Name: high resolution quantification of crystalline cellulose accumulation in arabidopsis roots to monitor tissue-specific cell wall modifications
Uploaded: 2016-05-10T13:00:00
Description: Watch this Scientific Journal Video about High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications at JoVE.com

We thank Dr. M. Rosenberg for her advice and help with anatomical sectioning. We also thank D. Eisler for his technical assistance. This research was supported by grants from FP7-PEOPLE-IRG-2008, Binational Agriculture research and Development (BARD; IS-4246-09), and Israel Science Foundation (ISF; 592/13).

1. Plant Growth
<ol>
	<li>Surface sterilize seeds.
	<ol>
		<li>Sterilize seeds (up to 30 mg) by wet or dry method. As an example, for dry sterilization method, add 1 ml glacial HCl 37% in 50 ml bleach, in a glass beaker in a closed desiccator for at least 4 hr and up to overnight. Perform this step in a chemical hood.</li>
	</ol>
	</li>
	<li>Spread the sterile seeds on plates with 0.8% plant agar in 0.5x Murashige &#38; Skoog (MS) medium. Wrap plates with Parafilm or porous tape and cover with aluminum foil.</li>
	<li>...

<ol>
	<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+the+plant+cell+wall.">Growth of the plant cell wall.</a> Nat Rev Mol Cell Biol. 6, 850-861 (2005).</li><li>Wolf, S., Hematy, K., Hofte, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+control+and+cell+wall+signaling+in+plants.">Growth control and cell wall signaling in plants.</a> Annu Rev Plant Biol. 63, 381-407 (2012).</li><li>Mazeau, K., Heux, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Dynamics+Simulations+of+Bulk+Native+Crystalline+and+Amorphous+Structures+of+Cellulose.">Molecular Dynamics Simulations of Bulk Native Crystalline and Amorphous Structures of Cellulose.</a> J. Phys. Chem. B. 107, 2394-2403 (2003).</li><li>Zhao, H., 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=Studying+cellulose+fiber+structure+by+SEM,+XRD,+NMR+and+acid+hydrolysis.">Studying cellulose fiber structure by SEM, XRD, NMR and acid hydrolysis.</a> Carbohydr Polym. 68, 235-241 (2007).</li><li>Fujita, 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=Cortical+microtubules+optimize+cell-wall+crystallinity+to+drive+unidirectional+growth+in+Arabidopsis.">Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis.</a> Plant J. 66, 915-928 (2011).</li><li>Peng, F., 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=Fractional+separation+and+structural+features+of+hemicelluloses+from+sweet+sorghum+leaves.">Fractional separation and structural features of hemicelluloses from sweet sorghum leaves.</a> Bioresources. 7, (2012).</li><li>Xu, S. L., Rahman, A., Baskin, T. I., 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=Two+leucine-rich+repeat+receptor+kinases+mediate+signaling,+linking+cell+wall+biosynthesis+and+ACC+synthase+in+Arabidopsis.">Two leucine-rich repeat receptor kinases mediate signaling, linking cell wall biosynthesis and ACC synthase in Arabidopsis.</a> Plant Cell. 20, 3065-3079 (2008).</li><li>Anderson, C. T., Carroll, A., Akhmetova, L., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Imaging+of+Cellulose+Reorientation+during+Cell+Wall+Expansion+in+Arabidopsis+Roots.">Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots.</a> Plant Physiol. 152, 787-796 (2010).</li><li>Baskin, T. I., Beemster, G. T., Judy-March, J. E., Marga, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorganization+of+cortical+microtubules+stimulates+tangential+expansion+and+reduces+the+uniformity+of+cellulose+microfibril+alignment+among+cells+in+the+root+of+Arabidopsis.">Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis.</a> Plant Physiol. 135, 2279-2290 (2004).</li><li>Iyer, K. R. K., Neelakantan, P., Radhakrishnan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birefringence+of+native+cellulosic+fibers.+I.+Untreated+cotton+and+ramie.">Birefringence of native cellulosic fibers. I. Untreated cotton and ramie.</a> J. Polym. Sci. A-2: Polymer Physics. 6, 1747-1758 (1968).</li><li>Abraham, Y., Elbaum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+microfibril+angle+in+secondary+cell+walls+at+subcellular+resolution+by+means+of+polarized+light+microscopy.">Quantification of microfibril angle in secondary cell walls at subcellular resolution by means of polarized light microscopy.</a> New Phytol. 197, 1012-1019 (2013).</li><li>Petricka, J. J., Winter, C. M., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Arabidopsis+root+development.">Control of Arabidopsis root development.</a> Annu Rev Plant Biol. 63, 563-590 (2012).</li><li>Vanstraelen, M., Benkova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+Interactions+in+the+Regulation+of+Plant+Development.">Hormonal Interactions in the Regulation of Plant Development.</a> Annu Rev Cell Dev Biol. , (2012).</li><li>Singh, A. P., Savaldi-Goldstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+control:+brassinosteroid+activity+gets+context.">Growth control: brassinosteroid activity gets context.</a> J Exp Bot. 66, 1123-1132 (2015).</li><li>Fridman, 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=Root+growth+is+modulated+by+differential+hormonal+sensitivity+in+neighboring+cells.">Root growth is modulated by differential hormonal sensitivity in neighboring cells.</a> Genes Dev. 28, 912-920 (2014).</li><li>Vragovic, K., 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=Translatome+analyses+capture+of+opposing+tissue-specific+brassinosteroid+signals+orchestrating+root+meristem+differentiation.">Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.</a> Proc Natl Acad Sci U S A. 112, 923-928 (2015).</li><li>Hacham, 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=Brassinosteroid+perception+in+the+epidermis+controls+root+meristem+size.">Brassinosteroid perception in the epidermis controls root meristem size.</a> Development. 138, 839-848 (2011).</li><li>Gu, 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=Identification+of+a+cellulose+synthase-associated+protein+required+for+cellulose+biosynthesis.">Identification of a cellulose synthase-associated protein required for cellulose biosynthesis.</a> Proc Natl Acad Sci U S A. 107, 12866-12871 (2010).</li><li>Fendrych, 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=Programmed+cell+death+controlled+by+ANAC033/SOMBRERO+determines+root+cap+organ+size+in+Arabidopsis.">Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis.</a> Curr Biol. 24, 931-940 (2014).</li></ol>

