RESEARCHERS USING METALLIC GLASS, OTHER MATERIALS AT CELL BREAKAGE

FROM:  NATIONAL SCIENCE FOUNDATION
Materials, like metallic glass, can help us understand how cells break

Research could lead to faster wound recovery and prove valuable in constructing buildings, producing golf clubs and more

"Disordered" materials are so-called because they are made up of objects that are in total disarray. Their composition, whether of atoms, molecules, grains or cells, do not lie in a neat, orderly pattern, but, instead, are all jumbled up.

"They're like sand on a beach, or mayonnaise," says M. Lisa Manning, an assistant professor of physics at Syracuse University. "When you mix up the oil and water for mayonnaise, the oil droplets sit unordered. That's what makes the mayonnaise stiff, all the little oil droplets packed together."

Many of these disordered materials, metallic glass, for example, are exceptionally strong, stronger than other metals, which offers potential for many industrial uses. But they also are prone to failure, and often break. Manning is studying these materials, searching for the defects in each that produce a crack-like fissure called a shear band.

"If we can find and identify these defects, then we can understand what causes the shear band," Manning says. "My goal is to figure out how they break. I am looking for defects in these materials. Once we figure out how they break, we can then figure out how to prevent them from breaking."

If successful, these materials--because of their inherent strength--could prove valuable in manufacturing, from constructing buildings to producing golf clubs, and "would be extremely good for making precision objects, because they don't change shape when they cool down," Manning says.

Insights from her research also could have important applications for biology, ultimately leading to possible future medical treatments, because disordered cells also exist in tissues, in developing embryos and in certain cancers.

"If you look at the cells in these tissues, they are disordered and look almost identical to pictures of foams, or emulsions," Manning says. "Embryos look like this, and so do healing wounds, and some cancer tissues too."

Biologists have a good understanding of what happens when a single cell migrates, or moves, she says. "But what is not well studied is how cells in this dense packing order move through tissues, which is important for wound healing," she says. "A cell has to push its way through its neighbors to move.

"If I can understand how non-biological particles move, this can provide new and exciting insights as to how a cell can move through tissues," she adds. "How stiff are the cells around it, for example? If I want a cell to move faster in tissue, should I make it softer or stiffer? The goal is to answer this, and test it."

Understanding this process could speed wound healing and "help repair embryonic defects when cells don't move to the correct places," she says.

In cancer, "recent work has shown that cancer cells are softer than other cells, and have different mechanical properties," she says. "One question I hope to answer: if a cancer cell is softer, does that make it easier to move through tissue and metastasize? If I could stiffen up that cell with a drug, maybe it wouldn't move anymore."

To find the defects, Manning creates computer simulations of the materials and studies sound modes that vibrate within the structure, much like a specific musical note vibrates inside an organ pipe. When the researchers find "localized" vibrations, that is, a mode where the structure vibrates a lot more in a certain place, "that's where the defect is located," she says.

Manning is conducting her research under an NSF Faculty Early Career Development (CAREER) award, which she received in 2014. The award supports junior faculty who exemplify the role of teacher-scholars through outstanding research, excellent education and the integration of education and research within the context of the mission of their organization.

As part of the grant's educational component, Manning plans to develop tutorials for high school juniors and seniors in Syracuse University's Project Advance, a program that enables these students to earn college physics credits. Project Advance provides instructional materials to high school teachers, and sponsors extra training sessions for them at the university. Manning is designing teaching modules about current research in materials science that can be directly integrated into the introductory physics curriculum, as well as an online math tutorial to tune up students' math skills.

"The goal is to increase diversity and retention in STEM disciplines," she says, referring to science, technology, engineering and mathematics. "We need more engineers, and we want to keep the ones we have, and recruit a more diverse body of students."

-- Marlene Cimons, National Science Foundation
Investigators
M. Lisa Manning
Related Institutions/Organizations
Syracuse University

NSF EXAMINES HIV BUDDING FROM CELLS

FROM:  NATIONAL SCIENCE FOUNDATION 

Catching HIV budding from cells: it all comes down to ALIX

Study shows last-minute role of specific protein named ALIX

The secrets of the AIDS virus may all come down to a protein named ALIX.

Researchers have devised a way to watch newly forming AIDS particles emerging or "budding" from infected human cells without interfering with the process.

The method shows that a protein named ALIX (which stands for "alg-2 interacting protein x") gets involved during the final stages of virus replication, not early on, as was believed. ALIX assists in separating new virus buds from a cell. These buds repeat the replication process and further infect their host.

"We watch one cell at a time" and use a digital camera and special microscope to make movies and photos of the budding process, says virologist Saveez Saffarian, a scientist at the University of Utah, and co-author of a paper on HIV budding published this week in the journal PLOS ONE.

"We saw ALIX recruited into HIV budding for the first time," he says. "Everybody knew that ALIX was involved in HIV budding, but nobody could visualize the recruitment of ALIX into the process."

