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Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P₂) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P₂, or disabling the interaction between FMNL2 and PtdIns(4,5)P₂. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton. Refereed/Peer-reviewed

Formins are a diverse class of actin regulators that influence filament dynamics and organization. Several formins have been identified at epithelial adherens junctions, but their functional impact remains incompletely understood. Here, we tested the hypothesis that formins might affect epithelial interactions through junctional contractility. We focused on mDia1, which was recruited to the zonula adherens (ZA) of established Caco-2 monolayers in response to E-cadherin and RhoA. mDia1 was necessary for contractility at the ZA, measured by assays that include a FRET-based sensor that reports molecular-level tension across alpha E-catenin. This reflected a role in reorganizing F-actin networks to form stable bundles that resisted myosin-induced stress. Finally, we found that the impact of mDia1 ramified beyond adherens junctions to stabilize tight junctions and maintain the epithelial permeability barrier. Therefore, control of tissue barrier function constitutes a pathway for cadherin-based contractility to contribute to the physiology of established epithelia. Refereed/Peer-reviewed

Mechanical tension governs epithelial morphogenesis and homeostasis, but its regulation remains poorly understood. Tension is commonly contractile, arising when the actomyosin cortices of cells are mechanically coupled together by cadherin adhesion. Here we report that caveolae control levels of epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) in the surrounding epithelium, but not in the oncogene-expressing cells, blocked extrusion leading to the retention and proliferation of transformed cells within the monolayer. Tensile stress was aberrantly elevated in CAV1-depleted monolayers due to elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P2) causing increased recruitment of the formin, FMNL2. Oncogenic extrusion was restored to CAV1-deficient monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P2, or disabling the interaction between FMNL2 and PtdIns(4,5)P2. Thus, by controlling lipid signalling to the actin cytoskeleton, caveolae regulate mechanical tension for epithelial homeostasis.

Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. In all these circumstances, it is essential that epithelial integrity be preserved despite the application of tensile stress. In this study, we identify junctional RhoA as a mechanosensitive signaling pathway that responds to epithelial stress. The junctional specificity of this response is mediated by the heterotrimeric protein Gα12, which is recruited by E-cadherin and, in turn, recruits p114 RhoGEF to activate RhoA. Further, we identify Myosin VI as a key mechanosensor, based on its intrinsic capacity to anchor E-cadherin to F-actin when exposed to tensile load. Tension-activated RhoA signaling was necessary to preserve epithelial integrity, which otherwise undergoes fracture when monolayer stress is acutely increased by calyculin. Paradoxically, this homeostatic RhoA signaling pathway increases junctional actomyosin, a contractile response that might be expected to itself promote fracture. Simulations of a vertex-based model revealed that the protective effect of RhoA signaling can be explained through increased yield limit at multicellular vertices, where experiments showed p114 RhoGEF was necessary to increase E-cadherin and promote actin assembly and organization.

Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. Here, we report a mechanosensitive signaling pathway that activates RhoA at adherens junctions to preserve epithelial integrity in response to acute tensile stress. We identify Myosin VI as the force sensor, whose association with E-cadherin is enhanced when junctional tension is increased by mechanical monolayer stress. Myosin VI promotes recruitment of the heterotrimeric G alpha 12 protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA. Despite its potential to stimulate junctional actomyosin and further increase contractility, tension-activated RhoA signaling is necessary to preserve epithelial integrity. This is explained by an increase in tensile strength, especially at the multicellular vertices of junctions, that is due to mDia1-mediated actin assembly. Refereed/Peer-reviewed

