Cells Send Signals Via Membrane Nanotubes

Cells Send Signals Via Membrane Nanotubes

  8:30:00 PM    Science Lover

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Most of the body's cells communicate with each other by sending electrical signals through nano-thin membrane tubes. A sensational Norwegian research discovery may help to explain how cells cooperate to develop tissue in the embryo and how wounds heal.

Here a cell has coupled with another cell by growing 
a long nanotube which enables it to exchange 
electrical signals. (Credit: UiB)

For nearly ten years, researchers have known that cells can "grow" ultra-thin tubes named tunnelling nanotubes (TNTs) between one another. These nanotubes -- the length of two to three cells and just 1/500th the thickness of a human hair -- are connections that develop between nearly all cell types to form a communication channel different from any previously known mechanisms.

In 2010, Dr. Xiang Wang and Professor Hans-Hermann Gerdes -- colleagues at the University of Bergen's Department of Biomedicine -- discovered that electrical signals were being passed through nanotubes from one cell to another at high speed (roughly 1-2 m/sec). Their research receives funding under the Research Council's large-scale research programme Nanotechnology and New Materials (NANOMAT).

The breakthrough

In their key experiment, Dr Wang used fluorescent dye that changes in intensity as the electric potential of the cell membrane changes. When two cells connected by forming a nanotube, he poked into one of them with a microinjection needle to depolarise that cell's membrane potential. This caused the fluorescent indicator on the cell membrane to light up like a firework, and it was soon followed by a similar light display in the cell on the other end of the nanotube.

The breakthrough discovery began with an experiment demonstrating intercellular transmission of electrical signals via nanotubes in 2007. The researchers then carried out similar trials with a number of other cell types, observing similar occurrences.

"We confirmed that this is a common phenomenon between cells," explains Professor Gerdes. "Still, this characteristic is not in every cell type."

The experiment was replicated a number of times to obtain statistically reliable data. The electrophysiology group at the University of Bergen took precise conductivity measurements of the cell systems to determine the strength of the electrical coupling. In autumn 2010 the results were published in Proceedings of the National Academy of Sciences (PNAS).

Short lifespan

Intercellular nanotubes are far from permanent. Most of them last only a few minutes. This means the researchers cannot predict where and when the cells will form nanotube connections.

"It is truly painstaking work," says Professor Gerdes. "You may sit there examining cells for hours through a microscope without seeing a single tube. If you are lucky, however, you catch sight of a nanotube being created and can film the event."

To raise the likelihood of finding nanotubes, the researchers developed a micro-matrix consisting of thousands of points and bridges on a plate surface. Smaller than a postage stamp, the plate is covered by a nano-structured material to which the cells adhere. The researchers place one cell onto each point and hope that nanotubes will form along the bridges between the points. The camera is focused on these bridges.

Once the nanotubes have been established, the researchers manipulate the cells at specified times; meanwhile the microscope is programmed to photograph, say, 50 preselected points every five minutes. The team can thus obtain data about many nanotube connections in a short time.

How do cells do this?
Dr. Wang quickly discovered that the mere presence of a nanotube was not sufficient to transmit an electrical signal. There had to be another mechanism involved as well.

Many cells form tiny membrane pores with each other called gap junctions, which are made up of ring-shaped proteins. Back in the 1960s it was discovered that directly adjacent cells could exchange electrical impulses through these gap junctions. What Dr Wang found was that one end of the nanotube was always connected to cells by a gap junctions before it transmitted its electrical impulses.

He also found that in some coupled cells voltage-gated calcium channels were involved in the forwarding of the incoming signals. When the electrical signal being sent through the nanotube reaches the membrane of the receiving cell, the membrane surface is depolarised, opening the calcium channel and allowing calcium -- a vital ion in cell signalling -- to enter.

"In other words," explains Professor Gerdes, "there are two components: a nanotube and a gap junction. The nanotube grows out from one cell and connects to the other cell through a gap junction. Only then can the two cells be coupled electrically."

Controls embryonic cells?

Now the scientists are seeking answers as to why the cells send signals to each other in this way.

"It's quite possible that the discovery of nanotubes will give us new insight into intercellular communication," asserts Professor Gerdes. "The process could explain how cells are coordinated during embryo growth. In that phase cells travel long distances -- yet they demonstrate a kind of collective behaviour, and move together like a flock of birds can."

Nanotubes may also be a factor in explaining cell movement associated with wound healing, since cells move toward a wound in order to close it. We already know that electrical signals are somehow involved in this process; scientists can only speculate as to whether nanotubes are involved here as well, stresses Professor Gerdes.

