In regime II, as we previously observed,13 cell alignment decreased with increasing (see Equation?1 in the Experimental Procedures and Figure?S1), clearly showing that the order transitions at (Figure?1D). mechanics framework for living cells, we show that contact guidance emerges from anisotropic cell shape fluctuation and gap avoidance, i.e., the energetic penalty of cell adhesions on non-adhesive gaps. Our findings therefore point to general biophysical mechanisms underlying cellular contact guidance, without the necessity of invoking specific molecular pathways. and inter-line spacing ranged from 2 to 200?m and were initially chosen to be equal (i.e., m) of fibronectin (red) stained for the FAs (magenta), actin cytoskeleton (green), and nucleus (blue). (B) Schematic diagram showing the analysis of cell orientation based on the best-fitted ellipse (dashed yellow). (C) The length of a cell on a homogeneous substrate (control). (D) The cell, actin fiber, nucleus, and FA orientation, where 0 represents the direction of the lines. The boxes of the boxplots represent the quartiles of the distributions, with the whiskers indicating the outliers in the experiments and the 5th and 95th percentiles of the distributions. Note that, with this data representation, the median is at 0 and the box ranges from C45 to 45 when the distribution of cell orientation is perfectly isotropic. The data reported are results from three independent samples; at least 60 cells were considered per condition. Images obtained 24?h after seeding showed that cell morphology and orientation are strongly influenced by the width of the lines (Figure?1A). On the thinnest lines?(was increased up to 20?m, cells increasingly elongated and aligned parallel to the lines. The trend inverted when was further increased up to?200?m. We quantified the changes in cell shape and orientation for more than Faropenem daloxate 600 cells on the substrates using an automated morphometric analysis of the immunofluorescence images.17 Briefly, we fitted an ellipse to the cell outline and Faropenem daloxate defined the orientation angle as the angle between the major axis of the best-fitted ellipse and direction of the lines (Figure?1B). The analysis revealed that, with increasing 20?m, where cell alignment was induced by multiple lines, and regime II for 20?m, where cell alignment was influenced by the spatial confinement within single lines. In regime II, Faropenem daloxate as we previously observed,13 cell alignment decreased with increasing (see Equation?1 in the Experimental Procedures and Figure?S1), clearly showing that the order transitions at (Figure?1D). This trend is similar to, but Faropenem daloxate weaker than, the orientation response of the cells. Therefore, our data show that, at length scales larger Faropenem daloxate than FA size, increasing the adhesive area for FAs leads to the counterintuitive increase of FA and cell alignment in the direction of the lines. This suggests that contact guidance at these length scales does not arise from spatially constrained alignment of FAs, which is an underlying mechanism of contact guidance at smaller scales.9,12 To further confirm this observation, we investigated in more detail the morphology and organization of FAs in regime I. The analysis showed that lines of resulted in more aligned, elongated FAs in the direction of the lines (Figure?2A). We further characterized the size and shape of individual FAs by determining their length and aspect ratio, respectively. The length and aspect ratio of FAs on 2-m lines RAB21 were significantly smaller compared to those on homogeneous substrates, whereas the long axes of FAs on 5- and 10-m lines were equal to those on the homogeneous substrates (Figures 2C and 2D). The aspect ratio of FAs on 5-m lines was smaller compared to FAs on 10-m lines, meaning that FAs on 5-m lines are long and wide, although FAs on 10-m lines are thinner. Open in a separate window Figure?2 Constrained FAs Do Not Guide Cellular Alignment (A) Representative.