1. Plant growth
<ol>
	<li>Sow Arabidopsis seeds expressing microtubule binding domain fused with green fluorescent protein (MBD-GFP)10 on soil and keep in long day (16 h day /8 h night), 20 &#176;C/6 &#176;C conditions for 1 week for germination.</li>
	<li>After germination, transfer seedlings to new pots with sufficient growth space to allow robust vegetative growth. Keep plants in short day (8 h day /16 h night), 20 &#176;C/16 &#176;C conditions for 3-5 weeks.</li>
	<li>Transfer plant...

<ol>
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E., Doring, A., Persson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+cellulose+synthesis.">The cell biology of cellulose synthesis.</a> Annual Reviews in Plant Biology. 65, 69-94 (2014).</li><li>Burgert, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+micromechanical+design+of+plant+cell+walls.">Exploring the micromechanical design of plant cell walls.</a> American Journal of Botany. 93 (10), 1391-1401 (2006).</li><li>Paredez, A. R., Somerville, C. R., Ehrhardt, D. 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G., 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=Alignment+between+PIN1+polarity+and+microtubule+orientation+in+the+shoot+apical+meristem+reveals+a+tight+coupling+between+morphogenesis+and+auxin+transport.">Alignment between PIN1 polarity and microtubule orientation in the shoot apical meristem reveals a tight coupling between morphogenesis and auxin transport.</a> PLoS Biology. 8 (10), e1000516 (2010).</li><li>Louveaux, M., Rochette, S., Beauzamy, L., Boudaoud, A., Hamant, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+mechanical+compression+on+cortical+microtubules+in+Arabidopsis:+a+quantitative+pipeline.">The impact of mechanical compression on cortical microtubules in Arabidopsis: a quantitative pipeline.</a> Plant Journal. 88 (2), 328-342 (2016).</li><li>Nakayama, 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=Mechanical+regulation+of+auxin-mediated+growth.">Mechanical regulation of auxin-mediated growth.</a> Current Biology. 22 (16), 1468-1476 (2012).</li><li>Marc, J., 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+GFP-MAP4+reporter+gene+for+visualizing+cortical+microtubule+rearrangements+in+living+epidermal+cells.">A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells.</a> Plant Cell. 10 (11), 1927-1940 (1998).</li><li>Strauss, S., Sapala, A., Kierzkowski, D., Smith, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Plant+Growth+and+Cell+Proliferation+with+MorphoGraphX.">Quantifying Plant Growth and Cell Proliferation with MorphoGraphX.</a> Methods in Molecular Biology. 1992, 269-290 (2019).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Erguvan, O., Louveaux, M., Hamant, O., Verger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+SurfCut:+a+user-friendly+pipeline+for+high-throughput+extraction+of+cell+contours+from+3D+image+stacks.">ImageJ SurfCut: a user-friendly pipeline for high-throughput extraction of cell contours from 3D image stacks.</a> BMC Biology. 17 (1), 38 (2019).</li><li>Boudaoud, A., 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=FibrilTool,+an+ImageJ+plug-in+to+quantify+fibrillar+structures+in+raw+microscopy+images.">FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images.</a> Nature Protocols. 9 (2), 457-463 (2014).</li><li>Sampathkumar, A., 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=Primary+wall+cellulose+synthase+regulates+shoot+apical+meristem+mechanics+and+growth.">Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.</a> Development. 146 (10), (2019).</li><li>Hervieux, 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=A+Mechanical+Feedback+Restricts+Sepal+Growth+and+Shape+in+Arabidopsis.">A Mechanical Feedback Restricts Sepal Growth and Shape in Arabidopsis.</a> Current Biology. 6 (8), P1019-P1028 (2016).</li><li>Sampathkumar, A., 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=Subcellular+and+supracellular+mechanical+stress+prescribes+cytoskeleton+behavior+in+Arabidopsis+cotyledon+pavement+cells.">Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells.</a> Elife. 3, e01967 (2014).</li><li>Sampathkumar, A., 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=Primary+wall+cellulose+synthase+regulates+shoot+apical+meristem+mechanics+and+growth.">Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.</a> Development. 146 (10), dev179036 (2019).</li><li>Uyttewaal, 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=Mechanical+stress+acts+via+katanin+to+amplify+differences+in+growth+rate+between+adjacent+cells+in+Arabidopsis.">Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis.</a> Cell. 149 (2), 439-451 (2012).</li><li>Hamant, O., 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=Developmental+patterning+by+mechanical+signals+in+Arabidopsis.">Developmental patterning by mechanical signals in Arabidopsis.</a> Science. 322 (5908), 1650-1655 (2008).</li></ol>

The assessment of mechanical signal transduction events is crucial to identify molecular regulators involved in the mechano-perception and transduction pathways. The protocol described here provides a quantitative view of such events by using the cortical microtubule response as a readout for such a process in Arabidopsis SAMs. The procedure described here is routinely used in several studies in various tissue types16,17,18</...

