Name: live cell imaging of microtubule cytoskeleton and micromechanical manipulation of the arabidopsis shoot apical meristem
Uploaded: 2020-05-23T14:30:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem at JoVE.com

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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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 ...

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