Here, we present a method for determination of the accumulation of crystalline cellulose in the different tissues composing the Arabidopsis roots, while maintaining anatomical information. As such, it provides an additional step towards understanding growth processes in plants at a cellular resolution. This method can be also applied for the study of additional plant species and organs.
A number of points must be considered when applying the method. First, crystalline cellulose accumu...

The plant cell wall is a dynamic structure. Growing cells are surrounded by a primary cell wall, the organization of which allows cells to expand. Cells that cease to grow deposit a more rigid secondary wall that enhances the mechanical support of the plant. Both cell walls are composed of cellulose microfibrils embedded in a matrix of polysaccharides of different structures (e.g., hemicellulose and pectin) that vary across the different developmental stage and tissues1,2. Cellulose is synthesized as chains of (1,4)-&#946;-D-glucan that are tightly aligned to form the microfibril of a crystalline structure. Amorphous cellulose refers to the regions where the glucan chains are less ordered. The ratio between the crystalline and the amorphous domains is one parameter thought to affect the mechanical properties of the cell wall, by providing mechanical strength and viscoelastic characteristic, respectively3. Several methods have been developed to detect and quantify the two forms of cellulose arrangement, among them X-ray diffraction and cross polarization/magic angle spinning solid-state NMR4. X-ray diffraction can be used to determine the proportion of crystalline versus amorphous cellulose domains in the sample5. An alternative method uses fractionation of cell wall content into acid-insoluble and acid-soluble material, to distinguish between the crystalline and amorphous cellulose or other polymers, respectively. In this approach, incorporation of labeled glucose ([14C]Glucose) is used to quantify the cellulose6,7. These methods require large volumes of plant material for whole organ analyses, at best, and hence, are inadequately sensitive to tissue-specific variation in cell wall structure. Visualization of cellulose microfibrils at a cellular resolution can be achieved in live imaging studies combined with fluorescent dyes8,9, that can identify changes in the orientation of the cellulose microfibrils. However, these dyes are not used for quantification, they are not specific to crystalline cellulose and may interfere with the normal structure of the cell wall8. Polscope is an imaging technique that relies on the ability of crystalline cellulose to split light beams and retard part of the light10. Light retardation is strongest for microfibrils that lie perpendicularly to the direction of light propagation. For microfibrils with similar orientation, the higher the degree of crystallinity, the larger the light retardance11. Hence, polscope is used to study both the relative levels and orientation of the cellulose microfibrils.
Roots exhibit linear growth, during which cells originating at the stem cell niche, at the tip of the root, undergo a series of cell divisions, before they rapidly expand12. The cells comprising the root expand in a unidirectional (anisotropic) manner, as dictated by small molecule signaling hormones that impact the properties of the cell wall13. Differential responses to hormones, in time and space, provide a means of ensuring balanced organ growth14. Hence, high resolution analysis of cell wall structure can provide important information necessary to better understand the connection between cell type-specific responses to whole organ growth. Here, we report the implementation of polscope to study tissue-specific accumulation of crystalline cellulose in Arabidopsis roots, as observed in high quality anatomical sections. This method recently uncovered cell type-specific accumulation of crystalline cellulose in response to spatial perturbation of hormonal activity15.

