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

Abstract

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.

Towards an understanding of spiral patterning in theSargassum muticumshoot apex

“…Based on these data, we could suggest that the area around the apical cell and the apical cell itself have stiffer walls. This is similar to the situation observed in plants, where the stem cells have been shown to be stiffer than the surrounding peripheral cells (Milani et al, 2014). It may be interesting to explore whether this increased stiffness regulates cell division rates, in both plants and algae.…”

“…In plants, new organs form in the peripheral zone after auxin maxima lead to wall softening (Braybrook and Peaucelle, 2013). The central zone, containing the meristematic stem cells, exhibits stiffer cell walls than the peripheral zone or new primordia (Milani et al, 2014). In most walled organisms, it is assumed that cell growth is limited by the cell wall and its mechanical properties, in turn linked to its biochemical composition (Peaucelle et al, 2013; Braybrook and Jönsson, 2016).…”

“…This niche serves as a reservoir for production of cells which then give rise to the lateral organs (Steeves and Sussex, 1989; Meyerowitz, 1997). Phyllotactic patterning occurs independent from division patterns within the meristematic niche and evidence exists for a position/morphogen-based patterning mechanism: organs emerge due to local softening of tissues at specific positions at the shot apex (Braybrook and Peaucelle, 2013; Milani et al, 2014); local maxima of the phytohormone auxin dictate the position of new organs (Reinhart et al, 2003); auxin maxima are positioned by the cell-cell dynamics of polar auxin transporters whose direction relates to auxin concentration of neighbouring cells (Reinhart et al, 2003); stochastic fluctuations in auxin concentration can lead to coordinated polarisation of auxin transporters and result in a self-organising pattern of organs (Jönsson et al, 2006). Here again, ablation of the meristematic niche leads to re-establishment of a new niche and organised phyllotaxis about the new centre lending weight to a robust self-organising mechanism rooted in the morphogen auxin (Steeves and Sussex, 1989; Reinhardt et al, 2003).…”

Diffuse Growth of Plant Cell Walls

“…Peaucelle et al, 2011;Milani et al, 2014;Sampathkumar et al, 2014a). This is a complex topic beyond the scope of this review, but readers are referred to reviews that assess the varied approaches and interpretations of these stiffness measurements (Milani et al, 2013;Mosca et al, 2017).…”

Gradient in cytoplasmic pressure in the germline cells controls overlying epithelial cell morphogenesis

“…Second, as cytoplasmic pressure contributes to tissue stiffness, we used an Atomic Force Microscope (AFM), a standard approach to measure non-invasively stiffness of biological samples (Beauzamy et al, 2015;Kim et al, 2014;Milani et al, 2014). The stiffness quantifies how easily a body is deformed when a force is applied to it; a high value corresponds to a stiff material.…”