Raymond Wightman

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

The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl

Complex shapes in biology depend on the ability of cells to shift from isotropic to anisotropic growth during development. In plants, this growth symmetry breaking reflects changes in the extensibility of the cell walls. The textbook view is that the direction of turgor-driven cell expansion depends on the cortical microtubule (CMT)-mediated orientation of cellulose microfibrils. Here, we show that this view is incomplete at best. We used atomic force microscopy (AFM) to study changes in cell-wall mechanics associated with growth symmetry breaking within the hypocotyl epidermis. We show that, first, growth symmetry breaking is preceded by an asymmetric loosening of longitudinal, as compared to transverse, anticlinal walls, in the absence of a change in CMT orientation. Second, this wall loosening is triggered by the selective de-methylesterification of cell-wall pectin in longitudinal walls, and, third, the resultant mechanical asymmetry is required for the growth symmetry breaking. Indeed, preventing or promoting pectin de-methylesterification, respectively, increased or decreased the stiffness of all the cell walls, but in both cases reduced the growth anisotropy. Finally, we show that the subsequent CMT reorientation contributes to the consolidation of the growth axis but is not required for the growth symmetry breaking. We conclude that growth symmetry breaking is controlled at a cellular scale by bipolar pectin de-methylesterification, rather than by the cellulose-dependent mechanical anisotropy of the cell walls themselves. Such a cell asymmetry-driven mechanism is comparable to that underlying tip growth in plants but also anisotropic cell growth in animal cells.

S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization

Location, location, S-acylation Cellulose synthase is a large, multisubunit machine that “swims” along the plant cell membrane as it spins out cellulose fibers. Kumar et al. show that the cellulose synthase complex is heavily modified through S-acylation. Subsets of the acylation sites were required for the complex to integrate into the plasma membrane. A single functional complex could bear as many as 100 modification sites, potentially changing its biophysical characteristics and helping it to associate with the membrane. Science, this issue p. 166 The large, multisubunit complex that makes cellulose fibers alters its own membrane environment to keep it in the correct location. Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here, we demonstrate that all catalytic subunits of the CSC, known as cellulose synthase A (CESA) proteins, are S-acylated. Analysis of Arabidopsis CESA7 reveals four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment.

Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche

Significance How does a cell decide when to divide or initiate DNA replication? How does it regulate its own growth? These fundamental questions are not well understood in most organisms; this lack of understanding is particularly true for multicellular eukaryotes. Following classical studies in yeast, we have quantified the key aspects of cell growth and division dynamics in the Arabidopsis apical stem cell niche. Our results disprove various theories for plant stem cell size/cell cycle regulation, such as that cell cycle progression is triggered when a prefixed critical size is attained, and constitute the necessary first step in the development of integrative mechanistic theories for the coordinated regulation of cell cycle progression, cell growth, and cell size in plants. Cell size and growth kinetics are fundamental cellular properties with important physiological implications. Classical studies on yeast, and recently on bacteria, have identified rules for cell size regulation in single cells, but in the more complex environment of multicellular tissues, data have been lacking. In this study, to characterize cell size and growth regulation in a multicellular context, we developed a 4D imaging pipeline and applied it to track and quantify epidermal cells over 3–4 d in Arabidopsis thaliana shoot apical meristems. We found that a cell size checkpoint is not the trigger for G2/M or cytokinesis, refuting the unexamined assumption that meristematic cells trigger cell cycle phases upon reaching a critical size. Our data also rule out models in which cells undergo G2/M at a fixed time after birth, or by adding a critical size increment between G2/M transitions. Rather, cell size regulation was intermediate between the critical size and critical increment paradigms, meaning that cell size fluctuations decay by ∼75% in one generation compared with 100% (critical size) and 50% (critical increment). Notably, this behavior was independent of local cell–cell contact topologies and of position within the tissue. Cells grew exponentially throughout the first >80% of the cell cycle, but following an asymmetrical division, the small daughter grew at a faster exponential rate than the large daughter, an observation that potentially challenges present models of growth regulation. These growth and division behaviors place strong constraints on quantitative mechanistic descriptions of the cell cycle and growth control.

