Whitman investigators


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By Wallace Marshall
Co-director, MBL Physiology Course

Last month, I had a problem. I was teaching in the MBL Physiology course, using the giant, single-celled organism Stentor as a model system for students to learn quantitative approaches in cell biology. Stentor, which live in ponds, eat by creating a vortex of water that drags food into the cell’s mouth. The flow is created by thousands of cilia—tiny, hair-like cell parts that swing back and forth pushing fluid around. (Cilia are also critical for making the mucus in your airway flow away from your lungs, and patients with defects in these cilia can be really sick. So the question of how cilia make fluid flow is very important from a medical perspective. )

Stentor is a genus of large, trumpet-shaped ciliates, commonly found in freshwater ponds. Credit: EOL / micro*scope

Stentor is a genus of large, trumpet-shaped ciliates, commonly found in freshwater ponds. Credit: EOL / micro*scope

One of the students in our class, Shashank Shekhar from the CNRS Institute, France, had become interested in how the cell generates this pattern of fluid flow. Shashank started tracking the flow by putting small plastic beads into the water around the Stentor and then taking video images of the beads moving. This is a pretty standard approach in fluid dynamics called particle image velocimetry (PIV). But it’s not that commonly used in biology, and we didn’t entirely know what we were doing. The software we had been trying to use to track these particles didn’t give really nice flow lines. So this was the problem: How to use the flow of these tiny beads to figure out the pattern of flow around the cell as it feeds.

Frustrated by this problem, I decided to go get some coffee from Woods Hole Market. On the way back, I ran into my colleague Magdalena Bezanilla, an MBL Whitman Investigator from University of Massachusetts, Amherst, who works on cell biology. She thinks a lot about things moving inside cells so I figured I could get her input into our PIV challenge.

We ended up chatting about the problem in the MBL’s Waterfront Park, and while we were talking, a couple of guys emerged from the harbor in full scuba gear, carrying a huge metal bracket upon which was mounted a video camera and a laser. (This would be quite weird back home but it’s business as usual in Woods Hole.) I asked the guys what they were up to and they said they were using PIV to study the flow of fluid around ctenophores! Ctenophores or comb jellies are jellyfish-like animals that swim using cilia. So at the exact moment that we were pondering how to use PIV to track cilia-generated fluid flow in our single-celled organisms in the Physiology course, a guy walks out of the water and announces that he is doing the exact same thing, for comb jellies! (Those people who say that Woods Hole is a magical place are telling the truth.)

The guys with the scuba gear and lasers were Jack Costello of Providence College and Sean Colin of Roger Williams University, Whitman Investigators working for the summer at the MBL. Jack offered to give us advice about how to analyze our data, so I sent Shashank over to Jack’s lab in the Rowe building. With Jack’s help and expertise, Shashank was able to get beautiful flow lines from his data (see photo), which clearly reveal the pattern of cilia-generated flow around the Stentor cell while it feeds. Our big problem was solved in a single day due to a fortuitous combination of people, courses, coffee breaks, cells, beaches, marine organisms, and advanced technology. And that’s what summer at the MBL is all about.

Fluid flow around Stentor visualized through particle image velocimetry. Courtesy of Wallace Marshall.

Fluid flow around Stentor as it feeds, visualized by particle image velocimetry. Courtesy of Wallace Marshall.

Thank you to Wallace Marshall of the University of California, San Francisco, for contributing this post. All MBL scientists, students, community members, and visitors are invited to submit items for the MBL’s blog. Please contact Diana Kenney: dkenney@mbl.edu.

By Laurel Hamers

One of the brain’s amazing abilities is self-repair: Although injury or illness may disrupt neural circuits, many connections will reform over time.

Artur Llobet, an MBL Research Awardee from the University of Barcelona, is spending his second consecutive summer in the Whitman Center for Visiting Research investigating olfactory neuron repair in Xenopus laevis, the African clawed frog.

Calcium labeling of olfactory sensory neurons' presynaptic terminals in a Xenopus laevis tadpole. Photo credit: Artur Llobet

Labelling of presynaptic terminals of olfactory sensory neurons in a Xenopus laevis tadpole using calcium green dextran. Image is pseudocolored so that yellow represents higher and blue lower calcium concentration.
Photo and caption: Artur Llobet

“One of the advantages of working with frogs is that they have fantastic regenerative capabilities,” says Llobet. Tadpoles are able to repair damaged neural circuits in a few days, making them ideal test subjects.

Llobet is working with a transgenic line of Xenopus tadpoles that express green fluorescent protein (GFP) in their neurons, allowing him to easily see the neural connections. Last year, he studied the timeframe of Xenopus neural repair by measuring how long snipped olfactory nerves took to regrow. Now, he is trying to understand in greater detail the mechanisms behind the repair process.

