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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

Published: April 26th, 2021

DOI:

10.3791/62352

1W. M. Keck Science Department, Scripps, Pitzer, and Claremont McKenna of The Claremont Colleges, 2W. M. Keck Science Department, Pitzer College, 3W. M. Keck Science Department, Scripps College, 4Department of Biochemistry and Biophysics, School of Medicine, University of California at San Francisco

We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genesmany not associated with the oral apparatus (OA)—that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

Stentor coeruleus (Stentor) is a well-known model organism that has been used to study unicellular regeneration owing to its large size, ability to withstand several microsurgical techniques, and ease of culturing in a laboratory setting1,2,3. Early regeneration studies focused on the largest and most morphologically distinct feature in Stentor—the OA—which is shed completely upon chemical shock4,5,6. De novo replacement of a lost OA be....

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NOTE: A population of approximately one hundred S. coeruleus cells were cultured in accordance with a previously published JoVE protocol8.

1. Sample preparation

  1. Cut a piece of 250 µm-thick silicone spacer sheet (Table of Materials) slightly smaller in both height and width than a microscope slide. Using a 5/16" hole punch, create circular wells. Be mindful of leaving sufficient space between neighboring wells to ensure a go.......

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The goal of this assay is to quantify the gradual change of movement patterns and phased increase in movement speed from cells within a large (N ~100) regenerating Stentor population. To aid interpretation of results, the custom code included in this protocol generates two types of plots: an overlay of all cell movement traces in a set of video data (Figure 1C-F and Figure S1), and a plot of swim speed by hour since the start of reg.......

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Many particle and cell tracking algorithms currently exist, some entirely free. Cost and user-friendliness are often trade-offs requiring compromise. Additionally, many of the existing cell-tracking programs are designed to track slow crawling motion of tissue culture cells, rather than the fast swimming motion of Stentor, which rotates while swimming and can undergo sudden changes of direction. After testing many of these options, the protocol presented here is intended to be a one-stop solution to go all the w.......

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This work was supported, in part, by Marine Biological Laboratory Whitman Early Career Fellowship (JYS). We acknowledge Evan Burns, Mit Patel, Melanie Melo, and Skylar Widman for helping with some of the preliminary analysis and code testing. We thank Mark Slabodnick for discussion and suggestions. WFM acknowledges support from NIH grant R35 GM130327.

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Name Company Catalog Number Comments
0.25 mm-thick silicone sheet Grace Bio-Labs CWS-S-0.25
24 x 50 mm, #1.5 coverglass Fisher Scientific NC1034527 As noted in Discussion, smaller coverglass can be used if fewer sample wells are placed on one slide.
CCD camera We used Nikon D750
Chlamydomonas 137c WT strain Chlamydomonas Resource Center CC-125
MATLAB MATHWORKS
MATLAB Image Processing Toolbox MATHWORKS needed for TrackCells.m and CleanTraces.m
MATLAB Statistics and Machine Learning Toolbox MATHWORKS needed for TrackCells.m
Microscope with camera port We used Zeiss AxioZoom v1.6 and Leica S9E
Pasteurized Spring Water Carolina 132458
TAP Growth Media ThermoFisher Scientific A1379801 Can also be made for much cheaper following recipe from Chlamy Resource Center

  1. Lillie, F. R. On the smallest parts of stentor capable of regeneration; a contribution on the limits of divisibility of living matter. Journal of Morphology. 12 (1), 239-249 (1896).
  2. Morgan, T. H. Regeneration of proportionate structures in Stentor. The Biological Bulletin. 2 (6), 311-328 (1901).
  3. Tartar, V., Kerkut, G. A. . The Biology of Stentor. , (1961).
  4. Tartar, V. Reactions of Stentor coeruleus to certain substances added to the medium. Experimental Cell Research. 13 (2), 317-332 (1957).
  5. Kelleher, J. K. A kinetic model for microtubule polymerization during oral regeneration in Stentor coeruleus. Biosystems. 9 (4), 269-279 (1977).
  6. Slabodnick, M. M., et al. The kinase regulator Mob1 acts as a patterning protein for Stentor morphogenesis. PLOS Biology. 12 (5), 1001861 (2014).
  7. Wan, K. Y., et al. Reorganization of complex ciliary flows around regenerating Stentor coeruleus. Philosophical Transactions of the Royal Society B: Biological Sciences. 375 (1792), 20190167 (2020).
  8. Lin, A., Makushok, T., Diaz, U., Marshall, W. F. Methods for the study of regeneration in Stentor. Journal of Visualized Experiments JoVE. (136), e57759 (2018).
  9. Sood, P., McGillivary, R., Marshall, W. F. The transcriptional program of regeneration in the giant single cell, Stentor coeruleus. bioRxiv. , 240788 (2017).
  10. Onsbring, H., Jamy, M., Ettema, T. J. G. RNA sequencing of Stentor cell fragments reveals transcriptional changes during cellular regeneration. Current Biology. 28 (8), 1281-1288 (2018).

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