Spatio-temporal registration of 3D microscopy image sequences of arabidopsis floral meristems

The shoot apical meristem (SAM) is at the origin of all the plant above-ground organs (including stems, leaves and flowers) and is a biological object of interest for the understanding of plant morphogenesis. The quantification of tissue growth at a cellular level requires the analysis of 3D microscopic image sequences of developing meristems. To address inter-individual variability, it is also required to compare individuals. This obviously implies the ability to process inter-individual registration, i.e. to compute spatial and temporal correspondences between sequences from different meristems. In the present work, we propose a spatial registration method dedicated to microscopy floral meristem (FM) images, and the identification, for a given still image of a meristem, of its best corresponding time-point in a sequence of an other individual (temporal registration).Recent microscopy techniques allow imaging temporal 3D stacks of developing organs or embryos with a cellular level of resolution and with a sufficient acquisition frequency to accurately track cell lineages. Imaging multiple organs or embryos in different experimental conditions may help decipher the impact of genetic backgrounds and environmental inputs on the developmental program. For this, we need to precisely compare distinct individuals and to compute population statistics. The first step of this procedure is to develop methods to register individuals. From a previous work of cell segmentation from microscopy images, we here demonstrate how to extract the symmetry plane of embryos at early stages, and how to use this information as a geometrical constraint to both register these embryos and obtain a cell-to-cell mapping.

Live Cell Imaging of the Cytoskeleton and Cell Wall Enzymes in Plant Cells

The use of live imaging techniques to visualize the dynamic changes and interactions within plant cells has given us detailed information on the function and organization of the cytoskeleton and cell wall associated proteins. This information has grown with the constant improvement in imaging hardware and molecular tools. In this chapter, we describe the procedure for the preparation and live visualization of fluorescent protein fusions associated with the cytoskeleton and the cell wall in Arabidopsis.

Live Imaging of Developing Xylem In Planta

The xylem vessel develops from a long cylindrical cell that deposits a patterned secondary wall and ending with programmed cell death. The dynamic arrangement of cell wall enzymes, membranes and the cytoskeleton can be recorded using live fluorescence imaging. The protocol presented here focuses upon the microscopy of intracellular components in developing vessels of the root using either epifluorescent or confocal microscopes.

A 3-dimensional fibre scaffold as an investigative tool for studying the morphogenesis of isolated plant cells

BackgroundCell culture methods allow the detailed observations of individual plant cells and their internal processes. Whereas cultured cells are more amenable to microscopy, they have had limited use when studying the complex interactions between cell populations and responses to external signals associated with tissue and whole plant development. Such interactions result in the diverse range of cell shapes observed in planta compared to the simple polygonal or ovoid shapes in vitro. Microfluidic devices can isolate the dynamics of single plant cells but have restricted use for providing a tissue-like and fibrous extracellular environment for cells to interact. A gap exists, therefore, in the understanding of spatiotemporal interactions of single plant cells interacting with their three-dimensional (3D) environment. A model system is needed to bridge this gap. For this purpose we have borrowed a tool, a 3D nano- and microfibre tissue scaffold, recently used in biomedical engineering of animal and human tissue physiology and pathophysiology in vitro.ResultsWe have developed a method of 3D cell culture for plants, which mimics the plant tissue environment, using biocompatible scaffolds similar to those used in mammalian tissue engineering. The scaffolds provide both developmental cues and structural stability to isolated callus-derived cells grown in liquid culture. The protocol is rapid, compared to the growth and preparation of whole plants for microscopy, and provides detailed subcellular information on cells interacting with their local environment. We observe cell shapes never observed for individual cultured cells. Rather than exhibiting only spheroid or ellipsoidal shapes, the cells adapt their shape to fit the local space and are capable of growing past each other, taking on growth and morphological characteristics with greater complexity than observed even in whole plants. Confocal imaging of transgenic Arabidopsis thaliana lines containing fluorescent microtubule and actin reporters enables further study of the effects of interactions and complex morphologies upon cytoskeletal organisation both in 3D and in time (4D).ConclusionsThe 3D culture within the fibre scaffolds permits cells to grow freely within a matrix containing both large and small spaces, a technique that is expected to add to current lithographic technologies, where growth is carefully controlled and constricted. The cells, once seeded in the scaffolds, can adopt a variety of morphologies, demonstrating that they do not need to be part of a tightly packed tissue to form complex shapes. This points to a role of the immediate nano- and micro-topography in plant cell morphogenesis. This work defines a new suite of techniques for exploring cell-environment interactions.