Matching Patterns of Gene Expression to Mechanical Stiffness at Cell Resolution through Quantitative Tandem Epifluorescence and Nanoindentation

Cell differentiation has been associated with changes in mechanical stiffness in single-cell systems, yet it is unknown whether this association remains true in a multicellular context, particularly in developing tissues. In order to address such questions, we have developed a methodology, termed quantitative tandem epifluorescence and nanoindentation, wherein we sequentially determine cellular genetic identity with confocal microscopy and mechanical properties with atomic force microscopy. We have applied this approach to examine cellular stiffness at the shoot apices of Arabidopsis (Arabidopsis thaliana) plants carrying a fluorescent reporter for the CLAVATA3 (CLV3) gene, which encodes a secreted glycopeptide involved in the regulation of the centrally located stem cell zone in inflorescence and floral meristems. We found that these CLV3-expressing cells are characterized by an enhanced stiffness. Additionally, by tracking cells in young flowers before and after the onset of GREEN FLUORESCENT PROTEIN expression, we observed that an increase in stiffness coincides with this onset. This work illustrates how quantitative tandem epifluorescence and nanoindentation can reveal the spatial and temporal dynamics of both gene expression and cell mechanics at the shoot apex and, by extension, in the epidermis of any thick tissue.Morphogenesis is a complex process that results from the coordinated actions of many genes and gene products across developing tissues and organs. Because shape is a function of the structural elements of cells, the molecular and genetic control of growth and morphogenesis must rely on the regulation of the mechanics of these elements. In this context, cell differentiation has been linked with mechanical stiffness in animal single-cell systems (Collinsworth et al., 2002; Balland et al., 2006; Engler et al., 2006; Darling et al., 2008), although the direct measurement of cell mechanics in growing animal tissues remains elusive (Blanchard and Adams, 2011; Davidson, 2011).In plants, growth involves a delicate mechanical balance: it is powered by turgor pressure and contained by cell wall stiffness (Cosgrove, 1986). Several groups have recently achieved mechanical measurements made at a subcellular resolution in plants (Milani et al., 2011; Peaucelle et al., 2011; Fernandes et al., 2012; Radotić et al., 2012; Routier-Kierzkowska et al., 2012) using scaled-down indentation methods (Geitmann, 2006; Hayot et al., 2012; Milani et al., 2013; Routier-Kierzkowska and Smith, 2013), wherein one quantifies the force needed to push down on a sample to a prescribed depth. These studies have revealed spatiotemporal patterns of stiffness, notably in tissues (Milani et al., 2011; Peaucelle et al., 2011; Fernandes et al., 2012; Routier-Kierzkowska et al., 2012).However, these measurements have not been associated directly with cell identity. This association would become feasible if mechanical measurements were combined with optical imaging of fluorescent reporters. Such a combination, termed nanoindentation coupled to inverted optical microscopy, has already been developed for single animal cells and for thin plant tissues, (Rotsch and Radmacher, 2000; Routier-Kierzkowska and Smith, 2014), but it cannot be extended to thick tissues because they are opaque, making it impossible to simultaneously observe the tissue surface optically with an inverted microscope and probe it mechanically. To circumvent this difficulty, we have developed a methodology involving the use of three microscopes to image the same sample: (1) an atomic force microscope (AFM), which is a nanoindentation system for obtaining stiffness maps of the surface of a sample; (2) an AFM-coupled upright epifluorescence macroscope to precisely identify the points to be probed; and (3) a confocal microscope to determine cell fate at cellular resolution, which may in turn be correlated with the stiffness maps. We call this methodology quantitative tandem epifluorescence and nanoindentation (qTEN), and we use it to probe the shoot apical meristem (SAM) of Arabidopsis (Arabidopsis thaliana), which is a good model system in which to investigate morphogenesis.The SAM is located at the growing tip of the shoot and consists of distinct functional zones (Ha et al., 2010). One of these zones is the slow-dividing central zone (CZ), which can be defined by the expression of the CLAVATA3 (CLV3) signaling glycopeptide. Through cell division, cells exit the CZ into the surrounding peripheral zone (PZ). In the PZ, cells proliferate rapidly, and some become incorporated into organ primordia, thus yielding all aerial organs of the plant. Recent work on the SAM has revealed patterns of mechanical properties (Milani et al., 2011; Peaucelle et al., 2011; Kierzkowski et al., 2012; Braybrook and Peaucelle, 2013), but it is still unclear how these patterns are related to the activity of the SAM or to its functional zonation. Here, we analyze the dynamics of such a mechanical pattern in vivo and show that it is spatially and temporally related to stem cell fate.