Mechanical coherence of cell layers is essential for epithelia to function as tissue barriers and to control active tissue dynamics during morphogenesis. RhoA signaling at adherens junctions plays a key role in this process by coupling cadherin-based cell-cell adhesion together with actomyosin contractility. Here we propose and analyze a mathematical model representing core interactions involved in the spatial localization of junctional RhoA signaling. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. This dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behavior of the system when contractility is inhibited are in qualitative agreement with the predictions of the model., Author summary Mathematical models play a key role in uncovering mechanisms responsible for the formation of patterns in cells and tissues. The well known Turing mechanism based on nonlinear reaction kinetics and differential diffusion explains the formation of static patterns, while positive feedback interactions can generate dynamical structures such as propagating fronts and excitable pulses. Recent studies have demonstrated the importance of mechanical forces that can lead to novel mechanisms of pattern formation such as clustering and oscillations in contractile systems. Here we investigate how contractile forces in mechanically active media can affect bistable front propagation. We found that contraction regulates the front speed or can fully suppress its propagation in space to create a static localized zone. The proposed model provides a new mechanism for cross-talk between mechanical activity of cells and biochemical signaling.

Microtubule-Targeting agents (MTA) are indispensable for cancer therapeutics. We here report thymoquinone (TQ) as a new MTA that already has been appreciated for its anticancer effects. TQ induced G2/M cell cycle arrest in human non-small lung epithelial cells (A549) and majority of arrested cells were in mitosis. TQ depolymerized the microtubule (MT) network and disrupted mitotic spindle organization of A549 cells. MT depolymerization by TQ was followed by apoptosis and subsequent loss in cell viability (IC50 value of ∼10 μM). Interestingly, TQ didn't affect the MT network of normal HUVEC cells at and below the IC50 concentration for A549 cells. TQ also inhibited tubulin polymerization in cell-free system with an IC50 of 27 μM and bound to tubulin heterodimers at a single site with a dissociation constant of 1.19 μM at 25 °C. Binding of TQ to tubulin quenched the tryptophan fluorescence of protein in a time-dependent manner. The TQ-tubulin binding kinetics was biphasic in nature and equilibrated in 30 min. TQ competed with colchicine for tubulin binding with a Ki of 1.9 μM as determined by modified Dixon plot analysis, this suggests TQ may bind tubulin at or near the colchicine binding site and in silico modeling study supported that. Our results establish a novel antimitotic mechanism of TQ by its direct binding to tubulin-MT network in A549 cells.

KIF17 is a kinesin-2 family motor that interacts with EB1 at microtubule (MT) plus-ends and contributes to MT stabilization in epithelial cells. The mechanism by which KIF17 affects MTs and how its activity is regulated are not yet known. Here, we show that EB1 and the KIF17 autoinhibitory tail domain (KIF17-Tail) interacted competitively with the KIF17 catalytic motor domain (K370). Both EB1 and KIF17-Tail decreased the K0.5MT of K370, with opposing effects on MT-stimulated ATPase activity. Importantly, K370 had independent effects on MT dynamic instability, resulting in formation of long MTs without affecting polymerization rate or total polymer mass. K370 also inhibited MT depolymerization induced by dilution in vitro and by nocodazole in cells, suggesting that it acts by protecting MT plus-ends. Interestingly, KIF17-Tail bound MTs and tubulin dimers, delaying initial MT polymerization in vitro and MT regrowth in cells. However, neither EB1 nor KIF17-Tail affected K370-mediated MT polymerization or stabilization significantly in vitro, and EB1 was dispensable for MT stabilization by K370 in cells. Thus, although EB1 and KIF17-Tail may coordinate KIF17 catalytic activity, our data reveal a novel and direct role for KIF17 in regulating MT dynamics.

In this study we sought to identify how contractility at adherens junctions influences apoptotic cell extrusion. We first found that the generation of effective contractility at steady-state junctions entails a process of architectural reorganization whereby filaments that are initially generated as poorly organized networks of short bundles are then converted into co-aligned perijunctional bundles. Reorganization requires coronin 1B, which is recruited to junctions by E-cadherin adhesion and is necessary to establish contractile tension at the zonula adherens. When cells undergo apoptosis within an epithelial monolayer, coronin 1B is also recruited to the junctional cortex at the apoptotic/neighbor cell interface in an E-cadherin-dependent fashion to support actin architectural reorganization, contractility, and extrusion. We propose that contractile stress transmitted from the apoptotic cell through E-cadherin adhesions elicits a mechanosensitive response in neighbor cells that is necessary for the morphogenetic event of apoptotic extrusion to occur. Refereed/Peer-reviewed

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.

