Cytoskeletal-transcriptional feedback prevents excessive focal adhesion maturation to enable persistent motility in human ECFCs.

A. Model of YAP/TAZ-mediated transcriptional feedback regulation of the cytoskeleton, as established in (Mason et al., 2019; van der Stoel et al., 2020). B-D. ECFCs were treated with the transcription inhibitor, Actinomycin D, and stained for vinculin, phalloidin (F-actin), and DAPI (nuclei) after 24 hours. E-G. ECFCs were depleted of both YAP and TAZ by RNAi and stained for vinculin, phalloidin (F-actin), and DAPI (nuclei) after 28 hours. Both transcription inhibition and YAP/TAZ depletion induced motile arrest, indicated by loss of fan-shaped lamellipodial migration and acquisition of an oval shape, ringed by mature focal adhesions (B, E). Vinculin+ focal adhesions (C, F) and mature vinculin+ focal adhesions, defined as greater than 1 µm (D, G). N = 20-22 cells per condition. **** p < 0.0001, Student’s two-tailed unpaired t-test. Data are shown as mean ± S.E.M.

Extracellular matrix rigidity regulates mechanotransduction and motility dynamics in human ECFCs.

A. Schematic of adhesion-spreading-polarization-migration (ASPM) assay. B. Methacrylated hyaluronic acid (MeHA) hydrogels were cross-linked with varying concentrations of DTT to form 1 kPa (soft), 8 kPa (moderate), and 18 kPa (stiff) hydrogels. Atomic force microscopy-measured elastic moduli are shown as mean ± S.D. n = 4, p < 0.0002, one-way ANOVA with Tukey’s post hoc test. ECFCs were then seeded on the MeHA hydrogels and assayed at 4 hours post-attachment. C. Cell area. n = 80 cells, p < 0.0001, Kruskal-Wallis with Dunn’s post hoc test. D-E. Quantification of the nuclear-to-cytosolic ratio of fluorescent intensity for YAP (D) and TAZ (E) at 4 hours post-attachment. n = 40 cells, p < 0.0001, one-way ANOVA with Tukey’s post hoc test. F. Representative immunofluorescent images visualizing F-actin (magenta), YAP or TAZ (yellow), and nuclei (blue) at 4 hours post-attachment. G. qPCR for the YAP/TAZ-target gene, CTGF. n = 3, p < 0.0054, one-way ANOVA with Tukey’s post hoc test. H. CTGF mRNA expression at 0, 1, 4, and 8 hours post-seeding, compared to unattached cells at t0 (dotted line). n = 3, * p < 0.01, *** p < 0.0008, two-way ANOVA with Sidak’s post hoc test. I-K. Next, motility of mTomato-expressing ECFCs was tracked over 20 hours post-attachment. Instantaneous migration speed, cell area, and circularity were calculated at 15-minute intervals until hour 24. Soft (n = 88 cells), moderate (n = 86 cells), and stiff (n = 89 cells). Data are shown as mean ± S.E.M. in error bars or shaded bands.

De novo gene expression and RhoA-YAP/TAZ signaling regulate zebrafish ISV morphogenesis in vivo.

A. Bright field and spinning disc confocal imaging of the (Tg(fli:egfp) y1) zebrafish transgenic line at 29, 32, and 35 hpf outlining the timeline of ISV morphogenesis in the zebrafish trunk. B. Schematic of experimental design. C. Representative spinning disc confocal images of DMSO, Actinomycin D (Act. D) 10 µg/ml (low), Act. D 25 µg/ml (high) and Puromycin treated zebrafish embryos. Compounds were added at 29 hpf and ISV morphogenesis imaged at 32 hpf (top) and 35 hpf (bottom). D,E. Quantification of ISV length, tracking individual fish over time, with data plotted as mean ± S.D. (D), vs comparative analysis at isolated time points with data plotted as mean ± S.E.M. * p < 0.05, two-way ANOVA with Sidak’s post hoc test (E). DMSO n = 8; Act. D (low) n = 8; Act. D (high) n = 8; and Puro n = 6. F. Representative spinning disc confocal images of DMSO, Rockout, Blebbistatin (Blebb), and Verteporfin (VP) treated zebrafish embryos. Compounds were added at 29 hpf and ISV morphogenesis imaged at 32 hpf (top) and 35 hpf (bottom). G,H. Quantification of ISV length, tracking individual fish over time, with data plotted as mean ± S.D. (G), vs comparative analysis at isolated time points with data plotted as mean ± S.E.M. * p < 0.05, two-way ANOVA with Sidak’s post hoc test (H). DMSO n = 8; Rockout n = 7; Blebb n = 10; and VP n = 8. Data points represent the ratio of ISV sprout length at the indicated time point to the total possible sprout length (dotted white line). Five ISVs were averaged per embryo to generate a single data point. ISV length ratio of ‘0’ indicates no ISV formation and ratio of ‘1’ indicates completion of ISV morphogenesis.

