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

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 genes—many 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 setting^1,2,3. Early regeneration studies focused on the largest and most morphologically distinct feature in Stentor—the OA—which is shed completely upon chemical shock^4,5,6. De novo replacement of a lost OA be....

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.......

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.......

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.......

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.