Stentor coeruleus

Summary

Working with Dr. Janet Sheung at Scripps College, I pipetted single-celled organism Stentor coeruleus between wells with sucrose or urea solutions to pacified spring water to remove the oral apparatus, edited MATLAB code to track cell motion on video, and did extensive background research on regulation of genes during cell regeneration.

This work has been published in the Journal of Visualized Experiments, and was presented at the 65th Biophysical Society Annual Meeting, summarized below.

Full Review

Stentor coeruleus has long been used as a model organism to understand unicellular regeneration. After a chemical shock, the oral apparatus is shed in its entirety and regenerated over the course of eight hours (Figure 1). As the oral apparatus regenerates, the newly formed cilia undergo reproducible changes in position and activity, which in turn affects the swimming motility of the cell. By analyzing motility, we implemented an assay for “functional regeneration”, that quantifies regeneration by quantifying the function of the regenerated structures. This assay can, in principle, be used to study development and functional regeneration in other swimming organisms.

We shocked cells in 10% sucrose or 2% urea to force regeneration of the oral apparatus, and then recorded 10-second videos of groups of 10 Stentor at each timepoint through a microscope. To track cell motion in the videos, we used MATLAB to identify and plot cell trajectories.

We observed a gradual and sustained increase in movement range from the most motile cells within the regenerating Stentor population. At two hours, the earliest time point, no cells move more than 1mm during the ten second video, while at nine hours many cells traversed more than 5mm during the same length of time (Figure 2).

Over 10 hours, the cells went through four behaviorally distinct phases of motion:

1. From 0-3 hours, most cells display little to no visible motion, while the range of movement of the most motile cells in the population gradually increases, as seen in the scatter overlay of Figure 3.
2. From 4-7 hours, the range of movement continues to increase as cell speeds drop across the population.
3. From 8-10 hours, we observed a statistically significant increase in speed. The fastest cells often swam across the well or followed the wall all the way around during a ten second video, interpreted as an intentional exploration of the environment, before settling into a small colony of cells.
4. After 10 hours, the cell population transitioned from exploring the environment to securely anchored with their holdfast and extended in Stentor’s characteristic trumpet shape. The cells at this phase behaviorally resemble healthy cells.

So why does Stentor coeruleus transition through four behaviorally distinct phases of movement patterns? Proteomic studies had shown Stentor coeruleus to be repressing transcription factors, including aurora kinases required for microtubule polymerization, during phase 1 (Sood, P. et al. 2017, Wang, L. et al. 2008). This suggests that the cell has recognized that it is missing the oral apparatus and is deconstructing its cytoskeleton in order to rebuild, consequently decreasing movement.

Further Reading

Sood, P., McGillivary, R., Marshall, W. F. The transcriptional program of regeneration in the giant single cell, Stentor coeruleus. bioRxiv. , 240788 (2017)

Wang, L. et al. Requirement of Aurora-A kinase in astral microtubule polymerization and spindle microtubule flux. Cell Cycle. 7 (8), 1104–1111, doi: 10.4161/cc.7.8.5738 (2008)