Blebbistatin Treatment Research Articles

RACK1 contributes to the upregulation of embryonic genes in a model of cardiac hypertrophy

Receptors for activated C kinases (RACKs) have been shown to coordinate PKC-mediated hypertrophic signalling in mice. However, little information is available on its participation in embryonic gene expression. This study investigated the involvement of RACK1 in the expression of embryonic genes in a zebrafish (ZF) ex vivo heart culture model by using phenylephrine (PE) or a growth factors cocktail (GFs) as a prohypertrophic/regeneration stimulus. Blebbistatin (BL) inhibition has also been studied for its ability to block the signal transduction actions of some PEs. qRT‒PCR and immunoblot analyses confirmed the upregulation of RACK1 in the PE- and GFs-treated groups. BL administration counteracted PE-induced hypertrophy and downregulated RACK1 expression. Immunohistochemical analyses of the heart revealed the colocalization of RACK1 and embryonic genes, namely, Gata4, Wt1, and Nfat2, under stimulation, whereas these genes were expressed at lower levels in the BL treatment group. Culturing ZF heart cells activated via GFs treatment increased the expression of RACK1. The overexpression of RACK1 induced by the transfection of recombinant RACK1 cDNA in ZF heart cells increased the expression of embryonic genes, especially after one week of GFs treatment. In summary, these results support the involvement of RACK1 in the induction of embryonic genes during cardiac hypertrophy/GFs stimulation in a fish heart model, which can be used as an alternative study model for mammals.

Force re-development after shortening reveals a role for titin in stretch-shortening performance enhancement in skinned muscle fibres.

Stretch-shortening cycles (SSCs) involve muscle lengthening (eccentric contractions) instantly followed by shortening (concentric contractions). This combination enhances force, work and power output compared with pure shortening contractions, which is known as the SSC effect. Recent evidence indicates both cross-bridge (XB)-based and non-XB-based (e.g. titin) structures contribute to this effect. This study analysed force re-development following SSCs and pure shortening contractions to gain further insight into the roles of XB and non-XB structures regarding the SSC effect. Experiments were conducted on rat soleus muscle fibres (n=16) with different SSC velocities (30%, 60% and 85% of maximum shortening velocity) and constant stretch-shortening magnitudes (18% of optimum length). The XB inhibitor blebbistatin was used to distinguish between XB and non-XB contributions to force generation. The results showed SSCs led to significantly greater [mean±s.d. 1.02±0.15 versus 0.68±0.09 (ΔF/Δt); t62=8.61, P<0.001, d=2.79) and faster (75 ms versus 205ms; t62=-6.37, P<0.001, d=-1.48) force re-development compared with pure shortening contractions in the control treatment. In the blebbistatin treatment, SSCs still resulted in greater [0.11±0.03 versus 0.06±0.01 (ΔF/Δt); t62=8.00, P<0.001, d=2.24) and faster (3010±1631 versus 7916±3230ms; t62=-8.00, P<0.001, d=-1.92) force re-development compared with pure shortening contractions. These findings deepen our understanding of the SSC effect, underscoring the involvement of non-XB structures such as titin in modulating force production. This modulation is likely to involve complex mechanosensory coupling from stretch to signal transmission during muscle contraction.

Interaction between membranous EBP50 and myosin 9 as a favorable prognostic factor in ovarian clear cell carcinoma.

Ezrin-binding phosphoprotein 50 (EBP50) is a scaffold protein that is required for epithelial polarity. Knockout (KO) of membranous EBP50 (Me-EBP50) in ovarian clear cell carcinoma (OCCC) cells induced an epithelial-mesenchymal transition (EMT)-like phenotype, along with decreased proliferation, accelerated migration capability, and induction of cancer stem cell (CSC)-like properties. Shotgun proteomics analysis of proteins that co-immunoprecipitated with EBP50 revealed that Me-EBP50 strongly interacts with myosin 9 (MYH9). Specific inhibition of MYH9 with blebbistatin phenocopied Me-EBP50 knockout, and blebbistatin treatment potentiated the effects of Me-EBP50 knockout. In OCCC cells from clinical samples, Me-EBP50 and MYH9 were co-localized at the apical plasma membrane. Patients with a combination of Me-EBP50-high and MYH9-high scores had the best prognosis for overall and progression-free survival. Our data suggest that Me-EBP50 has tumor-suppressive effects through the establishment and maintenance of epithelial polarization. In contrast, loss of Me-EBP50 expression induces EMT-like phenotypes, probably due to MYH9 dysfunction; this results in increased cell mobility and enhanced CSC-like properties, which in turn promote OCCC progression.

Effects of blebbistatin on force production, actin motility and the force-velocity relation produced by myosin II filaments and myofibrils.

