Amoebas get social when they hit hard times

Editor’s Note: This article was written by Rezina Siddique, a Ph.D. student in Biomedical Engineering at Johns Hopkins with an M.S. in Nanoscale Science and Engineering, and first appeared in the 2013 issue of Nano-Bio Magazine. 

How single-celled social amoebae respond to chemical signals is shedding light on the processes and behavior of more complex organisms, including mammals. A recent paper suggests that there is a mechanism by which amoeba amplify a desirable chemical stimulus in order to self-organize and collectively migrate.

Amoeba are single-celled organisms with the capability to aggregate to form a multi-cellular organism, and later to a fruiting body. Andre Levchenko, professor of biomedical engineering and affiliated faculty member of Johns Hopkins Institute for NanoBioTechnology, used the social amoeba, Dictyostelium discoideum, because as a multicellular organism it contains cells with different genotypes. Levchenko’s team sought to clarify how external signals were amplified by the organism to facilitate aggregation.

Dictyostelium discoideum slugs (bottom) and stalks with spore masses on top. Photo credit: Owen Gilbert

Dictyostelium discoideum slugs (bottom) and stalks with spore masses on top. Photo credit: Owen Gilbert

“The way [these organisms] detect signals and move are similar to how neutrophils, a natural part of our immune system, detect and move to the site of infection…They share the ability to migrate in a very directed way to get where they are needed,” said Levchenko. When resources are plentiful, Levchenko’s team found that Dicty are content in remain alone. But when food supplies run low, they gather into a multicellular slug.

As a slug, he said, “they can move together to find a more favorable location,” said Levchenko. The cell-cell communication that takes place during the transition relies on chemotaxis, which is the movement towards or away from a chemical stimulus along a concentration gradient. This behavior is similar in mammalian cells, relevant in both healthy and pathological conditions. Their results, are published in volume 5, issue 213 of Science Signaling.

Levchenko’s team developed a microfluidic pattern generating device that allows the user to control the environment and stimulus duration in a highly tunable way, while still being able to visualize cells under a microscope. Historically, Levchenko explained, these types of experiments were done with pipettes, but with the device his group was able to perform their experiments with the dynamic signaling responses consistent with the known behavior of the amoeba.

Previously, a mathematical model was developed to explain the mechanism of signal amplification that occurred in the amoeba, but there was no way to test it. However, using their microfluidic pattern generator, Levchenko’s group was able to validate the model experimentally. “Understanding the dynamics of chemotaxis within this system can shed insight into how other multicellular organisms, as well as how mammalian cells interact,” Levchenko said.

During aggregation, cyclic AMP (cAMP), a molecule that stimulates hunger, serves as chemoattractant. A starving social amoeba secretes cAMP to attract other amoebae to it, which all travel towards the central amoeba. These other cells also start releasing cAMP in a periodic fashion in order to amplify the signal and attract additional amoebae, creating a pulsating and wave-like signal. An individual cell ends up seeing waves of activity. This is similar to pacemaker cells in the heart, where periodic activity regulates cell behavior.

In a population of cells, some cells are more sensitive while others are less sensitive. This discrepancy is not visible when averaging the response over the entire population or when examining a single representative cell. By applying the hunger stimulus to cells within their device, Levchenko’s group found that there is a large difference across cells in a given population. Some cells did not respond at all, while others responded very strongly to the same stimulus. They also found that at higher doses, the majority of cells responded, while at lower doses, smaller numbers of cells responded. This indicates that the cells that respond strongly must have some ability to amplify the signal.

Differential sensitivity in the cells helps them to organize. Adaptation allows them to transiently suppress their sensitivity long enough to be able to form a multi-cellular organism. The adaptive and amplification properties of the amoeba resemble what occurs in bacterial chemotaxis. The results have implications for the study of cell decision making versus commitment to behavior within cells of a given tissue, or different types of cells that work together.

Editor’s Note: This article was written by Rezina Siddique, a Ph.D. student in Biomedical Engineering at Johns Hopkins with an M.S. in Nanoscale Science and Engineering, and first appeared in the 2013 issue of Nano-Bio Magazine. 

