Water-Based ‘Engine’ Propels Tumor Cells Through Tight Spaces in the Body

Johns Hopkins researchers have discovered a new mechanism that explains how cancer cells spread through extremely narrow three-dimensional spaces in the body by using a propulsion system based on water and charged particles.

The finding, reported in the April 24 issue of the journal Cell, uncovers a novel way that the deadly cells use to migrate through a cancer patient’s body. The discovery may lead to new treatments that help keep the disease in check. The work also points to the growing importance of studying how cells behave in three dimensions, not just atop flat two-dimensional lab dishes.

Based on such lab dish studies, cancer researchers had concluded that tumor cells require actin and other proteins to form arm-like extensions to “crawl” across the flat surfaces. This type of travel was believed to be the primary means of how cancer spreads within a patient, a process called metastasis. Based on this conclusion, researchers have been working on ways to disable actin and its molecular helpers, hoping this can keep cancer from spreading.

This illustration depicts how a cell can use a water-based “engine” to migrate through confined spaces in the body. Graphic by Martin Rietveld.

But in a study published in 2012, a Johns Hopkins team led byKonstantinos Konstantopoulos, chair of the Department of Chemical and Biomolecular Engineering, found that tumor cells could move through narrow spaces without using actin and its biochemical partners. “That was a stunning discovery, not in line with the prevailing beliefs about how cells migrate,” Konstantopoulos said. “So we wanted to figure out exactly how the tumor cells were able to move through these spaces without relying on actin.”

He collaborated with Sean X. Sun, a Johns Hopkins associate professor of mechanical engineering with experience in math modeling and physics at microscopic levels. “The mystery we needed to solve,” Sun said, “was how the cells in these confined spaces could still move when you took away their usual ‘engine,’ the actin.”

Kostantopoulos said Sun and Hongyuan Jiang, a postdoctoral fellow working in Sun’s lab, “came up with a phenomenal mathematical model that provided insights into how the cells might use a different system to travel.” Then Konstantopoulos and other team members, including Kimberly Stroka, a postdoctoral fellow in his own lab, used a microfluidic lab-on-a-chip and imaging techniques to conduct experiments establishing the new mechanism of migration proposed by Sun and Jiang’s model. The tests utilized human and animal cancer cells. Stroka and Jiang were designated co-lead authors of the resulting journal article.

As reported in the article, the tumor cells’ new “engine” turned out to be a combination of sodium-hydrogen ions, cell membrane proteins called aquaporins, and water. The researchers found that within tight spaces, cancer cells create a flow of liquid that takes in water and ions at a cell’s leading edge and pumps them out the trailing edge, propelling the cell forward. In the actin-dependent migration model, the cell is pushed forward by the biochemical equivalent of a boat engine. The water-based mechanism, the researchers said, more closely resembles the way a sailboat is thrust ahead by gusts of wind. The team called this mechanism the Osmotic Engine Model.

“This discovery is important because it reveals one reason why some diseases like cancer don’t always respond to certain treatments,” Konstantopoulos said. Sun added, “It’s because these diseases have redundant mechanisms—more than one method—for migrating through the body.”

The Johns Hopkins researchers are applying for funds to conduct further research into physical and biological aspects of the Osmotic Engine Model. Their hope is that the work will uncover a way to shut down this biochemical engine and keep it from spreading tumor cells.

The multidisciplinary research at Johns Hopkins was conducted within the university’s Institute for NanoBioTechnology and itsPhysical Sciences-Oncology Center. These organizations and the departments of Chemical and Biomolecular Engineering and Mechanical Engineering are based in the Whiting School of Engineering.

Co-lead author of the Cell article Stroka will join the faculty of the University of Maryland College Park as an assistant professor later this year. Whiting School postdoctoral fellow Jiang, the other lead author, also is now a professor at the University of Science and Technology of China. Kostantopoulos and Sun supervised the research and served as senior authors of the paper. Other co-authors, all from Johns Hopkins, were Shih-Hsun Chen, Ziqiu Tong and Denis Wirtz.

This work was supported by National Science Foundation grant NSF-1159823, National Cancer Institute grants U54-CA143868, RO1GM075305, RO1CA174388, T32-CA130840 and F32-CA177756, a Kleberg Foundation grant and National Natural Science Foundation of China grant NSFC 11342010.

