Molecular ‘muscles’ flex to external cell forces

If you have ever watched one of those weather people on television being buffeted about while trying to report on a hurricane, you might have some appreciation for what the life of a cell might be like inside a body.  New research from the cell biology laboratory of Doug Robinson, professor at the Johns Hopkins School of Medicine, reveals how the cell uses certain proteins to react and respond to these extreme external forces at the molecular level.

Cytoskeletal proteins move to different areas of a cell in response to the different forces created by suctioning with a thin glass tube. Robinson Lab

Cytoskeletal proteins move to different areas of a cell in response to the different forces created by suctioning with a thin glass tube. Robinson Lab

Graduate student Tianzhi Luo from the Robinson lab studied the cells experimentally by pulling on the cell’s outer membrane (or cytoskeleton) with a tiny glass vacuum tube. Images of fluorescently tagged membrane proteins were captured. Working with Pablo Iglesias, professor of electrical and computer engineering at the Whiting School of Engineering, and his graduate student, Krithika Mohan, the team developed a computer model to predict how cytoskeletal proteins would behave under certain physical forces. The work is summarized in the journal Nature Materials.

“For the first time,” said Robinson, “we are able to explain what a cell can do through the individual workings of different proteins, and because all cells use information about the forces in their environments to direct decisions about migration, division and cell fate, this work has implications for a whole host of cellular disorders including cancer metastasis and neurodegeneration.”

Read the full article paper here.

Read a press release about this research here.

Watch several videos demonstrating the computer model below.

Cancer data stored in the cloud could improve treatments

These days, storing photos or music remotely in “the cloud”  has become common place. Now Johns Hopkins researchers are applying the concept to the storage of medical data in the hopes of predicting and improving cancer patient treatments and outcomes.

Images courtesy Denis Wirtz/JHU

“The long-range goal is to make these data available through the Internet to physicians who are diagnosing and treating cancer patients around the world,” said Denis Wirtz , associate director of the Johns Hopkins Institute for NanoBioTechnology and professor of chemical and biomolecular engineering. Using a $3.75 million grant over five years from the National Cancer Institute Common Fund Single Cell Analysis Program, Wirtz launched the program in October, with two colleagues from the Johns Hopkins School of Medicine, Anirban Maitra and Ralph Hruban.

Initially the database will focus on information from pancreatic cancer patient cell lines but will expand to other types of cancer, including ovarian.  Data gathered and stored will be at the single cell level, which Wirtz explains, provides better information for predicting how individual patients may respond to certain drugs. Drugs that work well for one patient may do nothing at all, or even be harmful, for another, Wirtz said. Understanding and predicting these outcomes before treatment is a step toward more personalized medicine, he added.

To read more about “cloud pathology,” go to the press release issued by John Hopkins University.

Johns Hopkins Institute for NanoBioTechnology

Johns Hopkins Engineering in Oncology Center

Johns Hopkins Kimmel Cancer Center

 

Cells studied in 3-D may reveal novel cancer targets

Stephanie Fraley

Stephanie Fraley, a doctoral student in chemical and biomolecular engineering, was lead author of the study. Photo by Will Kirk/HomewoodPhoto.jhu.edu

Showing movies in 3-D has produced a box-office bonanza in recent months. Could viewing cell behavior in three dimensions lead to important advances in cancer research? A new study led by Johns Hopkins University engineers indicates it may happen. Looking at cells in 3-D, the team members concluded, yields more accurate information that could help develop drugs to prevent cancer’s spread.

“Finding out how cells move and stick to surfaces is critical to our understanding of cancer and other diseases. But most of what we know about these behaviors has been learned in the 2-D environment of Petri dishes,” said Denis Wirtz, director of the Johns Hopkins Engineering in Oncology Center and principal investigator of the study. “Our study demonstrates for the first time that the way cells move inside a three-dimensional environment, such as the human body, is fundamentally different from the behavior we’ve seen in conventional flat lab dishes. It’s both qualitatively and quantitatively different.”

One implication of this discovery is that the results produced by a common high-speed method of screening drugs to prevent cell migration on flat substrates are, at best, misleading, said Wirtz, who also is the Theophilus H. Smoot Professor of Chemical and Biomolecular Engineering at Johns Hopkins. This is important because cell movement is related to the spread of cancer, Wirtz said. “Our study identified possible targets to dramatically slow down cell invasion in a three-dimensional matrix.”

When cells are grown in two dimensions, Wirtz said, certain proteins help to form long-lived attachments called focal adhesions on surfaces. Under these 2-D conditions, these adhesions can last several seconds to several minutes. The cell also develops a broad, fan-shaped protrusion called a lamella along its leading edges, which helps move it forward. “In 3-D, the shape is completely different,” Wirtz said. “It is more spindlelike with two pointed protrusions at opposite ends. Focal adhesions, if they exist at all, are so tiny and so short-lived they cannot be resolved with microscopy.”