Plant cells are surrounded by an extracellular matrix of polysaccharides and glycoproteins that mechanically resembles a fiber reinforced composite material capable of dynamically changing its mechanical properties1. Growth in plant cells is driven by the uptake of water into the cell, which results in a concomitant buildup of tensile forces on the cell wall. In response to such forces, modifications to the physical state of the cell wall allows for cell expansion. Cells with primary walls are capable of undergoing rapid growth compared to secondary cell wall containing cells mainly due to differences in the chemical composition of the polysaccharides within. Primary wall cells are composed of cellulose, hemicellulose, and pectin in addition to glycoproteins, and lack lignin, a component that is present in the secondary cell wall2. Cellulose, a glucose polymer linked via &#946;-1,4 bonds, is the major component of the cell walls. It is organized into fibrillar structures that are capable of withstanding high tensile forces experienced during cell growth3. In addition to withstanding tensile forces, mechanical reinforcement along a preferential direction results in turgor-driven expansion along an axis perpendicular to the net orientation of the cellulose microfibril. The organization of the cellulose microfibrils is influenced by the cortical microtubule cytoskeleton, as they guide the directional movement of the cellulose-synthesizing complexes located at the plasma membrane4. Therefore, monitoring cortical microtubule organization using a microtubule-associated protein or tubulin fused with a fluorescent molecule serves as a proxy for the observation of overlying patterns of cellulose in plant cells.
The patterning of the cortical microtubule cytoskeleton is under the control of cell and tissue morphology derived mechanical forces. Cortical microtubule organization does not have any preferential organization over time in cells located at the apex of the SAM, whereas cells in the periphery and the boundary between the SAM and the emerging organ have a stable, highly organized supracellular array of cortical microtubules5. Several approaches have been developed to physically perturb the mechanical status of the cells. Changes to osmotic status, as well as treatment with pharmacological and enzymatic compounds that influence the stiffness of the cell wall can result in subsequent changes in the tensile forces experienced by the cell6,7. The use of contraptions that allow for the gradual increase in compressive forces experienced by tissues is another alternative8. Application of centrifugal forces has also been shown to influence the mechanical forces without any physical contact with the cells9. However, the most widely used means of changing directional forces in a group of cells take advantage of the fact that all epidermal cells are under tension and physical ablation of cells will eliminate turgor pressure locally as well as disrupting cell-to-cell adhesion, thereby modifying the tensile forces experienced by the neighboring cells. This is performed either by targeting a high-powered pulsed ultraviolet laser or by means of a fine needle.
Here we elaborate on the process of imaging and assessing cortical microtubule behavior for mechanical perturbation at the SAM.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FibrilTool</td>
			<td></td>
			<td></td>
			<td>Boudaoud, A. et al., Nat Protoc. 2014</td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td>Schindelin, J. et al., Nat Methods. 2012</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Merck</td>
			<td>1.04201.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 confocal microscope</td>
			<td>Leica</td>
			<td>DM6000 CS</td>
			<td></td>
		</tr>
		<tr>
			<td>MAP4-GFP</td>
			<td></td>
			<td></td>
			<td>Marc, J. et al., Plant Cell 1998</td>
		</tr>
		<tr>
			<td>micropore tape</td>
			<td>Leukopor</td>
			<td>02482-00</td>
			<td></td>
		</tr>
		<tr>
			<td>MorphographX</td>
			<td></td>
			<td></td>
			<td>Strauss, S. et al., Methods Mol Biol. 2019</td>
		</tr>
		<tr>
			<td>myo-inositol</td>
			<td>Sigma</td>
			<td>I5125</td>
			<td></td>
		</tr>
		<tr>
			<td>N6-benzyladenine</td>
			<td>Sigma</td>
			<td>B3408</td>
			<td></td>
		</tr>
		<tr>
			<td>nicotinic acid</td>
			<td>Sigma</td>
			<td>N4126</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic hinged box</td>
			<td>Electron microscopy sciences</td>
			<td>64312</td>
			<td></td>
		</tr>
		<tr>
			<td>PPM (Plant Preservative Mixture)</td>
			<td>Plant Cell Technology</td>
			<td>PPM</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>pyridoxine hydrochloride</td>
			<td>Sigma</td>
			<td>P9755</td>
			<td></td>
		</tr>
		<tr>
			<td>SURFCUT</td>
			<td></td>
			<td></td>
			<td>Erguvan, O. et al., BMC Biol. 2019</td>
		</tr>
		<tr>
			<td>thiamine hydrochloride</td>
			<td>Sigma</td>
			<td>T4625</td>
			<td></td>
		</tr>
	</tbody>
</table>

live cell imaging of microtubule cytoskeleton and micromechanical manipulation of the arabidopsis shoot apical meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.

Figure 1 shows typical projection images obtained from MBD-GFP lines with cells at the center of the dome containing disorganized cortical microtubules, and cells at the periphery having a circumferential distribution (Figure 1A,B), whereas the boundary domain cells contain cortical microtubules aligned parallel to the cell&#39;s long axis. These observations show differences in the spatial distribution of cortic...

