what is happening here,” said Jan Liphardt, the biophysicist who led this research.

Liphardt holds a joint appointment with Berkeley Lab’s Physical Biosciences Division and UC Berkeley’s Physics Department. He is the principal author of a paper now available PLoS Biology. Co-authoring the paper with Liphardt were Derek Greenfield, Ann McEvoy, Hari Shroff, Gavin Crooks, Ned Wingreen and Eric Betzig.

Key to a cell’s survival is the manner in which its critical components - proteins, lipids, nucleic acids, etc. - are arranged. For cells to thrive, the organization of these components must be optimized for their respective activities and also reproducible for succeeding generations of cells. Eukaryotic cells feature distinct subcellular structures, such as membrane-bound organelles and protein transport systems, whose complex organization is readily apparent. However, there is also complex spatial organization to be found within prokaryotic cells, such as rod-shaped bacteria like E. coli.

“It has remained somewhat mysterious how bacteria are able to organize and spatially segregate their interiors and membranes,” said Liphardt. “Two cells which are biochemically identical can have very different behaviors, depending upon their spatial organization. With new technologies such as PALM, we are able to see exactly how cells are organized and relate spatial organization with biological function.”

PALM and the Chemotaxis Network

In the PALM technique, target proteins are labeled with tags that fluoresce when activated by weak ultraviolet light. By keeping the intensity of this light sufficiently low, researchers can photoactivate individual proteins.

“Since individual proteins are imaged one at a time, we can localize and count them, and then computationally assemble the locations of all proteins into a composite, high-precision image,” said Liphardt. “With other technologies, we have to choose between observing large clusters or observing single proteins. With PALM, we can examine a cell and see single proteins, protein dimers, and so forth, all the way up to large clusters containing thousands of proteins. This enables us to see the relative organization of individual proteins within clusters and at the same time see how clusters are arranged with respect to one-another.”

Liphardt and his colleagues applied the PALM technique to the E.coli chemotaxis network of signaling proteins, which direct the movement of the bacteria towards or away from sugars, amino acids, and many other soluble molecules in response to environmental cues. The E.coli chemotaxis network is one of the best-understood of all biological signaling systems and is a model for studying bacterial spatial organization because its components display a nonrandom, periodic distribution in the cell membrane.

“Chemotaxis proteins cluster into large sensory complexes that localize to the poles of the bacterial cell,” Liphardt said. “We wanted to understand how these clusters form, what controls their size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells.”

Using PALM, Liphardt and his colleagues mapped the cellular locations of three proteins central to the chemotaxis signaling network - Tar, CheY and CheW - with a mean precision of 15 nanometers. They found that cluster sizes were distributed with no one size being “characteristic.” For example, a third of the Tar proteins were part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of more than one million individual proteins from 326 cells determined that they are not actively distributed or attached to specific locations in cells, as had been hypothesized.

“Instead,” said Liphardt, “random lateral protein diffusion and protein-protein interactions are probably sufficient to generate the observed complex, ordered patterns. This simple stochastic self-assembly mechanism, which can create and maintain periodic structures in biological membranes without direct cytoskeletal involvement or active transport, may prove to be widespread in both prokaryotic and eukaryotic cells.”

Liphardt and his research group are now applying PALM to signaling complexes in eukaryotic membranes to see how widespread is stochastic self-assembly in nature. Given that biological systems are nature’s version of nanotechnology, the demonstration that stochastic self-assembly is capable of organizing thousands of proteins into complex and reproducible patterns holds promise for a wide range of applications in nanotechnology, including the fabrication of nanodevices and the development of nanoelectronic circuits.

This work was funded by the U.S. Department of Energy’s Office of Science, Energy Biosciences Program, the Sloan and Searle Foundations, and National Institutes of Health grants.

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