Name: 3-d time-lapse imaging of cell wall dynamics using calcofluor in the moss physcomitrium patens
Uploaded: 2023-02-10T14:10:00
Description: Watch this Scientific Journal Video about 3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens at JoVE.com

The authors thank Dr. Soucy Patricia and Betty Nunn at the University of Louisville for assistance with the confocal microscope. This work was funded by the National Science Foundation (1456884 to M.P.R.) and by a National Science Foundation Cooperative Agreement (1849213 to M.P.R.).

NOTE: See the Table of Materials for the list of materials and equipment and Table 1 for the list of solutions to be used in this protocol.
1. Preparation of plants for glass bottom dishes
<ol>
	<li>Grow the moss protonemal tissue in BCDAT agar medium in a 13 cm Petri dish under constant white light (~50 &#181;mol m-2s-1) for 7 days at 25 &#176;C in the growth chamber.</li>
	<li>Filter-sterilize the calcofluor white and...

<ol>
	<li>Anderson, C. T., Kieber, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+construction,+perception,+and+remodeling+of+plant+cell+walls.">Dynamic construction, perception, and remodeling of plant cell walls.</a> Annual Review of Plant Biology. 71, 39-69 (2020).</li><li>Vaahtera, L., Schulz, J., Hamann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+integrity+maintenance+during+plant+development+and+interaction+with+the+environment.">Cell wall integrity maintenance during plant development and interaction with the environment.</a> Naure Plants. 5 (9), 924-932 (2019).</li><li>Bao, L., 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+cellular+function+of+ROP+GTPase+prenylation+important+for+multicellularity+in+the+moss+Physcomitrium+patens.">The cellular function of ROP GTPase prenylation important for multicellularity in the moss Physcomitrium patens.</a> Development. 149 (12), (2022).</li><li>Colin, L., 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=Imaging+the+living+plant+cell:+From+probes+to+quantification.">Imaging the living plant cell: From probes to quantification.</a> The Plant Cell. 34 (1), 247-272 (2022).</li><li>Fang, Y., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+plants.">Live cell imaging of plants.</a> Cold Spring Harbor Protocols. 2010 (2), (2010).</li><li>Goh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live-cell+imaging+approaches+to+study+lateral+root+formation+in+Arabidopsis+thaliana.">Long-term live-cell imaging approaches to study lateral root formation in Arabidopsis thaliana.</a> Microscopy. 68 (1), 4-12 (2019).</li><li>Hamant, O., Das, P., Burian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+developing+shoot+meristems+using+a+confocal+laser+scanning+microscope.">Time-lapse imaging of developing shoot meristems using a confocal laser scanning microscope.</a> Methods in Molecular Biology. 1992, 257-268 (2019).</li><li>Rigal, A., Doyle, S. M., Robert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+FM4-64,+a+tool+for+tracing+the+endocytic+pathways+in+Arabidopsis+root+cells.">Live cell imaging of FM4-64, a tool for tracing the endocytic pathways in Arabidopsis root cells.</a> Methods in Molecular Biology. 1242, 93-103 (2015).</li><li>Yagi, N., 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=Advances+in+synthetic+fluorescent+probe+labeling+for+live-cell+imaging+in+plants.">Advances in synthetic fluorescent probe labeling for live-cell imaging in plants.</a> Plant &amp; Cell Physiology. 62 (8), 1259-1268 (2021).</li><li>Whitewoods, C. D., 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=CLAVATA+was+a+genetic+novelty+for+the+morphological+innovation+of+3D+growth+in+land+plants.">CLAVATA was a genetic novelty for the morphological innovation of 3D growth in land plants.</a> Current Biology. 28 (15), 2365-2376 (2018).</li><li>Bao, L., 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=A+PSTAIRE-type+cyclin-dependent+kinase+controls+light+responses+in+land+plants.">A PSTAIRE-type cyclin-dependent kinase controls light responses in land plants.</a> Science Advances. 8 (4), 2116 (2022).</li></ol>

Time-lapse 3-D reconstruction, or 4-D imaging, is a powerful tool for observing the dynamics of cellular morphology during developmental processes. In this protocol, by mixing the calcofluor white in the medium, the dynamics of 3-D cellular morphology can be observed in the moss P. patens. Using this method, we observed that the surface of cell walls in ggb mutants are torn apart during development3. Moreover, the reduced thickening of cell walls is correlated with the cell expan...

