We would like to thank the PLATIM team for their technical support, as well as Arezki Boudaoud and members of the Biophysic team at the RDP lab for helpful discussions.

<ol><li>Du, F., Guan, C., Jiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanisms+of+leaf+morphogenesis%5BTitle%5D)+AND+%22Molecular+Plant%22%5BJournal%5D)">Molecular mechanisms of leaf morphogenesis.</a> Molecular Plant. 11, 1117-1134 (2018).</li>
<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+of+the+plant+cell+wall%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Growth of the plant cell wall.</a> Nature Reviews Molecular Cell Biology. 6, 850-861 (2005).</li>
<li>Dumais, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Can+mechanics+control+pattern+formation+in+plants%3F%5BTitle%5D)+AND+%22Current+Opinion+in+Plant+Biology%22%5BJournal%5D)">Can mechanics control pattern formation in plants?</a> Current Opinion in Plant Biology. 10, 58-62 (2007).</li>
<li>Smyth, D. R., Bowman, J. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+flower+development+in+Arabidopsis%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">Early flower development in Arabidopsis.</a> The Plant Cell. 2, 755-767 (1990).</li>
<li>Routier-Kierzkowska, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+force+microscopy+for+in+vivo+measurements+of+plant+tissue+mechanics%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">Cellular force microscopy for in vivo measurements of plant tissue mechanics.</a> Plant Physiology. 158 (4), 1514-1522 (2012).</li>
<li>Corson, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Turning+a+plant+tissue+into+a+living+cell+froth+through+isotropic+growth%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Turning a plant tissue into a living cell froth through isotropic growth.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 8453-8458 (2009).</li>
<li>Hervieux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mechanical+feedback+restricts+sepal+growth+and+shape+in+Arabidopsis%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">A mechanical feedback restricts sepal growth and shape in Arabidopsis.</a> Current Biology. 26, 1019-1028 (2016).</li>
<li>Stanislas, T., Hamant, O., Traas, J. Chapter 11 - In-vivo analysis of morphogenesis in plants. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aStanislas%2C+T+%22Methods+in+Cell%22">Methods in Cell</a>. Lecuit, T. 139, Academic Press. 203-223 (2017).</li>
<li>Beauzamy, L., Derr, J., Boudaoud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+hydrostatic+pressure+in+plant+cells+using+indentation+with+an+atomic+force+microscope%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Quantifying hydrostatic pressure in plant cells using indentation with an atomic force microscope.</a> Biophysical Journal. 108 (10), 2448-2456 (2015).</li>
<li>Costa, K. D., Sim, A. J., Yin, F. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-Hertzian+Approach+to+Analyzing+Mechanical+Properties+of+Endothelial+Cells+Probed+by+Atomic+Force+Microscopy%5BTitle%5D)+AND+%22Journal+of+Biomechanical+Engineering%22%5BJournal%5D)">Non-Hertzian Approach to Analyzing Mechanical Properties of Endothelial Cells Probed by Atomic Force Microscopy.</a> Journal of Biomechanical Engineering. 128 (2), 176-184 (2006).</li>
<li>Beauzamy, L., Louveaux, M., Hamant, O., Boudaoud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanically%2C+the+shoot+apical+meristem+of+Arabidopsis+behaves+like+a+shell+inflated+by+a+pressure+of+about+1MPa%5BTitle%5D)+AND+%22Frontiers+in+Plant+science%22%5BJournal%5D)">Mechanically, the shoot apical meristem of Arabidopsis behaves like a shell inflated by a pressure of about 1MPa.</a> Frontiers in Plant science. 6 (1038), 1-10 (2015).</li>
<li>Majda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanochemical+polarization+of+contiguous+cell+walls+shapes+plant+pavement+cells%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Mechanochemical polarization of contiguous cell walls shapes plant pavement cells.</a> Developmental Cell. 43 (3), 290-304 (2017).</li>
<li>Torode, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Branched+pectic+galactan+in+phloem-sieve-element+cell+walls%3A+implications+for+cell+mechanics%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">Branched pectic galactan in phloem-sieve-element cell walls: implications for cell mechanics.</a> Plant Physiology. 176, 1547-1558 (2018).</li>
<li>Farahi, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasticity%2C+elasticity%2C+and+adhesion+energy+of+plant+cell+walls%3A+nanometrology+of+lignin+loss+using+atomic+force+microscopy%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Plasticity, elasticity, and adhesion energy of plant cell walls: nanometrology of lignin loss using atomic force microscopy.</a> Scientific Reports. 7, 152(2017).</li>
<li>Peaucelle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pectin-induced+changes+in+cell+wall+mechanics+underlie+organ+initiation+in+Arabidopsis%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis.</a> Current Biology. 21, 1720-1726 (2011).</li>
<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffuse+growth+of+plant+cell+walls%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">Diffuse growth of plant cell walls.</a> Plant Physiology. 176, 16-27 (2018).</li>
<li>Sader, J. E., Larson, I., Mulvaney, P., White, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+the+calibration+of+atomic+force+microscope+cantilevers%5BTitle%5D)+AND+%22Review+of+Scientific+Instruments%22%5BJournal%5D)">Method for the calibration of atomic force microscope cantilevers.</a> Review of Scientific Instruments. 66 (7), 3789-3798 (1995).</li>
<li>Sader, J. E., Chon, J. W. M., Mulvaney, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calibration+of+rectangular+atomic+force+microscope+cantilevers%5BTitle%5D)+AND+%22Review+of+Scientific+Instruments%22%5BJournal%5D)">Calibration of rectangular atomic force microscope cantilevers.</a> Review of Scientific Instruments. 70 (10), 3967-3969 (1999).</li>
<li>Sikora, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+Normal+Force+Measurements+by+Means+of+Atomic+Force+Microscopy+Towards+the+Accurate+and+Easy+Spring+Constant+Determination%5BTitle%5D)+AND+%22Nanoscience+and+Nanometrology%22%5BJournal%5D)">Quantitative Normal Force Measurements by Means of Atomic Force Microscopy Towards the Accurate and Easy Spring Constant Determination.</a> Nanoscience and Nanometrology. 2 (1), 8-29 (2016).</li>
<li>Schillers, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardized+Nanomechanical+Atomic+Force+Microscopy+Procedure+%28SNAP%29+for+Measuring+Soft+and+Biological+Samples%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples.</a> Scientific Reports. 7 (1), (2017).</li>
</ol>