The finding has no immediate clinical significance for AIDS patients because ALIX is involved in too many critical functions like cell division to be a likely target for new medications, Saffarian says.

"We know a lot about the proteins that help HIV get out of the cell, but we don't know how they come together to help the virus emerge," he says. "In the next 10 to 20 years, we will know a lot more about this mechanism."

Saffarian conducted the research with the paper's first author Pei-I Ku, as well as researchers Mourad Bendjennat, Jeff Ballew and Michael Landesman. All are with the University of Utah.

The research was funded by the National Science Foundation (NSF).

"This project has led to the development of an important technique in basic research in cell biology and virology," says Parag Chitnis, director of NSF's Division of Molecular and Cellular Biosciences.

"It's uncovering a new understanding of the viruses involved in human diseases," says Chitnis. "This is an excellent example of how purely basic research can lead to the fundamental understanding of topics of societal need."

Watch, don't touch, as HIV buds

Biochemical methods used for years involve collecting millions of viruses in lab glassware and conducting analyses to reveal the proteins that make up the virus--for example, by using antibodies that bind to certain proteins and using other proteins to make the first proteins fluoresce so they can be seen.

"You're not doing it one virus at a time," Saffarian says. "The problem is that you don't see the differences among similar viruses. And you don't see the timing of how various proteins come and go to help the virus get out of the cell."

Other methods freeze or otherwise fix cells as new HIV particles emerge, and use an electron microscope to photograph freeze-frame views of viral replication.

Saffarian employs technology known as "total internal reflection fluorescence microscopy" that looks at the dynamic processes in cells.

The method has been used to make images of the budding of HIV and a similar horse virus, EIAV.

But Saffarian says that the EIAV study didn't show ALIX becoming involved in HIV budding, and that it wrongly indicated that ALIX got involved early in the EIAV budding process, suggesting it did the same in HIV budding.

Ku, Saffarian and colleagues combined their microscopy method with an improved way of genetically linking a green fluorescent "label" to ALIX proteins in cloned cells so they could see the proteins without harming their normal function.

The researchers tried numerous so-called "linkers" and found the one that let them see the ALIX proteins as they became involved in HIV budding.

Neither the microscope technology nor labeling proteins with green fluorescence are new, but "what we did that is new is to connect these fluorescence proteins to ALIX using many different kinds of linkers," says Saffarian, to find one that let the ALIX protein function properly.

The problem with research that indicated ALIX was involved early in the budding process was that only one linker was used, and it impaired ALIX's normal function, the scientists say.

Looking at proteins forming HIV

When HIV replicates inside a human cell, a protein named Gag makes up most of the new particles--there are 4,000 copies of the Gag protein in one HIV particle--although other proteins get involved in the process, including ALIX.

Experiments like those by Saffarian use "virus-like particles," which are HIV particles stripped of their genetic blueprint or genome so they don't pose an infection risk in the lab.

"Virus-like particles maintain the same geometry and same budding process as infectious HIV," Saffarian says.

During budding, Gag proteins assemble on the inside of a cell membrane--along with ALIX in the late stages--and form a new HIV particle that pushes its way out of the cell--the process by which AIDS in an infected person spreads from cell to cell.

To look at the budding process, Ku and Saffarian placed human cells containing the particles in a small amount of liquid growth medium in a petri dish and placed it under the microscope, which is in a glass chamber kept at body temperature so the cells can remain alive for more than 48 hours.

A solid-state blue laser was aimed at the sample to make the green-labeled ALIX and red-labeled Gag proteins glow or fluoresce so they could be seen as they assembled into a virus particle.

With red-labeled Gag proteins and green-labeled ALIX proteins, "we could see ALIX come in at the end of the assembly of the virus particle," says Saffarian. Some 100 ALIX proteins converged with the roughly 4,000 Gag molecules and assembled into a new HIV particle.

Enter ALIX

ALIX then brought in two other proteins, which cut off the budding virus particle from the cell when it emerged. ALIX's position during the pinching off of new particles hadn't been recognized before.

The researchers watched the virus particles bud one cell at a time: about 100 particles emerged during a two-hour period. Most of the ALIX proteins left when HIV assembly was complete and returned to the liquid inside a cell.

Saffarian says the discovery that ALIX doesn't get involved until the late stages of HIV budding suggests the existence of a previously unrecognized mechanism that regulates the timing of ALIX and other proteins in assembling new HIV particles.

"We discovered that the cellular components that help with the release of the virus arrive in a much more complex timing scheme than predicted based on the biochemical data," he says.

"The outcome of this study is promising because it uncovers a new regulatory mechanism for recruitment of cellular components to HIV budding sites, and opens the door to exciting future studies on the mechanism of HIV budding."

