Vertical uniformity of cells and nuclei in epithelial monolayers

Abstract

Morphological variability in cytoskeletal organization, organelle position and cell boundaries is a common feature of cultured cells. Remarkable uniformity and reproducibility in structure can be accomplished by providing cells with defined geometric cues. Cells in tissues can also self-organize in the absence of directing extracellular cues; however the mechanical principles for such self-organization are not understood. We report that unlike horizontal shapes, the vertical shapes of the cell and nucleus in the z-dimension are uniform in cells in cultured monolayers compared to isolated cells. Apical surfaces of cells and their nuclei in monolayers were flat and heights were uniform. In contrast, isolated cells, or cells with disrupted cell-cell adhesions had nuclei with curved apical surfaces and variable heights. Isolated cells cultured within micron-sized square wells displayed flat cell and nuclear shapes similar to cells in monolayers. Local disruption of nuclear-cytoskeletal linkages resulted in spatial variation in vertical uniformity. These results suggest that competition between cell-cell pulling forces that expand and shorten the vertical cell cross-section, thereby widening and flattening the nucleus, and the resistance of the nucleus to further flattening results in uniform cell and nuclear cross-sections. Our results reveal the mechanical principles of self-organized vertical uniformity in cell monolayers.

Figure 1. Uniform vertical shapes of nuclei and cells in breast epithelial monolayers.

(A) Shows the x-y view of MCF 10A cells in a monolayer. Shown in (B) is the x-z view of the corresponding nuclei labelled in (A). Similarly (C) shows the x-y view of isolated MCF 10A cells and (D) shows the x-z view of the corresponding nuclei. (E) Histograms compare the frequency distribution of nuclear heights of cells in monolayers and in isolated cells. Variance in nuclear heights is higher in isolated cells than in monolayers (also confirmed by an F-test in Table 1), n = 63 for cells in monolayers and n = 42 for isolated cells (F) x-z views of nuclei, F-actin and overlays. Cell shapes in monolayers are flat with the nucleus tightly enclosed by the cell shape. Isolated cells do not have flat apical surfaces. Scale bar is 10 μm for all images.

Figure 2. E-cadherin adhesions but not myosin activity is required to maintain nuclear uniformity.

(A) Plot shows that the cells spread less in monolayers than in isolated cells.*P < 0.05. (B) Plot shows that the nucleus is flatter in the monolayer compared to isolated cells. *P < 0.05, ^#P < 0.05, for all the indicated comparisons, n = 63 for cells in monolayers and n = 42 for isolated cells. Values are the mean ± SEM. (C) No correlation is observed between nuclear height or aspect ratio and the area of cell spreading in monolayers. (D) x-z views of cells and nuclei in monolayers, monolayers treated with Cytochalasin-D (Cyto-D), anti E-cadherin (E-cadherin inhibited) or ML-7. Nuclear shapes lose their uniform flat appearance in cytochalasin-D treated and E-cadherin inhibited monolayers. The nuclei in ML-7 treated monolayers showed no change in shape. F-actin stained cell shapes are seen to closely correlate with nuclear shape. Scale bar is 10 μm in all images. (E) Frequency distribution of the nuclear heights for control, DMSO control, Cyto-D treated, E-cadherin inhibited and ML-7 treated cells. Cyto-D treatment and E-cadherin inhibition increases variance in nuclear shapes (also see F-test in Table 1).

Figure 3. Vertical cell shape determines vertical nuclear shape.

(A) x-y and corresponding x-z view of a cell cultured in a 5 micron deep square well (left) and corresponding outlines (right). Cells crawl up the sides as seen in the x-z view, resulting in flat x-z cell cross-sections. The nucleus is seen to be correspondingly flat. (B) Nuclei in wells are flatter than in isolated cells. *P < 0.05, ^#P < 0.05, n = 16 for wells and n >40 for isolated cells. Values are the mean ± SEM. (C) The frequency distribution of nuclear heights of isolated cells in wells is narrower than of isolated cells on flat surfaces (see also Table 1), n = 16 for wells and n >40 for isolated cells. (D) x-y and corresponding x-z views of cells and nuclei at the edge of a monolayer. Boxed insets are magnified in the x-z view on the right along with sketched outlines. Apical surfaces of cells and nuclei at the edge of the monolayer are curved. Scale bar for all images is 10 μm.

Figure 4. Local disruption of vertical uniformity.

(A) x-y and x-z view of cells in monolayers transfected with GFP-KASH4. Nucleus ‘a’ and ‘h’ are GFP-KASH4 expressing cells, ‘b’ and ‘g’ are non-transfected cells adjacent to KASH4 expressing cells and, ‘c–f’ are cells farther away from the KASH4 expressing cells(x-y view of the GFP-KASH4 ring and outlines are shown). The nucleus is taller in KASH4 expressing cells and also in immediately adjacent cells, but is flatter farther away. Scale bar is 10 μm for all images. (B) Statistical comparison of nuclear heights of KASH4 expressing cells, adjacent cells and cells farther away (distant). *P < 0.01, n >20. Values are the mean ± SEM. (C) Frequency distribution of nuclear heights in GFP-KASH4 expressing cells (KASH4), cells adjacent to KASH4 cells (adjacent) and cells farther away from the KASH4 cells (distant). Nuclei in cells adjacent and farther away had significantly lower variance compared to KASH4 cells, n >20.

Figure 5. Cartoon depicting the model for nuclear shape in isolated cells and in monolayers.

Nuclei are pulled laterally by tensile forces generated during lateral expansion (indicated by ‘tension lines’ in the cell). The simultaneous vertical compression of the cell reduces the nucleus height. Nuclei flatten until the excess lamina is fully unfolded then further flattening is resisted as it requires nuclear volume compression. (I) For isolated cells, the amount of tension is higher toward the cell base where lateral expansion is greatest, resulting in a concave apical surface. (II) For cells in a monolayer, cell-cell pulling expands cells more uniformly across the vertical dimension, resulting in a more rectangular cross-section, thus a shorter profile for the same nuclear volume. The pulling forces between cells cause the cell heights at the cell-cell junctions to be continuous in both position and slope; this enforces local uniformity in cell heights. (III) For a cell in a microfabricated well, the nucleus flattens as the cell spreads laterally then up the walls of the well. The forces on the nucleus and the resulting flat apical surface of the cell and nucleus are similar to those of cells in monolayers. (IV) If one cell in the monolayer cannot generate tension on the nucleus, as with KASH4-tranfected cells, the nucleus will resist lateral expansion, resulting in a taller nucleus. The cell-cell pulling forces enforce continuity thereby causing the nearby cells to be similar in height.