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.

Figure 1.

Experimental setup for determining stiffness patterns. A and B, Photograph (A) and schematic (B) of the customized coupling of an AFM to an upright epifluorescence macroscope that was used to image specific regions within dissected shoot apices. C, Fluorescence image of a pCLV3::GFPer Arabidopsis inflorescence, as viewed with the macroscope. The cantilever of the AFM appears as a long, dark rectangular object above the sample. D, Closeup of a different pCLV3::GFPer inflorescence, with the SAM and stage 2 (FM2) and stage 3 (FM3) floral meristems indicated. E, Plot of the Ea, extracted from the AFM force-displacement curves, as a function of the position along the arrow shown in D, with the fluorescent region highlighted in green. Bars = 50 µm in C and 15 µm in D.

Figure 2.

Mechanical measurements and cellular quantification of stiffness. A, Typical force-tip position approach (blue) and retraction (red) curves obtained on a SAM with a spherical probe tip. The retraction curve (red) is fitted using the DMT model (black dotted line) to obtain the value of the Ea. B, Diagrammatic representation of the AFM tip indenting an epidermal cell in the SAM, either on an outer (periclinal) wall or on walls normal to the surface (anticlinal). When indentation depth is greater than wall thickness, periclinal walls are expected to bend more and to appear softer than anticlinal walls. C and D, Analysis of stiffness in the SAM from a pCLV3::GFPer plant. Shown is the global map of the Ea of a region of the SAM (C), delimited by the white outline in the surface projection of the confocal image (D), with the white dots serving as reference landmarks and the arrows indicating stage 1 and 2 primordia. The plant was stained with the FM4-64 dye to detect cell contours (in red), while GFP expression is shown in green. E and F, Quantification of stiffness maps. Maps of anticlinal (E) and periclinal (F) walls were reconstituted after segmentation of one of the AFM stiffness maps from the global map shown in C (Supplemental Fig. S2). The dotted lines separate the GFP+ (CLV3-expressing zone) and GFP− regions, which are indicated with + and −. G and H, Box plots for anticlinal (G) and periclinal (H) walls obtained by analysis of the stiffness maps in E and F, respectively. The boxes extend from the first quartile to the third quartile and the whiskers from 10% to 90% of all the data set. Pixels were separated into two groups, GFP+ and GFP−. The number of pixels analyzed in each group is indicated. The ratio of median Ea values of GFP+ and GFP− is shown. Bars = 20 µm in C and D and 5 µm in E and F.

Figure 3.

Cell fate correlates with a higher stiffness in the floral meristem. A, Surface projection from a confocal image of a stage 3 flower carrying the pCLV3::GFPer reporter, showing CLV3 expression (green) and cell outlines (red). B, Global map of the Ea of the framed region of the same stage 3 flower (A), with white dots serving as landmarks. C, Arrangement of the individual stiffness maps that constitute the global stiffness map of the flower shown in B, with each map numbered and positioned from front (map 1) to back (map 5; Supplemental Fig. S3). D, Pixel-level quantification of the individual maps (left column) and their corresponding box-plot representations (right column), summarizing the Ea distributions of GFP+ (CLV3-expressing zone) and GFP− anticlinal pixels. In each map, the white dotted line separates the GFP+ and GFP− regions, which are indicated by + and −, respectively. The numbers of analyzed pixels and median Ea ratios are indicated in the box plots, which are constructed as in Figure 2. Bars = 20 µm in A and B.

Figure 4.

The stiffness pattern is established dynamically along with GFP expression. Analysis of stiffness in a single pCLV3::GFPer flower was imaged at 0 h (A, C, E, and G) and 24 h (B, D, F, and H). A and B, Projections of confocal images of the whole inflorescence observed, with white frames showing the stage 2 flower studied. C and D, Closeups of the stage 2 flower, with GFP expression visible at 24 h (D) but not at 0 h (C). Yellow and green asterisks indicate lineage for two cells within the emerging CZ. E and F, Global Ea maps of the same flower, with white frames delimiting the individual stiffness maps. G and H, Pixel-level quantification of the individual maps, summarizing the Ea distributions of anticlinal pixels from GFP−, GFP+, and 0-h mother cells of 24-h GFP+ cells. The numbers of analyzed pixels and median Ea ratios are indicated in the box plots, which are constructed as in previous figures. Bars = 50 µm in A and B and 20 µm in C to F.

Figure 5.

Distribution of Ea for all the flowers that were analyzed quantitatively. Box-plot representations of the Ea distribution for all maps and groups (GFP+ and GFP−) of each flower are shown. The boxes extend from the first quartile to the third quartile and the whiskers from 10% to 90% of each data set; the number of analyzed pixels is indicated. The ratio between median Ea of GFP+ and GFP− and the corresponding P values (Wilcoxon test; see “Materials and Methods”) are shown where appropriate; the Ea ratio of mother cells of GFP+ cells to mother cells of GFP− cells is also specified for flower II⁻₄ (Fig. 4G). [See online article for color version of this figure.]