Revisiting an Age-Question with New Imaging: How Does Stentor Regenerate?

Revisiting an Age-Question with New Imaging: How Does Stentor Regenerate?
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By Sarah Lawhun

The majestic Stentor is the largest single-celled animal we know, reaching 1-2 millimeters in length and visible to the naked eye. It is found in freshwater lakes and ponds all over the world. In fact, diverse Stentor are swimming in ponds in the MBL’s backyard.

When Janet Sheung took the MBL Physiology course in 2012, she became inspired to work on Stentor during a research rotation led by Wallace Marshall of University of California, San Francisco. Stentor’s ability to fully regenerate from a just tiny piece amazed her: “A human with the same capabilities would be able to regenerate in full from a single finger!” she says.

Although Stentor has a long research history at MBL – including studies of its impressive regenerative capacity by Thomas Hunt Morgan in the 1890s – many of its behaviors have only been described in illustrations.

Sheung came back to MBL over the following summers to develop imaging techniques for Stentor in collaboration with Marshall. And she came back this summer as a Whitman Center Early Career Fellow with the goal of statistically classifying various behaviors of Stentor coeruleus through a high-throughput imaging system she designed herself.

“I’m a physicist, so when I started to observe and try to classify Stentor behavior, I naturally started to ask questions about how much energy it was using,” says Sheung, a visiting assistant professor at Vassar College. “I thought determining how much energy it needed to feed or to regenerate would be an interesting calculation.”

In prior experiments in which she cut off portions of Stentor’s cell, the organism would only survive and regenerate if she fed it first. After stumbling upon a cartoon on an MBL lab whiteboard — calculating how many photons (light particles) a student needs to get a graduate degree — she realized maybe, without enough energy from food, Stentor can’t complete the demanding process of regeneration. Maybe it all came down to that one thing: energy.

Stentor coeruleus wound healing. A S. coeruleus cell immobilized in 2% methocellulose cut with a glass needle. The ruptured membrane causes a bleb of displaced cytoplasm, which within seconds is pulled back into the cell volume. (Credit: Janet Sheung, prior publication https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4110943/)

Stentor is trumpet shaped with, at the wide end, a feeding apparatus lined with cilia — tiny, finger-like projections that capture microscopic prey. Sheung had observed Stentor use its cilia to create vortices — large whirlpools of water that suck food particles into its feeding apparatus. If Stentor were scaled up to the size of a human, this vortice area would be equivalent to 5 Olympic-sized swimming pools. In other words, it likely requires a whole lot of energy to create.

Maybe that’s why Stentor, as Sheung observed, couldn’t feed during regeneration. While scientists haven’t yet calculated how much energy Stentor need to regenerate, it’s easy to imagine that re-growing one’s entire body is no easy task.

At MBL, Sheung used high-resolution microscopy to create time-lapse videos during Stentor regeneration. She wrote her own video analysis software to measure Stentor’s distance traveled, direction, and velocity as it regenerates from a tiny fragment. With these measurements, she can begin to classify its behavior during regeneration.

She also used time-lapse videos to trace the path of food particles around Stentor’s feeding apparatus. Her software uses these paths to measure how much water Stentor is moving in the feeding vortices. This measurement, in turn, will enable her to calculate how much energy a healthy Stentor uses to feed, giving her a baseline that may help her understand the energy they need to regenerate.

Video: Stentor coeruleus generate external flows for feeding. One micron diameter polystyrene spheres serve as tracers to visualize flow pattern at 47.5x magnification. Flow allows food particles and prey to be swept into the oral apparatus from multiple body lengths away. (Image credit: Evan Burns/Janet Sheung)

Sheung has also discovered an unusual Stentor, a bright green strain that contains a symbiotic alga. She’s sequencing this strain’s DNA, which will help determine what, exactly, the symbiont is doing — perhaps providing Stentor with energy. Sequencing Stentor will also help other Stentor scientists figure out which genes are causing what behavior and develop methods for gene editing in the organism.

As Sheung moves forward, she hopes her work will expand the field of Stentor biology. Teaching and mentoring in biology at a liberal arts college is also important to her. She is launching a new biophysics course at Vassar College this spring, and her Whitman Center fellowship “allowed me to meet many leading researchers who are teaching or developing related courses, and who have generously shared material,” she says.

She also hopes to bring more undergraduates to MBL. Her research assistant last summer, Vassar undergraduate Evan Burns, had an unforgettable experience. “I want more  of my undergad students to be able to experience this kind of collaborative scientific community,” she says.

“My summer at the MBL is one that I will cherish forever,” Burns says.  “It was my first true research experience and one that has undoubtedly changed the trajectory of my career path for the better. While I have long sought to one day have a career as a medical doctor, the incredibly positive experience I had in my first exposure to live science at the MBL has made me now want to pursue an MD/PhD advanced degree. Had it not been for the incredible conversations I shared with my fellow scientists, the cutting-edge microscopy machines, and my mentor, Janet Sheung, I would not have had such a transformative experience as a scientist. Thank you, MBL!”

“Anyone I’ve shown Stentor to can appreciate how amazing its regeneration process is,” Sheung says. “And I love when people start asking their own questions about Stentor.”

Rotation about anchor point (holdfast) allows S. coeruleus to optimize food intake. Video has been sped up 4.2x and post-processed with Flowtrace for clarity. (Credit: Janet Sheung)