Extended Data Fig. 8: The effects of extracellular fluid viscosity on RHOA activity, and its contribution to myosin II-, ARP2/3- and TRPV4-regulated actin dynamics.

a-c, Lifetimes (a) or epi-fluorescent FRET ratios (b,c) of the RHOA activity biosensor in cells on 2D in serum-free (a,b) or serum-containing (10% FBS; a-c) media at the indicated viscosities. Data are mean ± s.d. for n≥16 cells from ≥2 experiments. d, Segmentation strategy of cells on 2D for confocal FLIM-FRET analysis. The entire cell without the nucleus is defined as “whole”, while the “lamella” at cell edge is identified from DIC images. The “body” of the cell comprises of the remainder of the cell without the lamella and the nucleus. Scale bars: 20 µm. e, f, RHOA activity in wild-type cells in the presence of vehicle control or BAPTA (e) or in SC and shNHE1 cells (f) on 2D at prescribed viscosities. Data are mean ± s.d. for n≥27 cells from 3 experiments. g, RHOA lifetimes in confined MDA-MB-231 cells. Yellow arrowheads indicate regions of high activity. h, Spatial distribution of RHOA activity in different segments of confined MDA-MB-231 cells (n≥38) from 5 experiments. Data are mean ± s.d. i, Confocal images of cells inside confining channels immunostained for pMLC with intensity shown as a heatmap. Scale bar: 5 µm. j, Line-scan of pMLC intensity along normalized length of each cell migrating in confinement. Intensity is normalized to the lowest intensity along the scan. Data are moving average ± s.e.m. of n = 14 or 16 cells at 0.77 or 8 cP, respectively, pooled from 2 experiments. k, Confined migration speeds at prescribed viscosities in the presence of vehicle control, blebbistatin or Y27632. Data are mean ± s.d. for n≥139 cells from ≥3 experiments. l, Relative MIIA, MIIB and MIIC mRNA levels in wild-type cells. Data are normalized to the average levels of MIIA, and represent the mean ± s.d. from 3 experiments. m,n, Leading-edge lamella growth of Lifeact-GFP-tagged cells following dual MIIA/MIIB knockdown (m) or Y27632 treatment (n) on 2D relative to appropriate controls. Data are moving averages ± s.e.m. at each time point of n≥20 cells per condition from 3 experiments. *P < 0.05 for t ≥ 45 s between 0.77 versus 8 cP for SC (m) or vehicle control (n) samples. P > 0.05 for all time points between 0.77 and 8 cP for SC versus dual shMIIA/MIIB (m) and vehicle versus Y27632 (n). o,p, (Left) Confocal images showing the spatial localization of MIIA-GFP in cells migrating on 2D at 0.77 and 8 cP (o) or 8 cP only in the presence of vehicle control or Y27632 (p). (Right) Intensity profile along dashed red line in corresponding images. Intensity was normalized to the highest intensity along the scan. Scale bars: 10 µm. q–t, Retrograde actin flow rate (q) and uninterrupted protrusion growth rate (s) in wild-type cells expressing β-actin-mRFP-PAGFP on 2D at 8 cP in the presence of vehicle control, Y27632, GSK 2193874 (GSK2) or CK666. Similar measurements were made for SC and shMIIA cells (r,t). Data are mean ± s.d. for n≥21 cells from ≥2 experiments. Tests performed: Mann Whitney test (a,b,r), unpaired t-test (c) and after log transformation (t), Kruskal-Wallis followed by Dunn’s multiple comparisons (e,k,q,s), one-way ANOVA followed by Tukey’s multiple comparisons (f), one-way ANOVA followed by Tukey’s between segments in each group (h), and two-way ANOVA followed by Tukey’s multiple comparisons (m,n). Images in i,o,p are representative of 2 and in g of 5 biological replicas. Cell model: MDA-MB-231.

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