<table>
	<tbody>
		<tr>
			<td>Murashige &#38; Skoog (MS)</td>
			<td>Duchefa</td>
			<td>M0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Borax</td>
			<td>LOBA CHEMIE&#160;</td>
			<td>6038</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma</td>
			<td>M-9140</td>
			<td></td>
		</tr>
		<tr>
			<td>Azure</td>
			<td>Sigma</td>
			<td>861065</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Driven 0.22mm PVDF Filter&#160;</td>
			<td>MILLEX-GV</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>EMS</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate</td>
			<td>Sigma</td>
			<td>C-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica kit historesin&#160;</td>
			<td>Leica</td>
			<td>7022 18 500</td>
			<td></td>
		</tr>
		<tr>
			<td>embedding molds</td>
			<td>Agar Scientific</td>
			<td>AGG3530</td>
			<td></td>
		</tr>
		<tr>
			<td>film 100&#181; P.P.C.&#160;</td>
			<td>www.Jolybar.com</td>
			<td></td>
			<td>overhead film</td>
		</tr>
		<tr>
			<td>Knivemaker</td>
			<td>LKB</td>
			<td>7800</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultratome III</td>
			<td>LKB</td>
			<td>8800</td>
			<td>Ultratome</td>
		</tr>
		<tr>
			<td>Shandon immumount&#160;</td>
			<td>Thermo</td>
			<td>9990402</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>light microscope</td>
			<td>Nikon</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr>
			<td>Abrio imaging system</td>
			<td>CRI</td>
			<td>Abrio imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>Abrio V2.2 software</td>
			<td>CRI</td>
			<td>Abrio V2.2 software</td>
			<td></td>
		</tr>
		<tr>
			<td>Open access polarized light image analysis software &#160;</td>
			<td>OPS</td>
			<td>OpenPolScope</td>
			<td><a href="http://www.openpolscope.org/">http://www.openpolscope.org/</a></td>
		</tr>
	</tbody>
</table>

high resolution quantification of crystalline cellulose accumulation in arabidopsis roots to monitor tissue-specific cell wall modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

We study the impact of cell type-specific responses to brassinosteroids (BRs), using the Arabidopsis root as a model organ15-17. When the BR receptor BRI1 is targeted, in the background of bri1 mutant, to a subset of epidermal cells called non-hair cells (Figure 2A,B), it inhibits unidirectional cell expansion of neighboring cells, and whole-root growth15. Polscope analysis was performed to reveal the mechanism underlying this inhib...

Watch this Scientific Journal Video about High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications at JoVE.com