The authors have nothing to disclose.

The emergence of shapes in plants is mainly determined by the coordinated rate and direction of growth during time and space. Plant cells are encased in a rigid cell wall made of a polysaccharidic matrix, which glues them together. As a result, cell expansion is controlled by the equilibrium between turgor pressure pulling on the cell wall, and stiffness of the cell wall resisting to this pressure. In order to understand the mechanisms underlying development, it is important to be able to measure both cell wall mechanica...

Atomic force microscopy (AFM) belongs to the scanning probe microscopy (SPM) family, where a tip with a radius of usually a few nanometers scans the surface of a sample. The detection of a surface is not achieved via optical or electron-based methods, but via the interaction forces between the tip and the sample surface. Thus, this technique is not limited to topographic characterization of a sample surface (3D resolution which can go down to a few nanometers), but also allows the measurement of any type of interaction forces such as electrostatic, van der Waals or contact forces. Furthermore, the tip can be used to apply forces at the surface of a biological sample and measure the resulting deformation, the so-called &#34;indentation&#34;, in order to determine its mechanical properties (e.g., Young&#39;s modulus, viscoelastic properties).
Mechanical properties of plant cell walls are essential to be taken into account when trying to understand mechanisms underlying developmental processes1,2,3. Indeed, these properties are tightly controlled during development, in particular since cell wall softening is required to allow cells to grow. AFM can be used to measure these properties and study the way they change between organs, tissues or developmental stages.
In this paper, we describe how we use AFM to measure both cell wall mechanical properties and turgor pressure. These two applications are demonstrated on two different AFM microscopes and are detailed here after.

