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. 

 

Nanowires Deliver Biochemical Payloads to One Cell Among Many

Imagine being able to drop a toothpick on the head of one particular person standing among 100,000 people in a sports stadium. It sounds impossible, yet this degree of precision at the cellular level has been demonstrated by researchers affiliated with The Johns Hopkins University Institute for NanoBioTechnology. Their study was published online in June in Nature Nanotechnology.

Arrow points to nanowire placed on cell surface. (Image: Levchenko/Chien labs)

The team used precise electrical fields as “tweezers” to guide and place gold nanowires, each about one-two hundredth the size of a cell, on predetermined spots, each on a single cell. Molecules coating the surfaces of the nanowires then triggered a biochemical cascade of actions only in the cell where the wire touched, without affecting other cells nearby. The researchers say this technique could lead to better ways of studying individual cells or even cell parts, and eventually could produce novel methods of delivering medication.

Indeed, the techniques not relying on this new nanowire-based technology either are not very precise, leading to stimulation of multiple cells, or require complex biochemical alterations of the cells. With the new technique the researchers can, for instance, target cells that have cancer properties (higher cell division rate or abnormal morphology), while sparing their healthy neighbors.

“One of the biggest challenges in cell biology is the ability to manipulate the cell environment in as precise a way as possible,” said principal investigator Andre Levchenko, an associate professor of biomedical engineering in Johns Hopkins’ Whiting School of Engineering. In previous studies, Levchenko has used lab-on-a-chip or microfluidic devices to manipulate cell behavior. But, he said, lab-on-a-chip methods are not as precise as researchers would like them to be. “In microfluidic chips, if you alter the cell environment, it affects all the cells at the same time,” he said.

Such is not the case with the gold nanowires, which are metallic cylinders a few hundred nanometers or smaller in diameter. Just as the unsuspecting sports spectator would feel only a light touch from a toothpick being dropped on the head, the cell reacts only to the molecules released from the nanowire in one very precise place where the wire touches the cell’s surface.

With contributions from Chia-Ling Chien, a professor of physics and astronomy in the Krieger School of Arts and Sciences, and Robert Cammarata, a professor of materials science and engineering in the Whiting School, the team developed nanowires coated with a molecule called tumor necrosis factor-alpha (TNF?), a substance released by pathogen-gobbling macrophages, commonly called white blood cells. Under certain cellular conditions, the presence of TNF? triggers cells to switch on genes that help fight infection, but TNF? also is capable of blocking tumor growth and halting viral replication.

Exposure to too much TNF?, however, causes an organism to go into a potentially lethal state called septic shock, Levchenko said. Fortunately, TNF? stays put once it is released from the wire to the cell surface, and because the effect of TNF? is localized, the tiny bit delivered by the wire is enough to trigger the desired cellular response. Much the same thing happens when TNF? is excreted by a white blood cell.

Additionally, the coating of TNF? gives the nanowire a negative charge, making the wire easier to maneuver via the two perpendicular electrical fields of the “tweezer” device, a technique developed by Donglei Fan as part of her Johns Hopkins doctoral research in materials science and engineering. “The electric tweezers were initially developed to assemble, transport and rotate nanowires in solution,” Cammarata said. “Donglei then showed how to use the tweezers to produce patterned nanowire arrays as well as construct nanomotors and nano-oscillators. This new work with Dr. Levchenko’s group demonstrates just how extremely versatile a technique it is.”

To test the system, the team cultured cervical cancer cells in a dish. Then, using electrical fields perpendicular to one another, they were able to zap the nanowires into a pre-set spot and plop them down in a precise location. “In this way, we can predetermine the path that the wires will travel and deliver a molecular payload to a single cell among many, and even to a specific part of the cell,” Levchenko said.

During the course of this study, the team also established that the desired effect generated by the nanowire-delivered TNF? was similar to that experienced by a cell in a living organism.

The team members envision many possibilities for this method of subcellular molecule delivery. “For example, there are many other ways to trigger the release of the molecule from the wires: photo release, chemical release, temperature release. Furthermore, one could attach many molecules to the nanowires at the same time,” Levchenko said. He added that the nanowires can be made much smaller, but said that for this study the wires were made large enough to see with optical microscopy.

Ultimately, Levchenko sees the nanowires becoming a useful tool for basic research. “With these wires, we are trying to mimic the way that cells talk to each other,” he said. “They could be a wonderful tool that could be used in fundamental or applied research.” Drug delivery applications could be much further off. However, Levchenko said, “If the wires retain their negative charge, electrical fields could be used to manipulate and maneuver their position in the living tissue.”

The lead authors for this Nature Nanotechnology article were Fan, a former postdoctoral fellow in the departments of materials science and engineering and in physics and astronomy; and Zhizhong Yin, a former postdoctoral fellow in the Department of Biomedical Engineering. The co-authors included Raymond Cheong, a doctoral student in the Department of Biomedical Engineering; and Frank Q. Zhu, a former doctoral student in the Department of Physics and Astronomy.

Regarding the faculty members’ participation, Chien led the group that developed the electric tweezers technique and collaborated with Levchenko on its biological applications.

The research was funded by the National Science Foundation and the National Institutes of Health.

Johns Hopkins Institute for NanoBioTechnology

On new lab chip, heart cells display a behavior-guiding ‘nanosense’

Johns Hopkins biomedical engineers, working with colleagues in Korea, have produced a laboratory chip with nanoscopic grooves and ridges capable of growing cardiac tissue that more closely resembles natural heart muscle. Surprisingly, heart cells cultured in this way used a “nanosense” to collect instructions for growth and function solely from the physical patterns on the nanotextured chip and did not require any special chemical cues to steer the tissue development in distinct ways. The scientists say this tool could be used to design new therapies or diagnostic tests for cardiac disease.