 

Fraley nets $500K Burroughs Wellcome Fund award for microfluidics work

Stephanie Fraley (Photo: Homewood Photography)

Stephanie Fraley (Photo: Homewood Photography)

A Johns Hopkins research fellow who is developing novel approaches to quickly identify bacterial DNA and human microRNA has won the prestigious $500,000 Burroughs Wellcome Fund (BWF) Career Award at the Scientific Interfaces. The prize, distributed over the next five years, helps transition newly minted PhDs from postdoctoral work into their first faculty positions.

Stephanie Fraley is a postdoctoral fellow working with Samuel Yang, MD, in Emergency Medicine/Infectious Disease at the Johns Hopkins School of Medicine and Jeff Wang, PhD, in Biomedical Engineering with appointments in the Whiting School of Engineering and the medical school. The goal of her work is to develop engineering technologies that can diagnose and guide treatment of sepsis, a leading cause of death worldwide, while simultaneously leading to improved understanding of how human cells and bacterial cells interact.

“Sepsis is an out of control immune response to infection,” Fraley said. “We are developing tools that are single molecule sensitive and can rapidly sort and detect bacterial and host response markers associated with sepsis. However, our devices are universal in that they can be applied to many other diseases.”

Fraley is using lab-on-chip technology, also known as microfluidics, to overcome the challenges of identifying the specific genetic material of bacteria and immune cells. Her technology aims to sort the genetic material down to the level of individual sequences so that each can be quantified with single molecule sensitivity.

“Bacterial DNA is on everything and contamination is everywhere, so trying to find the ones associated with sepsis is like the proverbial search for the needle in the haystack,” Fraley said. “With microfluidics, we can separate out all the bacterial DNA, so instead of a needle in a haystack, we have just the needles.”

Another advantage to Fraley’s novel technology is that it will assess all the diverse bacterial DNA present in a sample, without presuming which genetic material is important. “Bacteria are constantly evolving and becoming drug resistant,” she said. “With this technology, we can see all the bacterial DNA that is present individually and not just the strains we THINK we need to look for.”

Fraley’s award will follow her wherever her career takes her. The first two years of the prize fund postdoctoral training and that last three years help launch her professional career in academia. During the application process, she had to make a short presentation on her proposal to BWF’s panel of experts. “It was like the television show ‘Shark Tank’ but for scientists,” she laughs. “ The panelists gave me many helpful suggestions on my idea.”

Fraley earned her bachelor’s degree in chemical engineering from the University of Tennessee at Chattanooga and her doctorate in chemical and biomolecular engineering with Denis Wirtz, professor and director of Johns Hopkins Physical Sciences-Oncology Center. Wirtz is associate director for the Institute for NanoBioTechnology and Yang and Wang also are INBT affiliated faculty members.

BWF’s Career Awards at the Scientific Interface provides funding to bridge advanced postdoctoral training and the first three years of faculty service. These awards are intended to foster the early career development of researchers who have transitioned or are transitioning from undergraduate and/or graduate work in the physical/mathematical/computational sciences or engineering into postdoctoral work in the biological sciences, and who are dedicated to pursuing a career in academic research. These awards are open to U.S. and Canadian citizens or permanent residents as well as to U.S. temporary residents.

Device with tiny ‘speed bumps’ sorts cells

These illustrations show magnetically labeled circulating tumor cells (shown as yellow spheres), together with red, white and platelet cells, attempting to travel over an array of slanted ramps. The ramps act as speed bumps, slowing the tumor cells.. (Illustration by Martin Rietveld)

In life, we sort soiled laundry from clean; ripe fruit from rotten. Two Johns Hopkins engineers say they have found an easy way to use gravity or simple forces to similarly sort microscopic particles and bits of biological matter—including circulating tumor cells.

In the May 25 online issue of Physical Review LettersGerman Drazer, an assistant professor of chemical and biomolecular engineering, and his doctoral student, Jorge A. Bernate, reported that they have developed a lab-on-chip platform, also known as a microfluidic device, that can sort particles, cells or other tiny matter by physical means such as gravity. By moving a liquid over a series of micron-scale high diagonal ramps—similar to speed bumps on a road—the device causes microscopic material to separate into discrete categories, based on weight, size or other factors, the team reported.