Perhaps brain cells, too?

In terms of electronic signal processing, the human brain surpasses all other organs. If this same signalling mechanism proves to be present in human brain cells, it could add a new dimension to understanding how the brain functions. Communication channels involving synapses and dendrites that are already identified differ widely from nanotubes.

The Bergen-based neuroscientists see this research as an opportunity to formulate better explanations for phenomena related to consciousness and electrical connections in the brain. In the project "Cell-to-cell communication: Mechanism of tunnelling nanotube formation and function," they are now studying precisely how nanotube mechanisms function in brain cells.

Professor Gerdes is currently conducting research at the European Molecular Biology Laboratory in Heidelberg. By studying the electrical connections in vivo he hopes to figure out how the mechanisms work in live subjects. The results could enhance understanding of diseases that occur when cell mechanisms fail to function properly.

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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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Bioengineers Discover How Particles Self-Assemble in Flowing Fluids

Bioengineers Discover How Particles Self-Assemble in Flowing Fluids

  10:10:00 AM    Science Lover

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From atomic crystals to spiral galaxies, self-assembly is ubiquitous in nature. In biological processes, self-assembly at the molecular level is particularly prevalent.

A simple microfluidic "filter" structure converts 
microparticle streams with smaller interparticle 
spacings to trains of larger spacing. The channel 
width is about half the diameter of a human hair 
at the expansion. (Credit: Image courtesy 
of University of California - Los Angeles)

Phospholipids, for example, will self-assemble into a bilayer to form a cell membrane, and actin, a protein that supports and shapes a cell's structure, continuously self-assembles and disassembles during cell movement.

Bioengineers at the UCLA Henry Samueli School of Engineering and Applied Science have been exploring a unique phenomenon whereby randomly dispersed microparticles self-assemble into a highly organized structure as they flow through microscale channels.

This self-assembly behavior was unexpected, the researchers said, for such a simple system containing only particles, fluid and a conduit through which these elements flow. The particles formed lattice-like structures due to a unique combination of hydrodynamic interactions.

The research, published online December 13 in the journal Proceedings of the National Academy of Sciences, was led by UCLA postdoctoral scholar Wonhee Lee and UCLA assistant professor of bioengineering Dino Di Carlo.

The research team discovered the mechanism that leads to this self-assembly behavior through a series of careful experiments and numerical simulations. They found that continuous disturbance of the fluid induced by each flowing and rotating particle drives neighboring particles away, while migration of particles to localized streams due to the momentum of the fluid acts to stabilize the spacing between particles at a finite distance. In essence, the combination of repulsion and localization leads to an organized structure.

Once they understood the mechanism, the team developed microchannels that allowed for "tuning" of the spatial frequency of particles within an organized particle train. They found that by simply adding short regions of expanded channel width, the particles could be induced to self-assemble into different structures in a controllable and potentially programmable way.

"Programmable control of flowing microscale particles may be important in opening up new capabilities in biomedicine, materials synthesis and computation, similar to how improved control of flowing electrons has enabled a revolution in computing and communication," Di Carlo said.

For example, controlling the positions of microscale bioparticles, such as cells in flowing channels, is important for the operation of blood analysis and counting diagnostic systems. In addition, improving the uniformity of cell concentrations entering the microscale volume of a print head can enable burgeoning fields such as "tissue printing," in which single cells in a polymer ink are sequentially positioned to form a functional tissue architecture, such as the cylindrical lumen of a blood vessel.

More complete control of lattices of particles may also allow tunable manufacturing of optical or acoustic metamaterials that interact uniquely with light and sound waves based on the arrangement of the embedded particles, the researchers said.

Disclaimer: Views expressed in this article do not necessarily reflect us.

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New Drug Delivery Technique: Nanoblasts from Laser-Activated Nanoparticles Move Molecules, Proteins and DNA Into Cells

New Drug Delivery Technique: Nanoblasts from Laser-Activated Nanoparticles Move Molecules, Proteins and DNA Into Cells

  2:39:00 PM    Science Lover

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Using chemical "nanoblasts" that punch tiny holes in the protective membranes of cells, researchers have demonstrated a new technique for getting therapeutic small molecules, proteins and DNA directly into living cells.

