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Figures
- Fig. 1.
SINK method and mechanisms of intracellular force propagation reduction. (A) Monolayer of NRK-52E epithelial cells showing two cells expressing GFP-UBF (green); DNA is stained blue with Hoechst 33342. (B) Magnified image of outlined nucleus in A showing only the GFP-UBF channel. (C) Processed frame of B after imaging nucleus at 3 min time intervals for 60 min. Tracks of points are shown as blue overlay. Nuclei were aligned prior to tracking of points. (D) Tracks from all points in all nuclei for the control monolayer data shown on linear coordinates. (E) MSD average of intranuclear movement versus lag time (τ) plotted on log-log coordinates for control cells in a monolayer (green), cells in a monolayer treated with Y-27632 (yellow), and cells in a monolayer transfected with DN-KASH (orange). Error bars represent s.e.m. Data are fitted to a power law of the form shown. (F) Comparison of the force generation exponent (β) for control cells in a monolayer, Y-27632 and DN-KASH monolayers. Reduction of either cell force generation (Y-27632) or nuclear connectivity (DN-KASH) resulted in decreased β. Asterisk denotes significant difference based on the curve fit (P<0.05). Error bars represent 95% confidence interval for the fitting of β. Monolayer control, n=84; Y-27632, n=101; DN-KASH, n=64.
- Fig. 2.
Reduction of intranuclear movement and β in isolated cells versus monolayers. (A) Confocal image of isolated cells and (B) cells in a monolayer, with DNA (blue), and actin (green, phalloidin). Top view (top) and side view (bottom) shown. (C) Image analysis of apical-basal (z-direction) distribution of actin and DNA shows colocalization in the monolayer but separation in the isolated cells. In isolated cells actin lies primarily along the basal region of cells, while in monolayer cells actin is primarily coplanar with the nucleus. (D) MSD of intranuclear movement versus lag time (τ) plotted on log-log coordinates, comparing monolayer (green) versus isolated (blue) cells. Error bars represent s.e.m. (E) Comparison of the force generation (β) for monolayer and isolated cells. *P<0.05 based on the curve fit; error bars represent 95% confidence interval for the fitting of β. Monolayer, n=84; Isolated, n=55.
- Fig. 3.
Effect of substrate stiffness on intranuclear movement via SINK in monolayers. (A) MSD of intranuclear movement versus lag time (τ) plotted on log-log coordinates for cells in monolayers seeded on glass (green), or collagen coated polyacrylamide gels with elastic moduli of 30, 10 or 2.5 kPa. Intranuclear movement increases as substrate stiffness increases. Error bars represent s.e.m. (B) Comparison of the force generation exponent (β) for monolayers of varying elastic moduli for curves shown in A. *P<0.05 based on the curve fit; error bars represent 95% confidence interval for the fitting of β. Glass, n=84; 30 kPa, n=235; 10 kPa, n=287; 2.5 kPa, n=173.
- Fig. 4.
SINK method to measure changes in force in heterogeneous monolayers. (A-C) GFP-UBF (green)-expressing nuclei (blue) with DN-KASH (red) being expressed in the same cell (A), a cell 0-10 µm away (B) or a cell 10-20 µm away from the GFP-UBF-expressing cell (C). Distances measured represent nearest nucleus to nucleus distance to a DN-KASH-expressing nucleus. (D) Schematic of target cells expressing GFP-UBF (green dots) at various distances (a) from a DN-KASH-expressing cell (red outline). (E) MSD versus lag time for DN-KASH-expressing cells, cells of varying distances from DN-KASH and control monolayer cells. Nearby cells have similar intranuclear motion to co-transfected cells. MSD increases as cells are further from a DN-KASH-expressing cell. Error bars represent s.e.m. (F) Comparison of the force generation exponent (β) for nuclei transfected with DN-KASH (orange), or at different distances away from a DN-KASH-expressing cell (shades of red) as well as monolayers not transfected with DN-KASH (green). Error bars represent 95% confidence interval for the fitting of β. (G) Plot of the normalized β value versus distance away from DN-KASH. The data were fitted as an exponential of the form β=1–exp(−a/n), where a is the distance (in µm) away from a DN-KASH-expressing nucleus. The parameter n is a spatial parameter such that forces at a distance n (in µm) no longer feel the majority of the effects of the DN-KASH-expressing cell, ∼50 µm based on the fit. The R2 for the fit was 0.93. Y error bars are 95% confidence intervals of β after normalization. X error bars are the s.d. of distance away from DN-KASH for the non-adjacent cells. Monolayer control, n=84; a=30-40 um, n=50; a=20-30 um, n=132; a=10-20 um, n=93; a=0-10 um, n=185; co-transfected, n=64.
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