Acute transcription inhibition of human ECFCs induces motility and morphodynamic inflection at 4 hours post-attachment, followed by progressive motility arrest, on both soft and stiff substrates.

A. Experiment schematic: mTomato-expressing ECFCs were treated with DMSO or Actinomycin D (Act. D; 0.1 µg/mL) at the time of seeding on 1, 8, or 18 kPa MeHA matrices. Cell morphology and migration were tracked for 24 hours post-attachment or fixed for immunofluorescent imaging at 4 hours post-attachment. B-D. Instantaneous migration speed, cell area, and circularity were calculated at 15-minute intervals until hour 24 in four groups: soft-DMSO (n = 48 cells), soft-Act.D (n = 63 cells), stiff-DMSO (n = 41 cells), and stiff-Act.D (n = 36 cells). Moderate stiffness groups are shown in Fig. S1. E. Immunofluorescent imaging of F-actin (magenta), vinculin (yellow), and nuclei (blue) at 4 hours post-attachment. F, G. Quantification of vinculin+ focal adhesions (F) and mature vinculin+ focal adhesions (G), defined as focal adhesions greater than 1 µm in length. n = 21-22, * p < 0.05, *** p < 0.0002, two-way ANOVA with Sidak’s post hoc test. Data are shown as mean ± S.E.M.

YAP/TAZ-depletion in human ECFCs induces motility and morphodynamic inflection at 4 hours post-attachment, followed by progressive motility arrest, on both soft and stiff substrates.

A. Experiment schematic: mTomato-expressing ECFCs were treated with siRNA targeting YAP and TAZ or non-targeting control siRNA for 24 hours prior to seeding on 1, 8, or 18 kPa MeHA matrices. Cell morphology and migration were tracked for 24 hours post-attachment or fixed for immunofluorescent imaging at 4 hours post-attachment. B-D. Instantaneous migration speed, cell area, and circularity were calculated at 15-minute intervals for 24 hours in four groups: soft-siControl (n = 40 cells), soft-siYAP/TAZ (n = 77 cells), stiff-siControl (n = 48 cells), and stiff-siYAP/TAZ (n = 33 cells). Moderate stiffness groups are shown in Fig. S2. E. Immunofluorescence analysis of F-actin (magenta), vinculin (yellow), and nuclei (blue) at 4 hours post-attachment. F, G. Quantification of vinculin+ focal adhesions (F) and mature vinculin+ focal adhesions (G), defined as focal adhesions greater than 1 µm. in length. n = 21-22, * p < 0.02, ** p < 0.003, two-way ANOVA with Sidak’s post hoc test. Data are shown as mean ± S.E.M. H. Schematic of Adhesion-spreading-polarization-migration assay illustrating persistent migration with intact feedback and migration arrest with feedback interruption by either transcription inhibition (Act. D) or YAP/TAZ depletion (siY/T). Characteristic time scales indicated for initial feedback loop closure (τic) and motile equilibrium (τeq).

Rescue of vascular morphogenesis by inhibitor washout depends on washout timing.