<table><tbody><tr><td>&lt;strong&gt;Growth medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1,000x vimatin stock solution</td><td></td><td></td><td>used to make ACM, composition see Stanislas et al., 2017. Add to ACM after autoclaving, before pouring.</td></tr><tr><td>1-N-Naphthylphthalamic acid (NPA)</td><td>Sigma-Aldrich/Merck</td><td>132-66-1</td><td>add to Arabidopsis medium, 10 &amp;mu;M. Add after autoclaving, before pouring.</td></tr><tr><td>Agar-agar</td><td>Sigma-Aldrich/Merck</td><td>9002-18-0</td><td>add to Arabidopsis medium, 1% w/v.</td></tr><tr><td>Agarose</td><td>Merck Millipore</td><td>9012-36-6</td><td>used to make solid ACM, 0.8% w/v.</td></tr><tr><td>&lt;em&gt;Arabidopsis&lt;/em&gt; medium</td><td>Duchefa Biochimie</td><td>DU0742.0025</td><td>For in vitro arabidopsis culture, 11.82g/L.</td></tr><tr><td>Calcium nitrate tetrahydrate</td><td>Sigma-Aldrich/Merck</td><td>13477-34-4</td><td>add to Arabidopsis medium, 2 mM.</td></tr><tr><td>MURASHIGE &amp;amp; SKOOG MEDIUM</td><td>Duchefa Biochimie</td><td>M0221.0025</td><td>Basal salt mixture, used to make ACM, 2.2 g/ L.</td></tr><tr><td>N6-benzyladenine (BAP)</td><td>Sigma-Aldrich/Merck</td><td>1214-39-7</td><td>used to make ACM, 555 nM. Add to ACM after autoclaving, before pouring.</td></tr><tr><td>Oryzalin</td><td>Sigma-Aldrich/Merck</td><td>19044-88-3</td><td>for oryzalin treatement, 10 &amp;mu;g/mL.</td></tr><tr><td>Plant preservation mixture (PPM)</td><td>Plant Cell Technology</td><td></td><td>used to make ACM, 0.1% v/v. Add to ACM after autoclaving, before pouring.</td></tr><tr><td>Potassium hydroxide</td><td>Duchefa Biochimie</td><td>1310-58-3</td><td>used to make Arabidopsis medium and ACM, both pH 5.8.</td></tr><tr><td>Sucrose</td><td>Duchefa Biochimie</td><td>57-50-1</td><td>used to make ACM, 1% w/v.</td></tr><tr><td>&lt;strong&gt;Tools for AFM&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BioScope Catalyst BioAFM</td><td>Bruker</td><td></td><td>The AFM used for turgor pressure measurement in this protocol.</td></tr><tr><td>Nanowizard III + CellHesion</td><td>JPK (Bruker)</td><td></td><td>The AFM used for measuring mechanical properties.</td></tr><tr><td>Patafix</td><td>UHU</td><td>D1620</td><td></td></tr><tr><td>Reference elasitic structure</td><td>NanoIdea</td><td>2Z00026</td><td></td></tr><tr><td>Reprorubber-Thin Pour</td><td>Flexbar</td><td>16135</td><td>biocompatible glue.</td></tr><tr><td>Spherical AFM tips</td><td>Nanoandmore</td><td>SD-SPHERE-NCH-S-10</td><td>Tips used for measuring mechanical properties.</td></tr></tbody></table>

use of atomic force microscopy to measure mechanical properties and turgor pressure of plant cells and plant tissues

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.

Figure 1A and Figure 1B show a screenshot illustrating the result of the steps 1.3.4 to 1.3.6 of the protocol, used to locate a region of interest where to acquire the QI map. It is worth mentioning that the region of interest has been chosen in order not to be on a tilted surface (i.e., as flat as possible). Actually, as noticed by Routier et al.5, if the indentation axis is not perpendicular to the surfa...

Watch this Scientific Journal Video about Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues at JoVE.com

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...

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Research

JoVE Journal

Developmental Biology

11.4K Views.  Université de Lyon. Mechanische eigenschappen van plantencellen zijn essentieel om rekening mee te houden bij het begrijpen van mechanismen die aan de ontwikkeling ten grondslag liggen. Atoomkracht microscopie kan worden gebruikt om deze eigenschappen te meten, en volg de manier waarop ze veranderen, tussen organen, weefsels, alle ontwikkelingsstadia. De belangrijkste voordelen van deze techniek, is dat het geen invasieve, het is relatief snel en het vereist geen behandeling, zodat het direct kan worden toegepas...

Video: Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) and the moment of the radiation of Angiosperms. This led to the hypothesis that alterations in GMF polarity may play a role in plant evolution. Here, we describe a method to test this hypothesis by exposing Arabidopsis thaliana to artificially reversed GMF conditions. We used a three-axis magnetometer and the collected data were used to calculate the magnitude of the GMF. Three DC power supplies were connected to three Helmholtz coil pairs and were controlled by a computer to alter the GMF conditions. Plants grown in Petri plates were exposed to both normal and reversed GMF conditions. Sham exposure experiments were also performed. Exposed plants were photographed during the experiment and images were analyzed to calculate root length and leaf areas. Arabidopsis total RNA was extracted and Quantitative Real Time-PCR (qPCR) analyses were performed on gene expression of CRUCIFERIN 3 (CRU3), copper transport protein1 (COTP1), Redox Responsive Transcription Factor1 (RRTF1), Fe Superoxide Dismutase 1, (FSD1), Catalase3 (CAT3), Thylakoidal Ascorbate Peroxidase (TAPX), a cytosolic Ascorbate Peroxidase1 (APX1), and NADPH/respiratory burst oxidase protein D (RbohD). Four different reference genes were analysed to normalize the results of the qPCR. The best of the four genes was selected and the most stable gene for normalization was used. Our data show for the first time that reversing the GMF polarity using triaxial coils has significant effects on plant growth and gene expression. This supports the hypothesis that GMF reversal contributes to inducing changes in plant development that might justify a higher selective pressure, eventually leading to plant evolution.

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Bioengineering

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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.

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.

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

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Biology

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

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