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

The overall goal of this anatomical cross section preparation is to quantify relative crystal and cellulose accumulation at the cellular resolution enabling direct comparisons between the different tissues in the primary Arabidopsis Root. This method can help answer key questions in the plant physiology and development field such as how cellular composition of a given cell type impinges on a whole organs growth. After sterilizing Arabidopsis seed according to the text protocol, spread the sterile seeds on plates containing 0.8%plant agar and 0.5XM medium.With parafilm wrap the plates and cover with aluminum foil. Store the plates at 4 degrees Celsius for one to four days to promote uniform germination. Then transfer the plates to a growth chamber suitable for growing Arabidopsis seedlings orienting the plates vertically to maintain root growth on top of the agar medium Prepare Richardson's solution according to the text protocol Filter through a syringe driven 0.22 micron PVD filter unit before use.Next under a chemical hood prepare 50 milliliters of fixative by combining 38 ml of 0.5M sodium cacodylate with 2.5 ml of glutaraldehyde. Then add 100 microliters of Richardson's solution to the fixation solution to generate the final fixation solution. Use a pre-marked plate, then place 2 to 3 ml of final fixation solution into the wells so that the solution will fully cover the seedlings.Using plastic forceps carefully transfer up to 10 seedlings to each well. Use parafilm to seal the plates before storing in the dark at 4 degrees Celsius for at least one night and up to one week. To dehydrate the fixed seedlings use forceps to pick up the plants by their an transfer them to the wells of new marked six well plates containing increasing concentrations of ethanol for the times listed here.Prepare infiltration medium in a small glass bottle by mixing the contents of one bag of Historesin Activator with 50ml of basic resin liquid. Insert a magnet and stir the solution on stir plate for 20 minutes. Fill the labeled wells of a six well plate with 2 to 3 ml of infiltration medium.Then when the seedlings are fully dehydrated use forceps to transfer them from the 95%ethanol solution to the infiltration medium ensuring that they are fully covered by the medium. Store the samples at 4 degrees Celsius for at least four days to ensure maximum penetration into the plant tissues. To prepare blocks place small labels pencil marked with the sample name inside each well embedding molds.Prepare embedding medium by adding 1ml of hardener to 15ml of infiltration medium. With about 200 microliters of embedding medium fill half of each well in the mold. And use an overhead piece of film to cover the mold with an extra 1ml on each side.Incubate the molds at room temperature for at least two hours to allow partial polymerization. Next, prepare an additional batch of embedding medium and keep it on nice to avoid polymerization while root tips are being arranged in the mold. Add embedding medium into a small plate and place one seedling in a medium.Under a dissecting scope use a scalpel to cut each root tip then very carefully position it in the mold about 2 to 3 mm away from the mold periphery either vertically for transfer sections or horizontally for longitudinal sections. After arranging the tissue use embedding medium to fill the mold completely, and cover with an overhead film. Then incubate at room temperature for a minimum of two hours To dry the blocks place them in a desiccator with dry silica gel and leave at room temperature overnight.Using a knife maker equipped with a diamond scoring tool prepare glass knives from glass rods, by cutting the glass road into a square and then cutting the square diagonally to produce two knives. Verify that both knives have sharp edges. Mount a block into a holder.Then with a single edge industrial blade remove the excess block material up to one mm from the root tip to obtain a pyramid shape. Section the shaped block into 2 to 3 micron width slices and transfer the slices to a marked glass slide. Then add water droplets on the slices.It is important to prepare sections that are uniform in width to minimize variability of analyzed data. Place the glass slides on the heat blocks set to low heat until the water evaporates. To prepare slides for viewing under a polarized microscope dilute Richardson's solution to 2%of the original solution and use about 1ml to cover the slices.Then place the samples on the heat block for 5 seconds and use distilled water to wash them. After drying the slides on the hot plate observe the slices under a dissecting microscope and use a marker to mark the back of a slide to indicate the position of the slices of interest. Add 100 microliters of mounting medium before covering with the cover slip.Image and analyze according to the text protocol. Shown here is a cross section of the Arabidopsis primary root with radial organization of its constituent cells and tissues including non-hair cells, hair cells and cortex. This confocal image shows BRI1-GFP expression in non-hair cells in a BRI1 mutant background which inhibits the unidirectional expansion of neighboring cells and whole root growth.As seen here longitudinal sections of the roots obtained from wild type and from lines expressing BRI1 in non-hair cells displayed a similar orientation of cellulose microfibrils with a high variability of angles present in meristematic cells as compared to cells in the transition zone. This enables comparison of relative accumulation of crystal in cellulose in the corresponding meristematic and elongating cells of the two genetic backgrounds as show here in cross sections and reveals a correlation between BRI1 expression in non-hair cells and high local accumulation of crystal in cellulose. Generally in the videos new to this method will struggle because it is challenging to obtain well oriented roots during the embedding procedure.Therefore several blocks should be prepared for sectioning. Once mastered, a single block section can be done in 2 to 3 hours. While attempting this procedure it is important to remember that the accumulation of crystal in cellulose is comparable among cells if they have similar orientation of its cellulose microfibrils, as determined by the longitudinal sections.After watching this video you should have a good understanding of how to fix Arabidopsis seedlings and how embed them in blocks and how to then prepare longitudinal and transverse root cross sections. You should also have a good insight into the data that can be obtained from using polarized light microscopy.

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Research

JoVE Journal

Biology

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...