A field of human prostate cancer cells is shown after exposure to laser-activated carbon nanoparticles. The cell membranes have been stained red to assist in visualization. Each of the red circles is a single cell. (Credit: Credit: Prerona Chakravarty)

Carbon nanoparticles activated by bursts of laser light trigger the tiny blasts, which open holes in cell membranes just long enough to admit therapeutic agents contained in the surrounding fluid. By adjusting laser exposure, the researchers administered a small-molecule marker compound to 90 percent of targeted cells -- while keeping more than 90 percent of the cells alive.

The research was sponsored by the National Institutes of Health and the Institute of Paper Science and Technology at Georgia Tech. It will be reported in the August issue of the journal Nature Nanotechnology.

"This technique could allow us to deliver a wide variety of therapeutics that now cannot easily get into cells," said Mark Prausnitz, a professor in the School of Chemical and Biomolecular Engineering at the Georgia Institute of Technology. "One of the most significant uses for this technology could be for gene-based therapies, which offer great promise in medicine, but whose progress has been limited by the difficulty of getting DNA and RNA into cells."

The work is believed to be the first to use activation of reactive carbon nanoparticles by lasers for medical applications. Additional research and clinical trials will be needed before the technique could be used in humans.

Researchers have been trying for decades to drive DNA and RNA more efficiently into cells with a variety of methods, including using viruses to ferry genetic materials into cells, coating DNA and RNA with chemical agents or employing electric fields and ultrasound to open cell membranes. However, these previous methods have generally suffered from low efficiency or safety concerns.

With their new technique, which was inspired by earlier work on the so-called "photoacoustic effect," Prausnitz and collaborators Prerona Chakravarty, Wei Qian and Mostafa El-Sayed hope to better localize the application of energy to cell membranes, creating a safer and more efficient approach for intracellular drug delivery.

Their technique begins with introducing particles of carbon black measuring 25 nanometers -- one millionth of an inch -- in diameter into the fluid surrounding the cells into which the therapeutic agents are to be introduced. Bursts of near-infrared light from a femotosecond laser are then applied to the fluid at a rate of 90 million pulses per second. The carbon nanoparticles absorb the light, which makes them hot. The hot particles then heat the surrounding fluid to make steam. The steam reacts with the carbon nanoparticles to form hydrogen and carbon monoxide.

The two gases form a bubble which grows as the laser provides energy. The bubble collapses suddenly when the laser is turned off, creating a shock wave that punches holes in the membranes of nearby cells. The openings allow therapeutic agents from the surrounding fluid to enter the cells. The holes quickly close so the cell can survive.

The researchers have demonstrated that they could get the small molecule calcein, the bovine serum albumin protein and plasmid DNA through the cell membranes of human prostate cancer cells and rat gliosarcoma cells using this technique. Calcein uptake was seen in 90 percent of the cells at laser levels that left more than 90 percent of the cells alive.

"We could get almost all of the cells to take up these molecules that normally wouldn't enter the cells, and almost all of the cells remained alive," said Prerona Chakravarty, the study's lead author. "Our laser-activated carbon nanoparticle system enables controlled bubble implosions that can disrupt the cell membranes just enough to get the molecules in without causing lasting damage."

To assess how long the holes in the cell membrane remained open, the researchers left the simulated therapeutics out of the fluid when the cells were exposed to the laser light, then added the agents one second after turning off the laser. They saw almost no uptake of the molecules, suggesting that the cell membranes resealed themselves quickly.

To confirm that the carbon-steam reaction was a critical factor driving the nanoblasts, the researchers substituted gold nanoparticles for the carbon nanoparticles before exposure to laser light. Because they lacked the carbon needed for reaction, the gold nanoparticles produced little uptake of the molecules, Prausnitz noted.

Similarly, the researchers substituted carbon nanotubes for the carbon nanoparticles, and also measured little uptake, which they explained by noting that the nanotubes are less reactive than the carbon black particles.

Experimentation further showed that DNA introduced into cells through the laser-activated technique remained functional and capable of driving protein expression. When plasmid DNA that encoded for luciferase expression was introduced into the cancer cells, production of luciferase increased 17-fold.

For the future, the researchers plan to study use of a less expensive nanosecond laser to replace the ultrafast femtosecond instrument used in the research. They also plan to optimize the carbon nanoparticles so that nearly all of them are consumed during the exposure to laser light. Leftover carbon nanoparticles in the body should produce no harmful effects, though the body may be unable to eliminate them, Prausnitz noted.

"This is the first study showing proof of principle for laser-activation of reactive carbon nanoparticles for drug and gene delivery," he said. "There is a considerable path ahead before this can be brought into medicine, but we are optimistic that this approach can ultimately provide a new alternative for delivering therapeutic agents into cells safely and efficiently."