Neurons pass electrochemical messages between each other at junctions called synapses; when a neuron fires, the voltage change propagates along the nerve fiber (axon) and calcium increases at the presynaptic terminal, which releases neurotransmitters. By labeling the tadpoles’ synaptic terminals with calcium indicators, Llobet can visualize the functionality of the re-grown connections and determine when during the repair process the new synapses start signaling.

“In a GFP animal, we can see that the nerve has re-grown, but we don’t know if that nerve is actually working or not,” says Llobet. “So we look at the synapses and see whether the calcium concentration increases when we stimulate olfactory sensory neurons.” This calcium accumulation indicates that the new nerve is not just present, but also functional.

By examining neural repair in frogs, scientists hope to gain insight into this process in more complex systems such as the human brain.

Llobet’s research is taking place through the National Xenopus Resource (NXR) at the MBL, a center that maintains breeding stocks of frogs and provides training on advanced imaging and experimental technologies. According to Llobet, the specialized resources offered by the NXR make this research project possible. He is one of six MBL Research Awardees in 2014 to be using the animals and research services of the NXR, which is one of 28 National Institutes of Health-funded Animal Resource Centers nationwide and a cornerstone facility of the MBL’s Bell Center for Regenerative Biology and Tissue Engineering.

By Aviva Hope Rutkin

Visiting scientist Guillermo Yudowski wants to make sea anemones happy.

Every morning, he arrives at his MBL laboratory and looks into a group of plastic tanks. Inside are samples of Aiptasia pallida, a hardy strain of anemone found in abundance near the University of Puerto Rico, where Yudowski conducts neurobiological research. Happy A. pallida, he says, are “colorful and open”; sad ones are closed and white. The white samples are near death and will only last three to four days in their containers.

Top view of a day-old spawned Porites spp. coral larvae. Composite image seen under a fluorescent microscope. Symbiotic zooxanthellae autofluorescence in red, larvae epidermis autofluorescence in green. Courtesy of Guillermo Yudowski.

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Turning white—becoming, in Yudowski’s words, “sad”—is called bleaching. The anemone’s tissues are home to zooxanthellae, vibrant photosynthetic algae that produce food for the anemone and give it a characteristic brown color. Bleaching expels this algae from their home. The bleaching process is thought to be triggered by stress: a decrease in light availability, for example, or changes in the water’s temperature or pH. And these changes don’t need to be dramatic. A difference of a couple degrees Celsius can be enough to effectively bleach an anemone.

Yodowski and his colleagues hope their research will point to a cost-effective treatment for bleaching, which poses a serious threat not only to anemones, but to the world’s coral reefs. Though anemones and corals are different, strategies that work for the one organism may be effective for another. The changing climate has already led to mass bleaching events in the Great Barrier Reef, as well as coral reefs in the Indian Sea, the Caribbean Sea, and the Florida Keys.

“If you read the literature, some say that all the coral is going to die in 50 years. Others say, maybe 50 to 100,” says Yudowski. “It doesn’t make a big difference.”

To move toward a solution, Yudowski wants to understand what’s happening to the anemones on a microscopic level. If we figure out why bleaching occurs on a cellular level, then perhaps we can discover how to stop it from happening altogether.

“We don’t really know much about the basic molecular mechanics of the process,” explains Yudowski. “We are trying to understand how stresses like increased ocean temperature and acidification induce the expulsion of the algae.”

Yudowski and his student, Michael Marty-Rivera, are treating anemones with antioxidant compounds found in red wine and green tea. Previous research shows that reactive oxygen species, a kind of chemically reactive molecule, can trigger the bleaching process. Yudowski and Marty-Rivera think that these antioxidants might be able to counteract the effects of these trigger molecules. They will test the efficacy of their treatments by measuring the amount of photosynthetic activity in the anemones, as well as the number of zooxanthellae present.

Yudowski and Marty-Rivera will spend two months at the MBL this summer before returning to the University of Puerto Rico where, in close collaboration with Professors Loretta Roberson and Joshua Rosenthal, they run several different coral research projects. They want to understand the mechanism of calcification in corals and how environmental variables, such as temperature and pH, impact corals’ ability to form reefs and maintain a healthy symbiosis with their zooxanthellae partners.

Funding for the research is provided by the Puerto Rico Center for Environmental Neuroscience and the National Science Foundation Center of Research Excellence in Science and Technology.

Who is the soft-spoken Nobelist who spends nearly every summer day at his lab bench at MBL? Avram Hershko, a Whitman investigator from the Technion-Israel Institute of Technology. Hershko, who appreciates what he has called MBL’s “creative” and “peaceful” environment for conducting research, was profiled yesterday in the Science section of the New York Times: http://nyti.ms/MBpVao

Avram Herskho in the MBL's Rowe Laboratory. Photo by Tom Kleindinst