A. Schematic diagram of continuous inhibitor treatment. Zebrafish embryos were treated with transcription inhibitor (Act. D), translation inhibitor (Puromycin), or YAP/TAZ inhibitor (Verteporfin), at 29 hpf and imaged at 32 hpf and 35 hpf. B. Schematic diagram of three-hour washout, in which inhibitors were washed out after 3 hours, prior to the time to feedback loop closure at τic = 4 hrs. Zebrafish embryos were treated with the indicated compounds at 29 hpf, had the inhibitors removed at 32 hpf, and were imaged as the ‘washout’ condition at 35 hpf. C. Representative spinning disc confocal images of DMSO, Act. D 25 ug/ml, Puromycin, and Verteporfin treated zebrafish embryos at 32 hpf (top), 35 hpf (middle), or at 35 hpf after 3 hours of incubation in the compounds followed by 3 hours of compound wash out (bottom). D,E,F. Quantification of ISV length, tracking individual fish over time. Plots indicate ISV growth rates under conditions of embryos continuously incubated in the indicated compound versus the associated washout condition. Act. D versus washout is shown in (D), Puromycin (Puro) versus washout is shown in (E), and Verteporfin (VP) versus washout shown in (F). G Aggregate bulk analysis of zebrafish embryos at isolated time points for each compound versus its associated washout condition. Data are plotted as mean ± S.E.M. DMSO n = 22; Act. D, n = 14 & 18; Puro, n = 11 & 11; and VP, n = 7 & 7 embryos each. * p < 0.05, two-way ANOVA with Sidak’s post hoc test. H. Schematic diagrams of our experimental scheme. Zebrafish embryos were treated with the indicated compounds at 26 hpf and imaged at 34 hpf and 38 hpf under conditions of continuous compound incubation versus compound ‘washout’ at 34 hpf. I. Representative spinning disc confocal images of DMSO, Act. D 25 ug/ml, Puromycin, and Verteporfin treated zebrafish embryos at 38 hpf either continuously maintained in compound (top) or after 8 hours of incubation in the compounds followed by 4 hours of compound washout (bottom). J. Aggregate bulk analysis of zebrafish embryos at 38 hpf for each compound versus its associated washout condition. Data are plotted as mean ± S.E.M. DMSO n = 15; Act. D n = 4 & 9; Puro n = 4 & 8; and VP n = 6 & 11. * p < 0.05, two-way ANOVA with Sidak’s post hoc test. Data points represent the ratio of ISV sprout length at the indicated time point to the total possible sprout length (dotted white line). Five ISVs were averaged per embryo to generate a single data point. A ratio of ‘0’ indicates no ISV formation and ‘1’ indicates completion of ISV morphogenesis.

Feedback history and cytoskeletal state prior to re-adhesion alter cytoskeletal and adhesion remodeling.

A. Transcription inhibition history experiment schematic: mTomato-expressing ECFCs were either pretreated with actinomycin D before plating (−20 hours) or at the time of attachment. All cells were fixed for immunofluorescence at 4 hours after attachment. B. Representative immunofluorescent images visualizing actin, vinculin, and nuclei with Alexa Fluor 488-conjugated phalloidin (magenta), Alexa Fluor 594-conjugated secondary (yellow), and DAPI (blue). C, D. Vinculin+ focal adhesions (C) and mature vinculin+ focal adhesions (D), defined as greater than 1 µm. n = 20-22, * p < 0.04, **** p < 0.0001, two-way ANOVA with Sidak’s post hoc test. E. YAP/TAZ depletion history experiment schematic: mTomato-expressing ECFCs were depleted of YAP and TAZ before plating (24 or 48 hours). All cells were fixed for immunofluorescence at 4 hours after attachment. F. Representative immunofluorescent images visualizing actin, vinculin, and nuclei with Alexa Fluor 488-conjugated phalloidin (magenta), Alexa Fluor 594-conjugated secondary (yellow), and DAPI (blue). G, H. Vinculin+ focal adhesions (C) and mature vinculin+ focal adhesions (D), defined as greater than 1 µm. n = 22, * p < .03, *** p < 0.0002, **** p < 0.0001 two-way ANOVA with Tukey’s post hoc test.

Transcriptional feedback suppresses myosin light chain phosphorylation, limits cell spreading through supranuclear membrane constriction, and promotes actin cap stress fiber maturation.