Leslie Tung, left, and Andre Levchenko, right, both of the Department of Biomedical Engineering, with Deok-Ho Kim, a doctoral student in Levchenko’s lab, who holds a nanopatterned chip able to cue heart cells to behave like natural heart tissue. Photo: Will Kirk/homewoodphoto.jhu.edu

Leslie Tung, left, and Andre Levchenko, right, both of the Department of Biomedical Engineering, with Deok-Ho Kim, a doctoral student in Levchenko’s lab, who holds a nanopatterned chip able to cue heart cells to behave like natural heart tissue. Photo: Will Kirk/homewoodphoto.jhu.edu

The device and experiments using it are described in this week’s online Early Edition issue of Proceedings of the National Academy of Sciences. The work, a collaboration with Seoul National University, represents an important advance for researchers who grow cells in the lab to learn more about cardiac disorders and possible remedies.

“Heart muscle cells grown on the smooth surface of a Petri dish would possess some, but never all, of the same physiological characteristics of an actual heart in a living organism,” said Andre Levchenko, an associate professor of biomedical engineering in Johns Hopkins’ Whiting School of Engineering. “That’s because heart muscle cells—cardiomyocytes—take cues from the highly structured extracellular matrix, or ECM, which is a scaffold made of fibers that supports all tissue growth in mammals. These cues from the ECM influence tissue structure and function, but when you grow cells on a smooth surface in the lab, the physical signals can be missing. To address this, we developed a chip whose surface and softness mimic the ECM. The result was lab-grown heart tissue that more closely resembles the real thing.”

Levchenko said that when he and his colleagues examined the natural heart tissue taken from a living animal, they “immediately noticed that the cell layer closest to the extracellular matrix grew in a highly elongated and linear fashion. The cells orient with the direction of the fibers in the matrix, which suggests that ECM fibers give structural or functional instructions to the myocardium, a general term for the heart muscle.” These instructions, Levchenko said, are delivered on the nanoscale—activity at the scale of one-billionth of a meter and a thousand times smaller than the width of a human hair.

Levchenko and his Korean colleagues, working with Deok-Ho Kim, a biomedical engineering doctoral student in Levchenko’s lab and the lead author of the PNAS article, developed a two-dimensional hydrogel surface simulating the rigidity, size and shape of the fibers found throughout a natural ECM network. This biofriendly surface made of nontoxic polyethylene glycol displays an array of long ridges resembling the folded pattern of corrugated cardboard. The ridged hydrogel sits upon a glass slide about the size of a U.S. dollar coin. The team made a variety of chips with ridge widths spanning from 150 to 800 nanometers, groove widths ranging from 50 to 800 nanometers and ridge heights varying from 200 to 500 nanometers. This allowed researchers to control the surface texture over more than five orders of magnitude of length.

“We were pleased to find that within just two days the cells became longer and grew along the ridges on the surface of the slide,” Kim said. Furthermore, the researchers found improved coupling between adjacent cells, an arrangement that more closely resembled the architecture found in natural layers of heart muscle tissue. Cells grown on smooth, unpatterned hydrogels, however, remained smaller and less organized, with poorer cell-to-cell coupling between layers. “It was very exciting to observe engineered heart cells behave on a tiny chip in two dimensions like they would in the native heart in three dimensions,” Kim said.

Collaborating with Leslie Tung, a professor of biomedical engineering in the Johns Hopkins School of Medicine, the researchers found that after a few more days of growth, cells on the nanopatterned surface began to conduct electric waves and contract strongly in a specific direction, as intact heart muscle would. “Perhaps most surprisingly, these tissue functions and the structure of the engineered heart tissue could be controlled by simply altering the nanoscale properties of the scaffold. That shows us that heart cells have an acute ‘nanosense,’” Levchenko said.

Johns Hopkins researchers developed this chip to culture heart cells that more closely resemble natural cardiac tissue. Photo: Will Kirk/homewoodphoto.jhu.edu

Johns Hopkins researchers developed this chip to culture heart cells that more closely resemble natural cardiac tissue. Photo: Will Kirk/homewoodphoto.jhu.edu

“This nanoscale sensitivity was due to the ability of cells to deform in sticking to the crevices in the nanotextured surface and probably not because of the presence of any molecular cue,” Levchenko said. “These results show that the ECM serves as a powerful cue for cell growth, as well as a supporting structure, and that it can control heart cell function on the nanoscale separately in different parts of this vital organ. By mimicking this ECM property, we could start designing better-engineered heart tissue.”

Looking ahead, Levchenko said that he anticipates that engineering surfaces with similar nanoscale features in three dimensions, instead of just two, could provide an even more potent way to control the structure and function of cultured cardiac tissue.

In addition to Kim, Levchenko and Tung, authors on this paper are postdoctoral fellow Elizabeth A. Lipke and doctoral students Raymond Cheong and Susan Edmonds Thompson, all from the Johns Hopkins School of Medicine Department of Biomedical Engineering; Michael Delannoy, assistant director of the Johns Hopkins School of Medicine Microscope Facility Center; and Pilnam Kim and Kahp-Yang Suh, both of Seoul National University.

Tung and Levchenko are affiliated faculty members of the Johns Hopkins Institute for NanoBioTechnology. Thompson is a member of INBT’s Integrative Graduate Education and Research Traineeship in nanobiotechnology. Funding for this research was provided by the National Institutes of Health and the American Heart Association.

Related Web sites

Andre Levchenko’s Lab

Leslie Tung’s Lab

Johns Hopkins Institute for NanoBioTechnology

Story by Mary Spiro