Pascale Milani

In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy.

Whereas the morphogenesis of developing organisms is relatively well understood at the molecular level, the contribution of the mechanical properties of the cells to shape changes remains largely unknown, mainly because of the lack of quantified biophysical parameters at cellular or subcellular resolution. Here we designed an atomic force microscopy approach to investigate the elastic modulus of the outer cell wall in living shoot apical meristems (SAMs). SAMs are highly organized structures that contain the plant stem cells, and generate all of the aerial organs of the plant. Building on modeling and experimental data, we designed a protocol that is able to measure very local properties, i.e. within 40-100 nm deep into the wall of living meristematic cells. We identified three levels of complexity at the meristem surface, with significant heterogeneity in stiffness at regional, cellular and even subcellular levels. Strikingly, we found that the outer cell wall was much stiffer at the tip of the meristem (5 ± 2 MPa on average), covering the stem cell pool, than on the flanks of the meristem (1.5 ± 0.7 MPa on average). Altogether, these results demonstrate the existence of a multiscale spatialization of the mechanical properties of the meristem surface, in addition to the previously established molecular and cytological zonation of the SAM, correlating with regional growth rate distribution.

Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells

Although it is a central question in biology, how cell shape controls intracellular dynamics largely remains an open question. Here, we show that the shape of Arabidopsis pavement cells creates a stress pattern that controls microtubule orientation, which then guides cell wall reinforcement. Live-imaging, combined with modeling of cell mechanics, shows that microtubules align along the maximal tensile stress direction within the cells, and atomic force microscopy demonstrates that this leads to reinforcement of the cell wall parallel to the microtubules. This feedback loop is regulated: cell-shape derived stresses could be overridden by imposed tissue level stresses, showing how competition between subcellular and supracellular cues control microtubule behavior. Furthermore, at the microtubule level, we identified an amplification mechanism in which mechanical stress promotes the microtubule response to stress by increasing severing activity. These multiscale feedbacks likely contribute to the robustness of microtubule behavior in plant epidermis. DOI: http://dx.doi.org/10.7554/eLife.01967.001

Mechanics of the IL2RA gene activation revealed by modeling and atomic force microscopy.

Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static.

An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis

To control morphogenesis, molecular regulatory networks have to interfere with the mechanical properties of the individual cells of developing organs and tissues, but how this is achieved is not well known. We study this issue here in the shoot meristem of higher plants, a group of undifferentiated cells where complex changes in growth rates and directions lead to the continuous formation of new organs. Here, we show that the plant hormone auxin plays an important role in this process via a dual, local effect on the extracellular matrix, the cell wall, which determines cell shape. Our study reveals that auxin not only causes a limited reduction in wall stiffness but also directly interferes with wall anisotropy via the regulation of cortical microtubule dynamics. We further show that to induce growth isotropy and organ outgrowth, auxin somehow interferes with the cortical microtubule-ordering activity of a network of proteins, including AUXIN BINDING PROTEIN 1 and KATANIN 1. Numerical simulations further indicate that the induced isotropy is sufficient to amplify the effects of the relatively minor changes in wall stiffness to promote organogenesis and the establishment of new growth axes in a robust manner.

Shrinking the hammer: micromechanical approaches to morphogenesis

Morphogenesis, the remarkable process by which a developing organism achieves its shape, relies on the coordinated growth of cells, tissues, and organs. While the molecular and genetic basis of morphogenesis is starting to be unravelled, understanding shape changes is lagging behind. Actually, shape is imposed by the structural elements of the organism, and the translation of cellular activity into morphogenesis must go through these elements. Therefore, many methods have been developed recently to quantify, at cellular resolution, the properties of the main structural element in plants, the cell wall. As plant cell growth is restrained by the cell wall and powered by turgor pressure, such methods also address the quantification of turgor. These different micromechanical approaches are reviewed here, with a critical assessment of their strengths and weaknesses, and a discussion of how they can help us understand the regulation of growth and morphogenesis.

Nucleosome positioning by genomic excluding-energy barriers

Recent genome-wide nucleosome mappings along with bioinformatics studies have confirmed that the DNA sequence plays a more important role in the collective organization of nucleosomes in vivo than previously thought. Yet in living cells, this organization also results from the action of various external factors like DNA-binding proteins and chromatin remodelers. To decipher the code for intrinsic chromatin organization, there is thus a need for in vitro experiments to bridge the gap between computational models of nucleosome sequence preferences and in vivo nucleosome occupancy data. Here we combine atomic force microscopy in liquid and theoretical modeling to demonstrate that a major sequence signaling in vivo are high-energy barriers that locally inhibit nucleosome formation rather than favorable positioning motifs. We show that these genomic excluding-energy barriers condition the collective assembly of neighboring nucleosomes consistently with equilibrium statistical ordering principles. The analysis of two gene promoter regions in Saccharomyces cerevisiae and the human genome indicates that these genomic barriers direct the intrinsic nucleosome occupancy of regulatory sites, thereby contributing to gene expression regulation.

Mechanochemical Polarization of Contiguous Cell Walls Shapes Plant Pavement Cells.