   An illustration depicting the Osmotic Engine Model is available; contact Phil Sneiderman.

A Tiny Pinch from a ‘Z-Ring’ Helps Bacteria Cells Divide New Mathematical Model Unravels the Mechanics of Microbe Reproduction

Sean Sun and Ganhui Lan

Johns Hopkins researchers Sean Sun and Ganhui Lan were part of a team that solved a small but important part of the mystery surrounding cell division in rod- shaped bacteria. Sun is an assistant professor of mechanical engineering. Lan is a doctoral student. Photo by Will Kirk

In a process that is shrouded in mystery, rod-shaped bacteria reproduce by splitting themselves in two. By applying advanced mathematics to laboratory data, a team led by Johns Hopkins researchers has solved a small but important part of this reproductive puzzle.

The findings apply to highly common rod-shaped bacteria such as E. coli, found in the human digestive tract. When these single-celled microbes set out to multiply, a signal from an unknown source causes a little-understood structure called a Z-ring to tighten like a rubber band around each bacterium’s midsection. The Z-ring pinches the rod-like body into two microbial sausages that finally split apart. To shed light on this process, the Johns Hopkins-led team developed a mathematical tool that computed the mechanical force exerted by the Z-ring when it helps these cells split.

The calculation will aid scientists who are trying to learn more about how these microbes live and reproduce. The work also may hasten the development of a new type of antibiotic that could disable the Z-ring to keep harmful bacteria in check.

Sean X. Sun

Sean X. Sun. Photo by Will Kirk

The bacteria research was reported in the Oct. 9 edition of Proceedings of the National Academy of Sciences. The work was led bySean X. Sun, an assistant professor ofmechanical engineering in Johns Hopkins’ Whiting School of Engineering.

“This type of bacteria is commonly found in the human body,” said Sun, a co-author of the journal article. “Understanding how organisms like this work can help us find new ways to treat bacterial illnesses, develop medications or do any type of bioengineering involving bacteria. If you want to target certain cellular activities, you need to know how single-celled creatures like this operate.”

Sun’s team brought a fresh perspective to the study of cell activity. While traditional biologists try to identify and learn the function of tiny bits of genetic material within cells, Sun studies how such proteins work together to form “molecular machines” that carry out tasks inside the cells. “Biologists are just beginning to understand that mechanical processes at the cellular level are also important,” he said. “I’m bringing the tools of mechanical engineering to bear on biological mysteries.”

rod-shaped microbe

This computer-generated image depicts a rod-shaped microbe in the midst of dividing. A little-understood structure called a Z-ring pinches the midsection during the division process. Image by Ganhui Lan

Toward this goal, Sun’s team’s sought to measure how much mechanical force the Z-ring applies to rod-shaped bacteria during cell division. The researchers knew that each rod-shaped bacterium possesses, around the inside of its midsection, a belt made of a filamentous protein called FtsZ. Most of the time, this ring is inactive. But when a bacterium cell is healthy and has sufficient food, it seeks to reproduce by dividing in two. When it is time for this to occur, the Z-ring receives a signal and begins to contract. This pinching continues until the rod breaks apart to form two daughter cells.

Sun’s team gathered data from microbiology labs that are studying cell division and then translated these observations into mathematical equations. The researchers used the equations to create computer simulations of the cell division process, models that yielded a prediction of the Z-ring force: 8 piconewtons. A piconewton is one- trillionth of a newton. One newton is approximately the amount of force needed to lift a baseball in Earth’s gravity.

“The surprise was that the amount of force generated by the Z-ring was so small,” Sun said. “Most researchers believed a lot more force would be required during the cell division process.”

Ganhui Lan

Ganhui Lan. Photo by Will Kirk

This information could be used, Sun said, by drug developers seeking a way to disable the Z-ring so that harmful bacteria can no longer reproduce. The research has wider implications as well. “Our mathematical equations could also be used to help understand how plant and animal cells divide, including human cells,” Sun said. “Human cells have an actin ring that behaves the same way as a Z-ring. It contracts during division. The mathematical formulas developed in this study could also be used in research concerning the division of human cells. The more we know about this process, the better we can affect the process through drugs or genetic manipulation.”