Watch this Scientific Journal Video about Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem at JoVE.com

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

This protocol it helps us understand how Microtubule Cytoskeleton behavior occurs in plants. At the same time, it also helps us understand how these structural components that influence cell and organ shape, respond to changes in mechanical forces. Although, this method requires little sophistication, it provides robust, quantitative read-after, the mechanical response differences between different genotypes and conditions.Begin by growing arabidopsis seeds expressing microtubule binding domains fused with green fluorescent protein in soil, at 20 and 6 degrees Celsius under long day conditions for one week. After germination, transfer the seedlings into new pots with sufficient growth space to allow robust vegetative growth, and place the plants at 20 and 16 degrees Celsius under short day conditions. After three to five weeks, transfer the plants back to long day conditions at the same temperature until the plants bolt, allowing the inflorescence to grow up to two to five centimeters long.For Shoot Apical Meristem dissection, putt the inflorescence, and use sharp forceps to peel the flowers at the base of the peduncles, until it is difficult to see the peduncles with the naked eye to remove the older flower buds. Use the forceps to create a slit in the agros in a dissecting dish, and plant the inflorescence base into the thick Augur. Place the dish under a dissecting microscope.Starting with the oldest flower bud, push with the forceps to remove the flower bud from the plant. The shoot apical meristem is usually exposed when older flowers up to stage six to seven are removed. And use the forceps to push each flower bud down until the shoot apical meristem is visible.As soon as the shoot apical meristem is exposed, let the freshly dissected sample in growth medium in an ethanol-sterilized rectangular plastic hinged culture box with the shoot apical meristem just exposed above the medium surface. Add a few drops of sterile deionized water to the edges of the culture box and close the lid to maintain the humidity inside the box. Wrap the box with micropore tape, and place the growth box under long or continuous day conditions at 22 degrees Celsius for 12 to 24 hours.When the plant has recovered from the dissection procedure, cover the sample with sterile deionized water and check for air bubbles under the dissecting microscope. Transfer the culture box to an upright confocal microscope stage, and select the 40 or 60X water dipping lens. Lower the objective into the water and check for air bubbles on the front lens of the object.To remove bubbles, lower the stage and gently wipe the lens with an optical tissue. Then use a Pasteur pipette to add a small volume of water to the front lens of the objective before re-immersing the lens in the water. Next, use the GFP filter and epi-illumination module of the confocal microscope to adjust the XY controller to locate the sample.Adjusting the position of the oculars, position the shoot apical meristem directly under the light source, and focus along the Z-axis until the apex is located. Use a laser capable of exciting GFP to illuminate the sample, and adjust the optical zoom of the microscope, so that the entire shoot apical meristem and the stage one floral primordia are in the field of view. Then, adjust the power of the laser output and the gain settings to obtain an optimal signal to noise ratio.After allowing the sample to settle for two to five minutes, acquire confocal Z stacks of the sample at 0.25 to 0.5 micrometers Z slice intervals, adding approximately 0.3 micro meter pixel size resolution. At the end of the acquisition, immediately remove the water and transfer the culture box back to the growth chamber. For Micromechanical Shoot Apical Meristem Perturbation, acquire pre-ablation image stacks of the cortical microtubule organization as just demonstrated, and decaf the water from the culture box into a culture dish.Transfer the shoot apical meristem under a dissecting microscope, and slowly approached the shoot apical meristem with a clean 0.4 by 20 millimeter syringe needle. Briefly contact the periphery of the dome of the shoot apical meristem with the needle tip to confirm that the ablation has been completed. And refill the culture dish with sterile deionized water.Then add 10 micrograms per milliliter of propidium iodide to the dish and immediately acquire image stacks as demonstrated every two hours for six hours, returning the culture dish to the incubation between each time point. For data analysis, generate surface projections of the image stacks using an appropriate image analysis software program, and perform extraction of the cortical microtubule anisotropy using the Fibril Tool Macro in Fiji. Here, typical projection images obtained from microtubule binding domain GFP lines with cells at the center of the dome containing disorganized cortical microtubules, cells at the periphery, having a circumferential distribution and boundary domain cells containing cortical microtubules aligned parallel to the cell's long axis can be observed.Time-lapse imaging, shows cortical microtubule alignment changing from a highly disordered array to a more organized array within six hours of ablation. Tensors could be superimposed on the cortical microtubule images, and the extracted information could be represented by plotting the mean anisotropy over time. When adjusting laser output power and gain settings, we should ensure it clear observation of the filaments and try to avoid overexposure, as over saturated signals could bias the tensile direction in later analysis.Atomic force microscopy can also be performed to assess how the changes in the mechanical forces actually affect the physical status of the cell wall.

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Developmental Biology

5.2K visualizações.  Max Planck Institute of Molecular Plant Physiology. Este protocolo nos ajuda a entender como o comportamento do Citoesqueleto microtúbulo ocorre nas plantas. Ao mesmo tempo, também nos ajuda a entender como esses componentes estruturais que influenciam a forma celular e do órgão, respondem a mudanças nas forças mecânicas. Embora, este método exija pouca sofisticação, fornece leitura robusta e quantitativa, as diferenças de resposta mecânica entre diferentes genótipos e condições.Comece cultivando sementes arabidopsis expr...