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Peptides May Hold 'Missing Link' to Life

Peptides May Hold 'Missing Link' to Life

  1:47:00 AM    Science Lover

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Emory scientists have discovered that simple peptides can organize into bi-layer membranes. The finding suggests a "missing link" between the pre-biotic Earth's chemical inventory and the organizational scaffolding essential to life.

Researchers tagged one end of peptide chains with an 
NMR label, and then allowed them to assemble to see 
if the ends would interact. The result was a bi-layer 
membrane with inner and outer faces and an additional, 
buried layer that localized functionality within the interior. 
(Credit: Image courtesy of Emory University)

"We've shown that peptides can form the kind of membranes needed to create long-range order," says chemistry graduate student Seth Childers, lead author of the paper recently published by the German Chemical Society's Angewandte Chemie. "What's also interesting is that these peptide membranes may have the potential to function in a complex way, like a protein."

Chemistry graduate student Yan Liang captured images of the peptides as they aggregated into molten globular structures, and self-assembled into bi-layer membranes. The results of that experiment were recently published by the Journal of the American Chemical Society.

"In order to form nuclei, which become the templates for growth, the peptides first repel water," says Liang, who is now an Emory post-doctoral fellow in neuroscience. "Once the peptides form the template, we can now see how they assemble from the outer edges."

In addition to providing clues to the origins of life, the findings may shed light on protein assemblies related to Alzheimer's disease, Type 2 diabetes, and dozens of other serious ailments.

"This is a boon to our understanding of large, structural assemblies of molecules," says Chemistry Chair David Lynn, who helped lead the effort behind both papers, which were collaborations of the departments of chemistry, biology and physics. "We've proved that peptides can organize as bi-layers, and we've generated the first, real-time imaging of the self-assembly process. We can actually watch in real-time as these nano-machines make themselves."

The ability to organize things within compartments and along surfaces underpins all of biology. From the bi-layer phospholipids of cell membranes to information-rich DNA helices, self-assembling arrays define the architecture of life.

But while phospholipids and DNA are complicated molecules, peptides are composed of the simple amino acids that make up proteins. The Miller-Urey experiment demonstrated in 1953 that amino acids were likely to be present on the pre-biotic Earth, opening the question of whether simple peptides could achieve supra-molecular order.

To test how the hollow, tubular structure of peptides is organized, the researchers used specialized solid-state nuclear magnetic resonance (NMR) methods that have been developed at Emory during the past decade. Working with Anil Mehta, a chemistry post-doctoral fellow, Childers tagged one end of peptide chains with an NMR label, and then allowed them to assemble to see if the ends would interact. The result was a bi-layer membrane with inner and outer faces and an additional, buried layer that localized functionality within the interior.

"The peptide membranes combine the long-range structure of cell membranes with the local order of enzymes," Childers said. "Now that we understand that peptide membranes are organized locally like a protein, we want to investigate whether they can function like a protein."

The goal is to direct molecules to perform as catalysts and create long-range order. "We'd really like to understand how to build something from the bottom up," Childers says. "How can we take atoms and make molecules? How can we get molecules that stick together to make nano-machines that will perform specific tasks?"

The research is part of "The Center for Chemical Evolution," a center based at Emory and Georgia Tech, for integrated research, education and public outreach focused on the chemistry that may have led to the origin of life. The National Science Foundation and the U.S. Department of Energy have funded the research.

Many groups studying the origins of life have focused on RNA, which is believed to have pre-dated living cells. But RNA is a much more complicated molecule than a peptide. "Our studies have now shown that, if you just add water, simple peptides access both the physical properties and the long-range molecular order that is critical to the origins of chemical evolution," Childers says.

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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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Tumors Feel The Deadly Sting Of Nanobees

Tumors Feel The Deadly Sting Of Nanobees

  1:59:00 AM    Science Lover

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When bees sting, they pump poison into their victims. Now the toxin in bee venom has been harnessed to kill tumor cells by researchers at Washington University School of Medicine in St. Louis. The researchers attached the major component of bee venom to nano-sized spheres that they call nanobees.

Bee on a finger. Researchers have recently harnessed the toxin in bee venom
to kill tumor cells. (Credit: iStockphoto/Tatiana Buzuleac)

In mice, nanobees delivered the bee toxin melittin to tumors while protecting other tissues from the toxin's destructive power. The mice's tumors stopped growing or shrank. The nanobees' effectiveness against cancer in the mice is reported in advance online publication Aug. 10 in the Journal of Clinical Investigation.

