Name: analysis of motility patterns of stentor during and after oral apparatus regeneration using cell tracking
Uploaded: 2021-04-26T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking at JoVE.com

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.

NOTE: A population of approximately one hundred S. coeruleus cells were cultured in accordance with a previously published JoVE protocol8.
1. Sample preparation
<ol>
	<li>Cut a piece of 250 &#181;m-thick silicone spacer sheet (Table of Materials) slightly smaller in both height and width than a microscope slide. Using a 5/16&#34; hole punch, create circular wells. Be mindful of leaving sufficient space between neighboring wells to ensure a go...

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

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...

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&#8212;the OA&#8212;which is shed completely upon chemical shock4,5,6. De novo replacement of a lost OA begins with the emergence of a new membranellar band&#8212;an array of cilia that gradually shift towards the anterior of the cell before forming a functional OA over eight morphological stages3. These stages have been observed sequentially, regardless of temperature, and provide a universal reference point for nearly all studies5.
Mechanistic analysis of Stentor regeneration requires tools for measuring the timing of regeneration that are robust and simple enough to be applied to multiple samples as part of a chemical or molecular screen. The standard method for performing a cell-based assay is imaging, in this case, imaging the formation of new OA during regeneration. However, such imaging-based assays are most effective when the regenerating structure contains distinct molecular components that can be used as markers, so that they would be easily detected in a fluorescence image. In the case of the Stentor OA, the known components (cilia, basal bodies) are also present on the rest of the cell surface; therefore, recognizing the restoration of the OA cannot be achieved simply by looking for the presence or absence of a component.
Rather, some form of shape recognition would be required to detect an OA, and this is potentially very challenging given the fact that Stentor cells often change shape via a rapid contractile process. This paper presents an alternative assay for regeneration that relies on the motile activity of the body and OA cilia. As the OA 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, it is possible to perform an assay for &#34;functional regeneration&#34; that quantifies regeneration by quantifying the function of the regenerated structures. Previous analysis of Stentor ciliary function during regeneration used particle image velocimetry, combined with tracer beads added to the external media, to observe changes in flow pattern at different stages of regeneration7; however, this approach requires laborious imaging of individual cells and their associated flow fields, one at a time.
By using the motion of the cell itself as a proxy for cilia-generated flow, it would be possible to analyze larger numbers of cells in parallel, using low-resolution imaging systems compatible with high-throughput screening platforms. This assay can, in principle, be used to study development and functional regeneration in other swimming organisms in the hundreds of microns to millimeters size scale. Section 1 of the protocol describes the construction of a multiwell sample slide, which allows for high-throughput imaging of a population of cells over up to an entire day. Details are provided for how to adjust for use with other cell types. Section 2 of the protocol covers the acquisition of video data for this assay, which can be accomplished on a dissection microscope with a digital single-lens reflex camera. Section 3 of the protocol provides a walk-through of cell tracking and cell speed calculation using MATLAB code (Supplemental Information). Section 4 of the protocol explains how to turn the numerical results into plots as shown in Figure 1C-F and Figure 2C for easy interpretation of results.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25 mm-thick silicone sheet</td>
			<td>Grace Bio-Labs</td>
			<td>CWS-S-0.25</td>
			<td></td>
		</tr>
		<tr>
			<td>24 x 50 mm, #1.5 coverglass</td>
			<td>Fisher Scientific</td>
			<td>NC1034527</td>
			<td>As noted in Discussion, smaller coverglass can be used if fewer sample wells are placed on one slide.</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td></td>
			<td></td>
			<td>We used Nikon D750</td>
		</tr>
		<tr>
			<td>Chlamydomonas 137c WT strain</td>
			<td>Chlamydomonas Resource Center</td>
			<td>CC-125</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MATHWORKS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB Image Processing Toolbox</td>
			<td>MATHWORKS</td>
			<td></td>
			<td>needed for TrackCells.m and CleanTraces.m</td>
		</tr>
		<tr>
			<td>MATLAB Statistics and Machine Learning Toolbox</td>
			<td>MATHWORKS</td>
			<td></td>
			<td>needed for TrackCells.m</td>
		</tr>
		<tr>
			<td>Microscope with camera port</td>
			<td></td>
			<td></td>
			<td>We used Zeiss AxioZoom v1.6 and Leica S9E</td>
		</tr>
		<tr>
			<td>Pasteurized Spring Water</td>
			<td>Carolina</td>
			<td>132458</td>
			<td></td>
		</tr>
		<tr>
			<td>TAP Growth Media</td>
			<td>ThermoFisher Scientific</td>
			<td>A1379801</td>
			<td>Can also be made for much cheaper following recipe from Chlamy Resource Center</td>
		</tr>
	</tbody>
</table>

analysis of motility patterns of stentor during and after oral apparatus regeneration using cell tracking

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes&#8212;many not associated with the oral apparatus (OA)&#8212;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.

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...

Watch this Scientific Journal Video about Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking at JoVE.com

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.

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

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds ...

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