A-C. ECFCs were treated for 24 hours with DMSO or Actinomycin D and lysed for relative protein quantification of MLC phosphorylation by immunoblot (A) and immunofluorescent imaging (B, C). Samples used for quantification of pMLC or MLC in (A) were run and transferred in parallel and GAPDH was used as internal loading control so that signal could be normalized between blots. n = 3, * p < 0.05, Student’s two-tailed unpaired t-test. Data shown as mean ± S.E.M. D-G. ECFCs were either pretreated with actinomycin D for 24 hours (D, E) or depleted of YAP and TAZ for 48 hours (F, G) prior to plating on glass coverslips. All cells were fixed for 3D confocal imaging at 10 minutes after attachment. D, F. Representative immunofluorescent images of basal (x-y) actin, pMLC, and nuclei with Alexa Fluor 647-conjugated phalloidin (magenta), Alexa Fluor 488-conjugated secondary (yellow), and DAPI (blue). Side views are orthogonal projections demonstrating cell height (y-z). E, G. Paired measurements of distance of apical-most pMLC signal above the nucleus after Act. D treatment (E) or YAP/TAZ depletion (G). n = 27-33, ** p < 0.003, **** p < 0.0001, student’s two-tailed t-test. Data are shown as mean ± S.E.M. H. Representative apical pMLC ring formation adjacent to the cell membrane in Y/T-depleted cell (cross-section corresponds to dotted line in panel H). I, J. ECFCs were depleted of YAP and TAZ for 48 hours and treated with low dose (15 µM) Blebbistatin for 1 hour. I. Representative immunofluorescent images of actin and nuclei with Alexa Fluor 488-conjugated phalloidin (grey) and DAPI (blue), respectively, depicting ECFC with and without an actin cap. J. Number of cells with an organized, disorganized, or no perinuclear actin either on the basal or apical side of the cell. n = 147-150, p < 0.04, Chi square test with Bonferroni’s post hoc test. Data are shown as mean ± S.E.M or as cell number separated into categories.

YAP and TAZ regulate transcriptional changes related to vascular development, endothelial cell migration, and RhoA signaling. A.

Volcano plot of differentially expressed genes (DEGs) from bulk RNA-seq data from cells cultured on 18 kPa substrates. Differential expression analysis was conducted for siYAP/TAZ vs siControl. B. Gene Ontology (GO) analysis of significantly enriched biological processes in downregulated (up) or upregulated (down) genes by YAP/TAZ depletion. (PANTHER 17.0 overrepresentation test, p < 0.05, FDR-adjusted Fisher’s exact). C-D. Volcano plots of DEGs from siYAP/TAZ vs siControl cells cultured on 18 kPa substrates corresponding to Rho protein signal transduction (GO:0007266) (C), direct RhoA regulatory molecules (compiled from Lawson and Ridley, 2017) (D), and regulation of endothelial cell migration (GO:0010594) (E) (FDR-adjusted Wald test, cutoff criteria p < 0.05 and Log2FC > 0.5).

siRNA sense and antisense sequences (Dupont et al Nature 2011, Mason et al JCB 2019)

Effects of transcription inhibition on ECFCs motility and morphodynamics after attachment to soft (1 kPa), moderate (8 kPa), and stiff (18 kPa) matrices.

(A-E) mTomato-expressing ECFCs were seeded on hydrogels with either DMSO or actinomycin D (0.1 µg/mL) and cell (A, B) motility, (C, D) area, and (E, F) circularity tracked as a function of time after attachment. Soft-DMSO (n = 48 cells), soft-Act.D (n = 63 cells), Moderate-DMSO (n = 97 cells), Moderate-Act.D (n = 39 cells), stiff-DMSO (n = 41 cells), and stiff-Act.D (n = 36 cells). Figures include soft and stiff data reproduced from Figure 4, for comparison. Data are shown as mean ± S.E.M.

Effects of YAP/TAZ depletion on ECFCs motility and morphodynamics after attachment to soft (1 kPa), moderate (8 kPa), and stiff (18 kPa) matrices.

(A-B) ECFCs were depleted of YAP and TAZ for 24 or 48 hours then lysed for relative protein quantification by Western Blot. n = 3, * p < 0.04, ** p < 0.007, *** p = 0.0001, one-way ANOVA with Sidak’s post-hoc test. Data are shown as mean ± S.E.M. (C-H) mTomato-expressing ECFCs depleted of YAP and TAZ were seeded on hydrogels and cell (C, D) motility, (E, F) area, and (G, H) circularity tracked as a function of time after attachment. Soft-siControl (n = 40 cells), soft-YAP/TAZ (n = 77 cells), Moderate-siControl (n = 47 cells), Moderate-siYAP/TAZ (n = 57 cells), stiff-Control (n = 48 cells), and stiff-siYAP/TAZ (n = 33 cells). Figures include soft and stiff data reproduced from Figure 5, for comparison. Data are shown as mean ± S.E.M.