NSF ON HOW CELLS FORM INTO INDIVIDUALS

FROM:  NATIONAL SCIENCE FOUNDATION 
Researcher seeks to identify the pathway that leads to cells forming into an individual body

By studying how genes influence cells to migrate and mutate, scientist hopes findings will lead to improved cancer treatments
All organisms begin life as a microscopic cluster of cells. What happens next, as they develop, is a source of endless fascination for scientists.

"How do you go from just a ball of cells into an organism that has a shape, and fingers, toes and brain?" says Traci Stevens, associate professor of biology at Randolph-Macon College. "Cells have to move during development. How do they do it? What is involved? How does that ball of cells become an organized individual?"

Stevens, a National Science Foundation (NSF)-funded scientist is trying to find out, specifically by learning more about the work of Abl, a gene responsible for regulating how cells migrate from that initial tiny collection of cells to form a shape and body parts. "It is a gene we all have, and it seems to work similarly in all organisms," she says.

Specifically, she is focusing on how Abl functions in Drosophila--the fruit fly--which has one Abl gene (humans--in fact, all vertebrates--have two related Abl genes, Abl 1 and Abl 2, whereas invertebrates have only one), making it easier to study. Moreover, "fruit flies have stages in development where we know exactly how the cells move and migrate in normal development," she says.

In her experiments, she activates a mutant form of Abl in the flies to see what happens as the altered gene disrupts the migration pattern. If she can see what goes wrong, she will understand what's supposed to go right. "When I can see what happens with a mutant form, it tells us what is supposed to happen normally," she says. "It seems like reverse logic, but that's how geneticists work."

Her research also involves using a genetic screen to search for other genes that interact with Abl. "We don't know all the details as to how Abl regulates cell migration, but we know Abl doesn't do it by itself, that it works with other proteins," she says.

Her work has potential applications in medicine, specifically for cancer treatment, since Abl "is misregulated in a lot of cancers," she says, adding that its dysfunction has an impact both on cell growth and how cancer cells metastasize. The gene is known to have a role in Chronic Myeloid Leukemia (CML), one of several types of leukemia, as well as in breast cancer, non small lung cancer and melanoma, she says.

To be sure, "we are many steps away, but by identifying not just Abl, but the things it interacts with, by understanding that pathway, it could help us control that pathway," she says. Ultimately, this could lead to the design of new drugs "that could inhibit or regulate this pathway in cases of cancer," she says.

She is conducting her research under an NSF Faculty Early Career Development (CAREER) award, which she received in 2009 as part of NSF's American Recovery and Reinvestment Act. The award supports junior faculty who exemplify the role of teacher-scholars through outstanding research, excellent education and the integration of education and research within the context of the mission of their organization. NSF is funding her work with $883,365 over four years.

As part of the grant's educational component, Stevens hosts two high school students and their biology teacher in her lab during the summer. They attend Cosby High School, a science-based high school in Chesterfield, Va. "They come into the lab for five weeks and work on a project or two," she says. "Then at the end of the summer, they present a poster at a college-wide conference."

Stevens is especially proud that one of the participating teachers had an opportunity to present her work at a national Drosophila meeting. "That was a great experience for her," Stevens says.

Stevens is studying Abl's role in two specific stages of fruit fly development. The first, the formation of the epithelium, or skin of the embryo, a process known as dorsal closure, where "there is a big opening on the skin and the cells migrate to close all the way around the embryo," she says. The second, head involution, is when "the inside of the head is on the outside, and those cells migrate inward to form the head," she explains.

"Those are the two stages we study," she adds. "They are both happening at nearly the same time, but at different places. They occur the first day you have a fertilized egg, before it hatches into a larva. So we are looking at the very beginning. We analyze their phenotypes, that is, we look at what developmental processes did not occur properly. For example, are there holes in the head indicating that head involution did not occur properly? Or are there holes in the dorsal surface indicating that there were cell migration problems during dorsal closure?"

In fruit flies, the Abl protein, which is made from the Abl gene, is found in the cytoplasm of the cell just under the cell membrane, where it can control cell migration, rather than in the nucleus, where it could regulate cell division.

In humans, and other vertebrates, Abl 2 is in the cytoplasm, as it is with fruit flies, but Abl 1 "shuttles back and forth between the nucleus and the cytoplasm," she says. "So Abl 1 binds to DNA in the nucleus and controls cell division, which Abl in Drosophila and Abl 2 in humans don't seem to do."

Scientists don't yet understand how the duties are divided between the two Abls in vertebrates and/or whether the two vertebrate genes might play unique roles, that is, roles that Drosophila Abl does not, according to Stevens.

Thus, "the fruit fly can't tell us everything," she says. "But it can tell us how the gene influences how cells move, and that can tell us something about what happens in humans since that is a function conserved in both fruit flies and humans."

--  Marlene Cimons, National Science Foundation
Investigators
Traci Stevens
Related Institutions/Organizations
Randolph-Macon College