Blebbistatin is a myosin II-specific inhibitor that is often used to study the relation between force generation and Pi release. The goal of this study was to investigate the effects of blebbistatin on force production and the actin motility while interacting with arrays of myosin motors. We used myosin filaments isolated from mammalian smooth muscles, rabbit psoas and the anterior byssus retractor (ABRM) muscles extracted from fresh Mytilus edulis in different experiments. We also used whole myofibrils isolated from the rabbit psoas. The force during myosin-actin interactions was measured with a system of micro-fabricated cantilevers, and the sliding velocity of actin was measured with a standard in-vitro motility assay. The force-velocity relation was measured in myofibrils with atomic force cantilevers. The force produced by the myosin filaments upon interaction with actin decreased with increasing concentrations of blebbistatin (5 μM-12.5 μM). The force produced by smooth and skeletal muscle myosin filaments were 95.93 ± 12.05 pN/μm before blebbistatin treatment, 71.45 ± 5.94 pN/μm with 5 μm blebbistatin, 62.05 ± 9.44 pN/μm with 7.5 μM blebbistatin, 38.28 ± 8.77 pN/μm with 10 μm blebbistatin and dropped to zero with 12.5 μM blebbistatin. All these differences were significantly different (p<0.0001). Sliding velocity of actin filaments also decreased consistently with increasing concentrations of blebbistatin. Before treatment, filaments slid over HMM at 3.68 ± 0.06 μm/s, decreasing to 2.30 ± 0.056 μm/s with 1 μM, 1.27 ± 0.043 μm/s with 5 μM, and 1.44 ± 0.032 μm/s with 30 μM. The maximal velocity decrease was observed at 5 μM of blebbistatin. These differences were significantly different (P<0.0001). There was a downward shift in the force-velocity relation in myofibrils treated with blebbistatin. These results suggest that blebbistatin regulates the mechanics of myosin motors working collectively in different experimental preparations.

Calpain-dependent degradation of cytoskeletal proteins as a key mechanism for a reduction in intrinsic passive stiffness of unloaded rat postural muscle.

In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical analysis and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle was significantly reduced after 7- and 14-day HS compared to the control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and -3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content, and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle.

Extracellular Vesicles Derived from SIPA1high Breast Cancer Cells Enhance Macrophage Infiltration and Cancer Metastasis through Myosin-9

Simple SummaryThe high expression of signal-induced proliferation-associated 1 (SIPA1) in breast cancer could aggravate cancer cell metastasis, but how the tumour microenvironment is involved in this incident is unknown. In this study, we investigated whether breast cancer cells with high SIPA1 expression recruited macrophages into the tumour microenvironment. We also found that extracellular vesicles (EVs) derived from MDA-MB-231 cells significantly enhanced macrophage migration, compared with that from SIPA1-knockdown MDA-MB-231 cells both in vitro and in vivo. In terms of the mechanism, SIPA1 in cancer cells modulated the key protein myosin-9 in EVs and promoted macrophage infiltration via EVs. We confirmed that either down-regulating SIPA1 expression or blocking myosin-9 by its inhibitor, blebbistatin, led to the suppression of macrophage infiltration. These findings contribute to a deep understanding of how SIPA1 regulates the tumour microenvironment in breast cancer to facilitate tumour metastasis and provide a basis for the development of therapeutics against breast cancer metastasis.Tumour cell metastasis can be genetically regulated by proteins contained in cancer cell-derived extracellular vesicles (EVs) released to the tumour microenvironment. Here, we found that the number of infiltrated macrophages was positively correlated with the expression of signal-induced proliferation-associated 1 (SIPA1) in invasive breast ductal carcinoma tissues and MDA-MB-231 xenograft tumours. EVs derived from MDA-MB-231 cells (231-EVs) significantly enhanced macrophage migration, compared with that from SIPA1-knockdown MDA-MB-231 cells (231/si-EVs) both in vitro and in vivo. We revealed that SIPA1 promoted the transcription of MYH9, which encodes myosin-9, and up-regulated the expression level of myosin-9 in breast cancer cells and their EVs. We also found that blocking myosin-9 by either down-regulating SIPA1 expression or blebbistatin treatment led to the suppression of macrophage infiltration. Survival analysis showed that breast cancer patients with high expression of SIPA1 and MYH9 molecules had worse relapse-free survival (p = 0.028). In summary, SIPA1high breast cancer can enhance macrophage infiltration through EVs enriched with myosin-9, which might aggravate the malignancy of breast cancer.

Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion.

Background & AimsLiver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis.MethodsIntracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model.ResultsIn our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets’ expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease.ConclusionsWe provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.