The kinesin KIF17 localizes at microtubule plus-ends where it contributes to regulation of microtubule stabilization and epithelial polarization. We now show that KIF17 localizes at cell-cell adhesions and that KIF17 depletion inhibits accumulation of actin at the apical pole of cells grown in 3D organotypic cultures and alters the distribution of actin and E-cadherin in cells cultured in 2D on solid supports. Overexpression of full-length KIF17 constructs or truncation mutants containing the N-terminal motor domain resulted in accumulation of newly incorporated GFP-actin into junctional actin foci, cleared E-cadherin from cytoplasmic vesicles and stabilized cell-cell adhesions to challenge with calcium depletion. Expression of these KIF17 constructs also increased cellular levels of active RhoA, whereas active RhoA was diminished in KIF17-depleted cells. Inhibition of RhoA or its effector ROCK, or expression of LIMK1 kinase-dead or activated cofilin(S3A) inhibited KIF17-induced junctional actin accumulation. Interestingly, KIF17 activity toward actin depends on the motor domain but is independent of microtubule binding. Together, these data show that KIF17 can modify RhoA-GTPase signaling to influence junctional actin and the stability of the apical junctional complex of epithelial cells.

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC50 value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC50 value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10(-7) M(-1) s(-1) and 4.0 × 10(-9) M(-1) s(-1) for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin-apigenin binding was 1:1 and binding the binding constant (Kd) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G2/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α-β-subunit of tubulin. The binding site is 19 A in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.

Genistein (4',5,7-trihydroxyisoflavone), an isoflavone, is a major constituent of soyfoods. It has potential antiproliferative activity against several tumor types. We have examined the effect of genistein on cellular microtubules as well as its binding with purified tubulin in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC(50) value for genistein is 72 microM. Flow cytometry experiments demonstrated that genistein arrested cell cycle progression at the G(2)/M phase, but mitotic index data showed that genistein did not arrest cell cycle progression at mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody demonstrated a significant depolymerization of the interphase microtubules in a dose-dependent manner, and this was confirmed by the Western blot experiment using genistein-treated A549 cells. In vitro polymerization of purified tubulin into microtubules was inhibited by genistein with an IC(50) value of 87 microM. Genistein binding to tubulin quenched protein tryptophan fluorescence in a time- and concentration-dependent manner. Binding of genistein to tubulin was slow, taking approximately 45 min for equilibration at 37 degrees C. The association rate constant was 104.64 +/- 20.63 M(-1) s(-1) at 37 degrees C. The stoichiometry of genistein binding to tubulin was nearly 1:1 (molar ratio) with a dissociation constant of 15 microM at 37 degrees C. It was interesting to note that genistein did not recognize either the colchicine site or the vinblastine binding site of tubulin. Surprisingly, genistein inhibited ANS binding and competed for its binding site of tubulin with a K(i) of 20 microM as determined from a modified Dixon plot. Hence, we conclude that one of the mechanisms of antiproliferative activity of genistein is depolymerization of microtubules through binding of tubulin.

Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.

The present study was aimed to elucidate the mechanism of stabilization of tubulin by deuterium oxide (D(2)O). Rate of decrease of tryptophan fluorescence during aging of tubulin at 4 degrees C and 37 degrees C was significantly lower in D(2)O than in H(2)O. Circular dichroism spectra of tubulin after incubation at 4 degrees C, suggested that complete stabilization of the secondary structure in D(2)O during the first 24 hours of incubation. The number of available cysteine measured by DTNB reaction was decreased to a lesser extent in D(2)O than in H(2)O. During the increase in temperature of tubulin, the rate of decrease of fluorescence at 335 nm and change of CD value at 222 nm was lesser in D(2)O. Differential Scanning calorimetric experiments showed that the T(m) values for tubulin unfolding in D(2)O were 58.6 degrees C and 62.17 degrees C, and in H(2)O those values were 55.4 degrees C and 59.35 degrees C.