"The nanobees fly in, land on the surface of cells and deposit their cargo of melittin which rapidly merges with the target cells," says co-author Samuel Wickline, M.D., who heads the Siteman Center of Cancer Nanotechnology Excellence at Washington University. "We've shown that the bee toxin gets taken into the cells where it pokes holes in their internal structures."

Melittin is a small protein, or peptide, that is strongly attracted to cell membranes, where it can form pores that break up cells and kill them.

"Melittin has been of interest to researchers because in high enough concentration it can destroy any cell it comes into contact with, making it an effective antibacterial and antifungal agent and potentially an anticancer agent," says co-author Paul Schlesinger, M.D., Ph.D., associate professor of cell biology and physiology. "Cancer cells can adapt and develop resistance to many anticancer agents that alter gene function or target a cell's DNA, but it's hard for cells to find a way around the mechanism that melittin uses to kill."

The scientists tested nanobees in two kinds of mice with cancerous tumors. One mouse breed was implanted with human breast cancer cells and the other with melanoma tumors. After four to five injections of the melittin-carrying nanoparticles over several days, growth of the mice's breast cancer tumors slowed by nearly 25 percent, and the size of the mice's melanoma tumors decreased by 88 percent compared to untreated tumors.

The researchers indicate that the nanobees gathered in these solid tumors because tumors often have leaky blood vessels and tend to retain material. Scientists call this the enhanced permeability and retention effect of tumors, and it explains how certain drugs concentrate in tumor tissue much more than they do in normal tissues.

But the researchers also developed a more specific method for making sure nanobees go to tumors and not healthy tissue by loading the nanobees with additional components. When they added a targeting agent that was attracted to growing blood vessels around tumors, the nanobees were guided to precancerous skin lesions that were rapidly increasing their blood supply. Injections of targeted nanobees reduced the extent of proliferation of precancerous skin cells in the mice by 80 percent.

Overall, the results suggest that nanobees could not only lessen the growth and size of established cancerous tumors but also act at early stages to prevent cancer from developing.

"Nanobees are an effective way to package the useful, but potentially deadly, melittin, sequestering it so that it neither harms normal cells nor gets degraded before it reaches its target," Schlesinger says.

If a significant amount of melittin were injected directly into the bloodstream, widespread destruction of red blood cells would result. The researchers showed that nanoparticles protected the mice's red cells and other tissues from the toxic effects of melittin. Nanobees injected into the bloodstream did not harm the mice. They had normal blood counts, and tests for the presence of blood-borne enzymes indicative of organ damage were negative.

When secured to the nanobees, melittin is safe from protein-destroying enzymes that the body produces. Although unattached melittin was cleared from the mice's circulation within minutes, half of the melittin on nanobees was still circulating 200 minutes later. Schlesinger indicates that is long enough for the nanobees to circulate through the mice's bloodstream 200 times, giving them ample time to locate tumors.

"Melittin is a workhorse," says Wickline, also professor of medicine in the Cardiovascular Division and professor of physics, of biomedical engineering and of cell biology and physiology. "It's very stable on the nanoparticles, and it's easily and cheaply produced. We are now using a nontoxic part of the melittin molecule to hook other drugs, targeting agents or imaging compounds onto nanoparticles."

The core of the nanobees is composed of perfluorocarbon, an inert compound used in artificial blood. The research group developed perfluorocarbon nanoparticles several years ago and have been studying their use in various medical applications, including diagnosis and treatment of atherosclerosis and cancer. About six millionths of an inch in diameter, the nanoparticles are large enough to carry thousands of active compounds, yet small enough to pass readily through the bloodstream and to attach to cell membranes.

"We can add melittin to our nanoparticles after they are built," Wickline says. "If we've already developed nanoparticles as carriers and given them a targeting agent, we can then add a variety of components using native melittin or melittin-like proteins without needing to rebuild the carrier. Melittin fortunately goes onto the nanoparticles very quickly and completely and remains on the nanobee until cell contact is made."

The flexibility of nanobees and other nanoparticles made by the group suggests they could be readily adapted to fit medical situations as needed. The ability to attach imaging agents to nanoparticles means that the nanoparticles can give a visible indication of how much medication gets to tumors and how tumors respond.

"Potentially, these could be formulated for a particular patient," Schlesinger says. "We are learning more and more about tumor biology, and that knowledge could soon allow us to create nanoparticles targeted for specific tumors using the nanobee approach."

Funding from the National Institutes of Health and the American Heart Association supported this research.

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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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