(A) Schematic summary of molecular mechanisms (green) associated with the ECM – adhesion – F-actin axis (gray) targeted here for perturbation to modulate implicated organizational features and, potentially, corresponding migration mode frequencies. Specifically, altering ECM ligand density (fibronectin concentration, [FN]) impacts mechanism 1) - integrin ligation and subsequent cell-matrix adhesion complex (CMAC) formation and maturation. RNAi-mediated knockdown of talin 1 limits both mechanism 2) - integrin activation and mechanism 3) - integrin-F-actin linkage, thereby also affecting CMAC formation, maturation and stability. Y-27632-mediated inhibition of ROCK disrupts mechanism 4) - actomyosin contractility, affecting F-actin dynamics and CMAC maturation. Images exemplifying the effects of each molecular perturbation are presented in Figure 6—figure supplement 1. (B) Boxplots summarizing the response of the same selection of organizational features to: ECM ligand modulation (2.5 µg/ml FN vs 10 µg/ml FN, upper row); talin 1 RNAi (control siRNA vs talin 1 siRNA, 5 µg/ml FN, middle row); or ROCK inhibition (DMSO vs 6 µM Y-27632, 5 µg/ml FN, lower row). All boxplots in B show median values per condition and inter-quartile ranges (IQR, 25% to 75%). Notches show the median +/−1.57 * IQR/ / √n (approximates 95% confidence interval of the median, n = number of cell observations, see 'Materials and methods'). In each case, statistically discernable differences were assessed by Wilcoxon rank sum testing, with resulting p values <0.001. (C) Migration mode frequency responses to each perturbation are depicted (Discontinuous, blue; Continuous, orange). Additional conditions were included for ROCK inhibition (low panel), showing a progressive response to 1 µM, 2 µM, and 6 µM Y-27632 as compared to DMSO vehicle control. Note: in addition to depicting specific perturbation-dependent trends, the matrix of results presented in B and C is used, together with spontaneous feature variations depicted in Figure 3D,E,G–I, to logically parse organizational features that are consistently coupled, or just occasionally correlated, with migration mode identity.