The lead author on the PNAS article was Ganhui Lan, a Johns Hopkins doctoral student in the Department of Mechanical Engineering, supervised by Sun. The other co- author was Charles Wolgemuth, an assistant professor in the Department of Cell Biology and the Center for Cell Analysis and Modeling at the University of Connecticut Health Center in Farmington, Conn.

Sun also is affiliated with the Institute for NanoBioTechnology at Johns Hopkins. His team’s research was supported by funding from the National Institutes of Health.

Color images of the researchers and a dividing microbe available; contact Phil Sneiderman.

Related Links
– Johns Hopkins Department of Mechanical Engineering

Mechanical engineers use magnets, nanobeads to measure DNA torque

Torque measures the tendency of a force to rotate something around an axis—think of a tether ball on a string. Torque also comes into play when the enzymes that read genetic code travel along a length of DNA. The segment behind the enzyme unwinds, while the portion ahead becomes more coiled and compact. Researchers from Johns Hopkins Institute for NanoBioTechnology have developed a method that uses magnets and a nanobead to measure, for the first time, single molecule rotational forces involved in the winding and unwinding of DNA fibers within the chromosome. Understanding these forces could help scientists predict gene regulation and provide important information on molecular targets for the development of disease-fighting drugs.

Mechanical engineering doctoral student Alfredo Celedon observed the torque of a 1-micron length of chromatin fiber. In this instance, torque is a measure of the rotational force required to twist the end of the fiber. Chromatin is the protein-rich protective structure that stuffs nearly six-feet of DNA into a package small enough to fit inside cells. DNA spools around proteins called histones within the chromatin fiber.

images

Alfredo Celedon, PhD

Celedon collaborated with principal investigator Sean X. Sun, associate professor of Mechanical Engineering and INBT affiliated faculty member. The team attached one end of the chromatin fiber to a glass slide. [See illustration.] The other end of the chromatin was attached to a 200-nanometer diameter rod coupled to a magnetic bead. This end of the fiber was pulled upward by weak forces exerted on the bead by the magnet tweezers positioned above the slide.

When the researchers rotated the magnetic tweezers, the nanorod-bead spun introducing twists but adding very little vertical pulling force on the fiber. As the chromatin fiber twisted clockwise or counter clockwise, Celedon obtained torque from the change in the angle of the nano-rod bead from its resting position. This work was published in the March 20, 2009 issue of Nano Letters.

“Because the forces pulling upward on the nanorod-bead are so small, we are able to measure chromatin torque without unraveling the structure of the fiber or melting DNA, something that has not been done before,“ says Sun. Applying low vertical pulling forces is physiologically relevant, Sun adds, because that state mimics what might be found in a living cell.

The researchers compared the torque of chromatin fibers with that of bare DNA strands, and found that DNA packed in the chromatin structure was able to absorb more twists with lower torque than bare DNA.

“This makes sense,“ Celedon says. “Because the histones act like springs and absorb rotational forces.“

The chromatin used in these experiments was reconstituted in vitro, Celedon added. “The next step is to study the effects on chromatin torque of modifications to the structure associated with different levels of gene expression This will allow us to understand how chromatin structure regulates transcription.“

Other contributors to this research include Ilana Nodelman, research scientist in Biophysics; Bridget Wildt, doctoral student in Materials Science; Rohit Dewan, junior in Chemical and Biomolecular Engineering; Peter Searson, Reynolds Professor of Materials Science and Engineering and INBT director; Denis Wirtz, professor of Chemical and Biomolecular Engineering and associate director of INBT; and Gregory Bowman, assistant professor of Biophysics. Celedon’s research was funded by the National Science Foundation and the Howard Hughes Medical Institute Graduate Training Program at the Johns Hopkins Institute for NanoBioTechnology. Bowman, Searson, Wirtz, and Sun are affiliated faculty members of INBT.

Links

Magnetic Tweezers Measurement of Single Molecule Torque, Alfredo Celedon, Ilana M. Nodelman, Bridget Wildt, Rohit Dewan, Peter Searson, Denis Wirtz, Gregory D. Bowman, Sean X. Sun, Nano Letters 2009 9 (4), 1720-1725.

Sean Sun’s INBT page

Greg Bowman’s INBT page

Story by Mary Spiro