Plant cell walls undergo dynamic changes during cell expansion and development1,2,3. Maintaining cell wall integrity is critical for plant cell adhesion during growth and development, as well as for the response to environmental signals. Although visualizing cell wall dynamics of living cells over a long period of time is critical to understanding how cell adhesion is maintained during development and adaptation to environmental changes, current methods for directly observing cell wall dynamics are still challenging.
Time-lapse imaging of cellular changes can provide informative developmental dynamics of an organism using a high-resolution fluorescence microscope4,5,6,7. While time-lapse 3-D imaging has a great deal of potential for studying dynamic changes of cell shape during growth and development, the technique normally requires transformation of a fluorescent protein4,5,6,7. However, for most systems, genetic transformations are either time-consuming or technically challenging. As an alternative, fluorescent dyes that attach to cellular components have long been available. The fluorescent dyes can emit fluorescent light after irradiation with light of a certain wavelength. Common examples are Edu, DAPI, PI, FM4-64, and calcofluor white8,9,10. One major drawback, however, is that these dyes can typically only be used in fixed tissue or for short experiments, in part due to the harm they cause to the cell8,9,10.
With the protocol presented here, calcofluor signals are stable when calcofluor white is mixed within the medium during time-lapse experiments in the moss P. patens. Using this method, the detachment of cells in ggb mutants using 3-D time-lapse imaging was observed over a 3 day period3 (Figure 1). This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-mm-thick red plastic light filter</td>
			<td>&#160;Mitsubishi</td>
			<td>&#160;no.102</td>
			<td></td>
		</tr>
		<tr>
			<td>27 mm diameter glass base dish&#160;</td>
			<td>Iwaki</td>
			<td>3930-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>&#160;Sigma</td>
			<td>A6924</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcofluor white&#160;</td>
			<td>&#160;Sigma</td>
			<td>18909-100ML-F</td>
			<td>Calcofluor White M2R, 1 g/L and Evans blue, 0.5 g/L</td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Nikon&#160;</td>
			<td>A1</td>
			<td>
			NIS element software; .nd2 file in NIS-elements Viewer, download from https://www.microscope.healthcare.nikon. 
			com/en_AOM/products/software/nis-elements/viewer
			</td>
		</tr>
		<tr>
			<td>Fluorescence microscope&#160;</td>
			<td>Nikon&#160;</td>
			<td>TE200&#160;</td>
			<td>Equipped with a DS-U3 camera;</td>
		</tr>
		<tr>
			<td>Gellan gum&#160;</td>
			<td>Nacali Tesque</td>
			<td>12389-96</td>
			<td></td>
		</tr>
		<tr>
			<td>Plant Growth Chambers</td>
			<td>SANYO&#160;</td>
			<td>Sanyo MLR-350H</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilized syringe 0.22 &#956;m filter</td>
			<td>Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
	</tbody>
</table>

3-d time-lapse imaging of cell wall dynamics using calcofluor in the moss physcomitrium patens

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

This method allows the observation of cell wall dynamics during development in wild type and ggb mutants (Figure 1). The results showed that regions with less thickening of the cell wall correlate with the cell expansion/branching sites, allowing for the prediction of expansion/branching sites in the wild type (Figure 1A). The surface of the cell walls in ggb mutants was torn apart during development due to uncontrolled cell expansion<sup class...

Watch this Scientific Journal Video about 3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens at JoVE.com

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In ...

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