Acute compressive stress activates RHO/ROCK-mediated cellular processes

<p>The ability to rapidly respond to applied force underpins cell/tissue homeostasis. This response is mediated by mechanotransduction pathways that regulate remodeling and tension of the actomyosin cytoskeleton to counterbalance external forces. Enhanced extracellular matrix tension hyper-activates mechanotransduction and characterizes diseased states such as cancer, but is also required for normal epidermal regeneration. While the impact of extracellular matrix tension on signaling and cell biology are well appreciated, that of acute compressive force is under-studied. We show here that acute compressive force applied to cells and tissues in a native 3-dimensional context elevates RHOA-GTP levels and increases regulatory myosin phosphorylation, actomyosin contractility and tension via ROCK. In consequence, cell proliferation was increased, as was the expression of regulators of epithelial-mesenchymal transition. Pharmacological inhibition of ROCK abrogated myosin phosphorylation, but not RHOA activation. Our results strongly suggest that acute compressive stress impairs cellular homeostasis in a RHO/ROCK-dependent manner, with implications for disease states such as cancer.</p

Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics

<div><p>The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and <i>in vivo</i> in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is <i>in vivo</i> or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.</p></div

Tpm3.1 maintains constant and rapid cycling on stress fibers in the presence of jasplakinolide.

<p>(A) Representative image and FRAP sequence of MEFs transfected with C-Tpm3.1. FRAP zone indicated by white arrow. Top panel: FRAP sequence of untreated control cells. Bottom panel: FRAP sequence after treatment with 7 μM jasplakinolide. (B) FRAP curves of C-Tpm3.1 in control and drug-treated conditions. (C) Mobile fraction of control and drug-treated condition (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s007" target="_blank">S5 Table</a>). (D,E) Curve fits for C-Tpm3.1 in control (D) and drug-treated condition (E). Data obtained from 3 separate experiments, 3–8 cells per experiment. Error bars are +/- <i>SEM</i>. Scale bars = 10 μm.</p

Intracellular intravital imaging of the kinetics of N- and C-terminal tagged Tpm3.1 constructs transfected into rat salivary gland acinar cells.

<p>(A) Confocal image of an acinus from rat submandibular salivary gland section stained with anti-Tpm3.1 (2G10) antibody. Tpm3.1 is enriched on the apical plasma membranes that form the canaliculi of acinar cells (white arrow). (B) Confocal image of a C-Tpm3.1 transfected cell (arrow) in a single acinus of a rat salivary gland <i>in situ</i>. Extracellular space outside the acinus was stained with 10kDa dextran Alexa 647 conjugate. (C) Illustration of the transfected acinar cell in (B), arrow shows apical membrane/canaliculi, arrowhead shows basolateral membrane. (D,E) Intravital microscopy and FRAP analysis of N- and C-Tpm3.1 constructs in live transfected rats. Numbers indicate time in sec. White arrows indicate FRAP zones on the canaliculi of rat acinar cells. (F) FRAP curves for N- and C-Tpm3.1. (G) Mobile fraction of N- and C-Tpm3.1. (H) Half-times for N- and C-Tpm3.1. (I, J) Curve fits for N- and C-Tpm3.1. 11–16 cells assayed from at least 3 animals per construct (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s004" target="_blank">S2 Table</a>). Error bars are +/- <i>SEM</i>. Scale bars = 5 μm.</p

The majority of actin in stress fibers is stable.

<p>(A) Representative image and FRAP sequence of MEFs transfected with GFP-beta-actin. FRAP zone indicated by white arrow. Top panel: FRAP sequence of untreated control cells. Bottom panel: FRAP sequence after treatment with 7 μM jasplakinolide. (B) FRAP curves for GFP-actin in control and jasplakinolide treated condition. (C) FRAP curves for Lifeact-RFP in control and jasplakinolide treated condition. (D) Mobile fractions of control and drug treated GFP-actin and Lifeact-RFP (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s005" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s006" target="_blank">S4</a> Tables). (E) Curve fits for GFP-Actin control. (F) Curve fit for GFP-Actin treated with jasplakinolide. Data obtained from 3 separate experiments, 2–8 cells per experiment. Error bars are +/- <i>SEM</i>. Scale bars = 5 μm.</p

N- and C-terminal tagged Tpm3.1 constructs have similar mobile fractions but dissimilar recovery rates.

<p>(A,B) Representative images of FRAP assay in MEFs transfected with either N- or C-Tpm3.1. FRAP zones (white arrows) were bleached and cells imaged at 1 fps for 2 min. (inset A,B). Enlarged images of FRAP zones over time (s). (C,D) FRAP curves of N- or C-Tpm3.1 transfected MEFs. (E) Half-times of N- and C-Tpm3.1 recovery (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s003" target="_blank">S1 Table</a>). Data obtained from 6 experiments, 3–15 cells per experiment. Error bars are +/- <i>SEM</i>. Scale bars = 10 μm.</p

Additional file 8: Figure S3. of MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

showing that MCL-1 antagonism by BIMs2A slows tumor growth in mice bearing MDA-MB-468-2A xenografts but not MDA-MB-231-2A xenografts. (A–F) Line graphs depicting the tumor growth curves of MDA-MB-468-2A xenografts (A, B) and MDA-MB-231-2A xenografts (C, D) from mice fed with DOX or control food. Linear regression of these curves shown in B and D respectively. A comparison of the growth rate of tumors in mice bearing MDA-MB-468-2A (black) and MDA-MB-231-2A (red) xenografts fed with control food (E, F). (JPG 1348 kb

Additional file 5: Figure S5. of MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

showing that MCL-1 antagonism resulted in changes in proteins involved in SRC family kinase signaling and phosphorylation at serine3 of Cofilin. (A) Normalized z-ratio (a measure of statistical significance) of phosphorylated and total proteins (as indicated) in MDA-MB-468-2A cells at 24 hours after treatment with DOX compared with control cells. (B) Western blots of serine 3 phosphorylated Cofilin, total Cofilin and Actin in xenografts of MDA-MB-468-2A and MDA-MB-231-2A fed DOX food as indicated. (C) Bar graphs depicting the ratio of serine 3 phosphorylated Cofilin to total Cofilin from (B). Bars indicate statistically significant groups, Mann–Whitney p value. (D) Immunofluorescence of Cofilin and p-Cofilin MDA-MB-231-2A cells grown on fibronectin 24 hours after DOX or vehicle treatment. (E) Proximity ligation assays using antibodies to MCL-1 and Cofilin (green), Phalloidin (red) and Dapi (blue) in MDA-MB-231-2A cells. (JPG 1419 kb

Additional file 6: Figure S6. of MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

showing that MCL-1 antagonism and Dasatinib treatment induced apoptosis in MDA-MB-468-2A cells but not MDA-MB-231-2A cells when grown in 2D monolayer cultures. Bar graphs depicting the average fraction of apoptotic cells (total Annexin V-positive by flow cytometry) as indicated after 24 hours after treatment with vehicle, DOX, 5 μM A1210477 and 5 μM UMI-77 alone and in combination with 1 μM dasatinib after 24 hours. All graphs and western blots are the average of three independent experiments. Bars indicate statistically significant groups, p value paired t tests. (JPG 1029 kb

Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imaging

Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Forster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival. </p