The epidermis of aerial plant organs is thought to be limiting for growth, because it acts as a continuous load-bearing layer, resisting tension. Leaf epidermis contains jigsaw puzzle piece-shaped pavement cells whose shape has been proposed to be a result of subcellular variations in expansion rate that induce local buckling events. Paradoxically, such local compressive buckling should not occur given the tensile stresses across the epidermis. Using computational modeling, we show that the simplest scenario to explain pavement cell shapes within an epidermis under tension must involve mechanical wall heterogeneities across and along the anticlinal pavement cell walls between adjacent cells. Combining genetics, atomic force microscopy, and immunolabeling, we demonstrate that contiguous cell walls indeed exhibit hybrid mechanochemical properties. Such biochemical wall heterogeneities precede wall bending. Altogether, this provides a possible mechanism for the generation of complex plant cell shapes.

Effect of Genomic Long-Range Correlations on DNA Persistence Length: From Theory to Single Molecule Experiments

Sequence dependency of DNA intrinsic bending properties has been emphasized as a possible key ingredient to in vivo chromatin organization. We use atomic force microscopy (AFM) in air and liquid to image intrinsically straight (synthetic), uncorrelated (hepatitis C RNA virus) and persistent long-range correlated (human) DNA fragments in various ionic conditions such that the molecules freely equilibrate on the mica surface before being captured in a particular conformation. 2D thermodynamic equilibrium is experimentally verified by a detailed statistical analysis of the Gaussian nature of the DNA bend angle fluctuations. We show that the worm-like chain (WLC) model, commonly used to describe the average conformation of long semiflexible polymers, reproduces remarkably well the persistence length estimates for the first two molecules as consistently obtained from (i) mean square end-to-end distance measurement and (ii) mean projection of the end-to-end vector on the initial orientation. Whatever the operating conditions (air or liquid, concentration of metal cations Mg(2+) and/or Ni(2+)), the persistence length found for the uncorrelated viral DNA underestimates the value obtained for the straight DNA. We show that this systematic difference is the signature of the presence of an uncorrelated structural intrinsic disorder in the hepatitis C virus (HCV) DNA fragment that superimposes on local curvatures induced by thermal fluctuations and that only the entropic disorder depends upon experimental conditions. In contrast, the WLC model fails to describe the human DNA conformations. We use a mean-field extension of the WLC model to account for the presence of long-range correlations (LRC) in the intrinsic curvature disorder of human genomic DNA: the stronger the LRC, the smaller the persistence length. The comparison of AFM imaging of human DNA with LRC DNA simulations confirms that the rather small mean square end-to-end distance observed, particularly for G+C-rich human DNA molecules, more likely results from a large-scale intrinsic curvature due to a persistent distribution of DNA curvature sites than from some increased flexibility.

Variations in basement membrane mechanics are linked to epithelial morphogenesis

The regulation of morphogenesis by the basement membrane (BM) may rely on changes in its mechanical properties. To test this, we developed an atomic force microscopy-based method to measure BM mechanical stiffness during two key processes in Drosophila ovarian follicle development. First, follicle elongation depends on epithelial cells that collectively migrate, secreting BM fibrils perpendicularly to the anteroposterior axis. Our data show that BM stiffness increases during this migration and that fibril incorporation enhances BM stiffness. In addition, stiffness heterogeneity, due to oriented fibrils, is important for egg elongation. Second, epithelial cells change their shape from cuboidal to either squamous or columnar. We prove that BM softens around the squamous cells and that this softening depends on the TGFβ pathway. We also demonstrate that interactions between BM constituents are necessary for cell flattening. Altogether, these results show that BM mechanical properties are modified during development and that, in turn, such mechanical modifications influence both cell and tissue shapes. Summary: Basement membrane fibril composition and stiffness vary temporally and spatially during Drosophila ovarian follicle development, influencing follicle rotation, egg elongation and cuboidal-to-squamous cellular transition dependent on TGFβ signalling.

A gradient in inner pressure of germline cells controls overlaying epithelial cell morphogenesis

It is unknown how growth in one tissue impacts morphogenesis in a neighboring tissue. To address this, we used the Drosophila ovarian follicle, where a cluster of 15 nurse cells and a posteriorly located oocyte are surrounded by a layer of epithelial cells. It is known that as the nurse cells grow, the overlying epithelial cells flatten in a wave that begins in the anterior. Here, we demonstrate that an anterior to posterior gradient of decreasing cytoplasmic pressure is present across the nurse cells and that this gradient acts through TGFß to control both the triggering and the progression of the wave of epithelial cell flattening. Our data indicate that intrinsic nurse cell growth is important to control proper nurse cell pressure. Finally, we reveal that nurse cell pressure and subsequent TGFß activity in the StC combine to increase follicle elongation in the anterior, which is crucial for allowing nurse cell growth and pressure control. More generally, our results reveal that during development, inner cytoplasmic pressure in individual cells has an important role in shaping their neighbors. Impact Statement Cell shape change depends on extrinsic forces exerted by cytoplasmic pressure in neighbouring cells.