The study’s lead author, Stephanie Fraley, a Johns Hopkins doctoral student in Chemical and Biomolecular Engineering, said that the shape and mode of movement for cells in 2-D are merely an “artifact of their environment,” which could produce misleading results when testing the effect of different drugs. “It is much more difficult to do 3-D cell culture than it is to do 2-D cell culture,” Fraley said. “Typically, any kind of drug study that you do is conducted in 2D cell cultures before it is carried over into animal models. Sometimes, drug study results don’t resemble the outcomes of clinical studies. This may be one of the keys to understanding why things don’t always match up.”

collagen fibers

Reflection confocal micrograph of collagen fibers of a 3D matrix with cancer cells embedded. Image by Stephanie Fraley/Wirtz Lab

Fraley’s faculty supervisor, Wirtz, suggested that part of the reason for the disconnect could be that even in studies that are called 3-D, the top of the cells are still located above the matrix. “Most of the work has been for cells only partially embedded in a matrix, which we call 2.5-D,” he said. “Our paper shows the fundamental difference between 3-D and 2.5-D: Focal adhesions disappear, and the role of focal adhesion proteins in regulating cell motility becomes different.”

Wirtz added that “because loss of adhesion and enhanced cell movement are hallmarks of cancer,” his team’s findings should radically alter the way cells are cultured for drug studies. For example, the team found that in a 3-D environment, cells possessing the protein zyxin would move in a random way, exploring their local environment. But when the gene for zyxin was disabled, the cells traveled in a rapid and persistent, almost one-dimensional pathway far from their place of origin.

Fraley said such cells might even travel back down the same pathways they had already explored. “It turns out that zyxin is misregulated in many cancers,” Fraley said. Therefore, she added, an understanding of the function of proteins like zyxin in a 3-D cell culture is critical to understanding how cancer spreads, or metastasizes. “Of course tumor growth is important, but what kills most cancer patients is metastasis,” she said.

To study cells in 3-D, the team coated a glass slide with layers of collagen-enriched gel several millimeters thick. Collagen, the most abundant protein in the body, forms a network in the gel of cross-linked fibers similar to the natural extracellular matrix scaffold upon which cells grow in the body. The researchers then mixed cells into the gel before it set. Next, they used an inverted confocal microscope to view from below the cells traveling within the gel matrix. The displacement of tiny beads embedded in the gel was used to show movement of the collagen fibers as the cells extended protrusions in both directions and then pulled inward before releasing one fiber and propelling themselves forward.

Fraley compared the movement of the cells to a person trying to maneuver through an obstacle course crisscrossed with bungee cords. “Cells move by extending one protrusion forward and another backward, contracting inward, and then releasing one of the contacts before releasing the other,” she said. Ultimately, the cell moves in the direction of the contact released last.

When a cell moves along on a 2-D surface, the underside of the cell is in constant contact with a surface, where it can form many large and long-lasting focal adhesions. Cells moving in 3-D environments, however, only make brief contacts with the network of collagen fibers surrounding them–contacts too small to see and too short-lived to even measure, the researchers observed.

“We think the same focal adhesion proteins identified in 2-D situations play a role in 3-D motility, but their role in 3-D is completely different and unknown,” Wirtz said. “There is more we need to discover.”

Fraley said her future research will be focused specifically on the role of mechanosensory proteins like zyxin on motility, as well as how factors such as gel matrix pore size and stiffness affect cell migration in 3-D.

Co-investigators on this research from Washington University in St. Louis were Gregory D. Longmore, a professor of medicine, and his postdoctoral fellow Yunfeng Feng, both of whom are affiliated with the university’s BRIGHT Institute. Longmore and Wirtz lead one of three core projects that are the focus of the Johns Hopkins Engineering in Oncology Center, a National Cancer Institute-funded Physical Sciences in Oncology Center. Additional Johns Hopkins authors, all from the Department of Chemical and Biomolecular Engineering, were Alfredo Celedon, a recent doctoral recipient; Ranjini Krishnamurthy, a recent bachelor’s degree recipient; and Dong-Hwee Kim, a current doctoral student.

Funding for the research was provided by the National Cancer Institute.  This study, a collaboration with researchers at Washington University in St. Louis, appeared in the June issue of Nature Cell Biology.

Related links:

Johns Hopkins Engineering in Oncology Center

Department of Chemical and Biomolecular Engineering

Watch a related video on YouTube

Story by Mary Spiro

Podcast: Nanotech method to study cell detachment could lead to improved cancer therapies

Peter Searson

Peter Searson

Cancer spreads from organ to organ when cells break free from one site and travel to another. Understanding this process, known as metastasis, is critical for developing ways to prevent the spread and growth of cancer cells. Peter Searson, Reynolds Professor of Materials Science and Engineering in the Whiting School of Engineering and director of the Institute for NanoBioTechnology, led a team of engineers who have developed a method to specifically measure detachment in individual cells.

The method, which uses lab-on-a-chip technology, allows researchers to observe and record the exact point when a cell responds to electrochemical cues in its environment and releases from the surface upon which it is growing. Better knowledge of the biochemistry of cell detachment could point the way to better cancer therapies. In this “Great Ideas” podcast, Elizabeth Tracey, communications associate for the School of Medicine, interviews Searson about this current research.

“…We know that processes like cell detachment are important in cancer metastasis, where cells become detached from tumors…” Peter Searson

Click here to listen:  Great Ideas Podcast: Peter Searson

Related links:

You can watch a video and read more about Searson’s method of studying cell detachment here.

Peter Searson’s INBT profile page.

This podcast was originally posted to the Johns Hopkins University “Great Ideas” web page. To view the original posting, click here.