As the tumor cells slow, the flow carries them along the length of the ramp, causing lateral displacement. After the tumor cells traverse an array of these ramps, they have sufficiently been displaced and can be continuously isolated from other cells in the sample. (Illustration by Martin Rietveld)

The process described in the journal article could be used to produce a medical diagnostic tool, the Whiting School of Engineering researchers say. “The ultimate goal is to develop a simple device that can be used in routine checkups by health care providers,” said doctoral student Bernate, who is lead author on the paper. “It could be used to detect the handful of circulating tumor cells that have managed to survive among billions of normal blood cells. This could save millions of lives.”

Ideally, these cancer cells in the bloodstream could be detected and targeted for treatment before they’ve had a chance to metastasize, or spread cancer elsewhere. Detection at early stages of cancer is critical for successful treatment.

How does this sorting process occur? Bernate explained that inside the microfluidic device, particles and cells that have been suspended in liquid flow along a “highway” that has speed-bump-like obstacles positioned diagonally, instead of perpendicular to, the path. The speed bumps differ in height, depending on the application.

“As different particles are driven over these diagonal speed bumps, heavier ones have a harder time getting over than the lighter ones,” the doctoral student said. When the particles cannot get over the ramp, they begin to change course and travel diagonally along the length of the obstacle. As the process continues, particles end up fanning out in different directions.

“After the particles cross this section of the ‘highway,’” Bernate said, “they end up in different ‘lanes’ and can take different ‘exits,’ which allows for their continuous separation.”

Gravity is not the only way to slow down and sort particles as they attempt to traverse the speed bumps. “Particles with an electrical charge or that are magnetic may also find it hard to go up over the obstacles in the presence of an electric or magnetic field,” Bernate said. For example, cancer cells could be “weighted down” with magnetic beads and then sorted in a device with a magnetic field.

The ability to sort and separate things at the micro- and nanoscale is important in many industries, ranging from solar power to bio-security. But Bernate said that a medical application is likely to be the most promising immediate use for the device.

He is slated to complete his doctoral studies this summer, but until then, Bernate will continue to collaborate with researchers in the lab of Konstantinos Konstantopoulos, professor and chair of the Department of Chemical and Biomolecular Engineering, and with colleagues at InterUniversity Microelectronics Center, IMEC, in Belgium. In 2011, Bernate spent 10 weeks at IMEC in a program hosted by Johns Hopkins’ Institute for NanoBioTechnology and funded by the National Science Foundation.

His doctoral adviser, Drazer, said, the research described in the new journal article eventually led Jorge down the path at IMEC to develop a device that can easily sort whole blood into its components. A provisional patent has been filed for this device.

The research by Bernate and Drazer was funded in part by the National Science Foundation and the National Institutes of Health.

Story by Mary Spiro.

Related links:

 

 

German Drazer’s Web page: http://microfluidics.jhu.edu/

Department of Chemical and Biomolecular Engineering: http://www.jhu.edu/chembe/

INBT researchers use LEGO to study what happens inside lab-on-a-chip devices

Johns Hopkins engineers are using a popular children’s toy to help them visualize the behavior of particles, cells and molecules in environments too small to see with the naked eye. These researchers are arranging little LEGO pieces shaped like pegs to recreate microscopic activity taking place inside lab-on-a-chip devices at a scale they can more easily observe. These lab-on-a-chip devices, also known as microfluidic arrays, are commonly used to sort tiny samples by size, shape or composition, but the minuscule forces at work at such a small magnitude are difficult to measure. To solve this small problem, the Johns Hopkins engineers decided to think big.

Led by Joelle Frechette and German Drazer, both assistant professors of chemical and biomolecular engineering in the Whiting School of Engineering, the team used beads just a few millimeters in diameter, an aquarium filled with goopy glycerol and the LEGO pieces arranged on a LEGO board to unlock mysteries occurring at the micro- or nanoscale level. Their observations could offer clues on how to improve the design and fabrication of lab-on-a-chip technology. Their study concerning this technique was published in the August 14 issue of Physical Review Letters. Both Drazer and Frechette are affiliated faculty members of Johns Hopkins Institute for NanoBioTechnology.

The idea for this project comes from the concept of “dimensional analysis,” in which a process is studied at a different size and time scale while keeping the governing principles the same. [Read more...]