Reducing Myosin II and ATP-Dependent Mechanical Activity Increases Order and Stability of Intracellular Organelles.

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca++ probe as a small and mobile tracer in live T cells. Ca++, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca++-rich features in length scale < 1.1 μm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca++-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and numerical simulations, we suggest that diffusion in the viscoelastic and confined medium of intracellular organelles may promote spatial dis-homogeneity and stability of their content. This may be revealed only after inhibiting intracellular mechanical work and related cell vibrations. Our described mechanisms may allow the cell to control its organization via balancing its viscoelasticity and mechanical activity, with implications to cell physiology in health and disease.

Endothelial Conditional Knockdown of NMMHC IIA (Nonmuscle Myosin Heavy Chain IIA) Attenuates Blood-Brain Barrier Damage During Ischemia-Reperfusion Injury.

In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Endothelial detachment contributes to BBB disruption and neurovascular dysfunction, but its regulation in stroke has yet to be clarified. We investigated the function of NMMHC IIA (nonmuscle myosin heavy chain IIA) in the endothelium on BBB breakdown and its potential mechanisms. Endothelial conditional knockdown NMMHC IIA (Myh9ECKD) was constructed in vivo and in vitro, and its role was explored in middle cerebral artery occlusion/reperfusion-injured mice and oxygen-glucose deprivation/reoxygenation-injured brain microvascular endothelial cells. The degree of brain injury was analyzed using staining (2,3,5-triphenyltetrazolium chloride, hematoxylin, and eosin) and electron microscopy. BBB breakdown was investigated with leakage of Evans Blue dye and expression of TJs (tight junctions) and MMP (matrix metallopeptidase)-2/9. Transcriptomics for enrichment analysis was adopted to explore the potential downstream signaling pathways of NMMHC IIA involved in middle cerebral artery occlusion/reperfusion-induced BBB dysfunction. NMMHC IIA expression was upregulated in endothelial cells after cerebral ischemia/reperfusion injury. Myh9ECKD mice exhibited improvement in endothelial barrier hyperpermeability and TJs integrity stimulated by cerebral ischemia/reperfusion. Blebbistatin (NMMHC II inhibitor) treatment exerted the same effect. Transcriptomics showed that NMMHC IIA was involved in regulating various BBB-related genomic changes in the middle cerebral artery occlusion/reperfusion model, and NMMHC IIA was confirmed to significantly modulate Hippo and peroxisome proliferator-activated receptor gamma/nuclear factor-kappa B signaling pathways, which are closely related to BBB damage. Our findings provide some new insights into how NMMHC IIA contributes to maintaining the integrity of the cerebral endothelial barrier. NMMHC IIA could be a potential therapeutic target for ischemic stroke.

Inhibition of apical domain formation does not block blastocyst development in bovine embryos.

The first event of cellular differentiation consists of the segregation of the trophectoderm and the inner cell mass. Studies in mice suggest that cell contractility and the formation of an apical domain play important roles in this event; however, this remains unknown in the bovine. We tested the hypothesis that blocking apical domain formation would halt subsequent trophectoderm differentiation in bovine embryos. We first assessed the formation of an apical domain by the presence of Par-6 Family Cell Polarity Regulator Beta (PARD6B) and Ezrin (EZR), which appeared after the 8-cell stage. We inhibited apical domain formation by blocking cell contractility with 25μM (-)-blebbistatin. Treatment from 90 to 186h after insemination did not reduce blastocyst development compared with the untreated control group or the group treated with inactive (+)-blebbistatin. Immunofluorescence staining after blebbistatin treatment revealed the absence of EZR and the trophectoderm marker Caudal Type Homeobox 2 (CDX2). Following blebbistatin treatment, Yes1 Associated Transcriptional Regulator (YAP), which is involved in the Hippo signalling pathway, exhibited cytoplasmic staining instead of nuclear localisation. Despite changes in protein expression and localisation, no difference in trophectoderm or total cell numbers was observed. In conclusion, inhibition of cell contractility inhibited apical domain formation without impairing blastocyst formation, suggesting that a different biological mechanism is involved in trophectoderm and inner cell mass differentiation in bovine embryos.

Inhibition of focal adhesion kinase increases myofibril viscosity in cardiac myocytes.

The coordinated generation of mechanical forces by cardiac myocytes is required for proper heart function. Myofibrils are the functional contractile units of force production within individual cardiac myocytes. At the molecular level, myosin motors form cross-bridges with actin filaments and use ATP to convert chemical energy into mechanical forces. The energetic efficiency of the cross-bridge cycle is influenced by the viscous damping of myofibril contraction. The viscoelastic response of myofibrils is an emergent property of their individual mechanical components. Previous studies have implicated titin-actin interactions, cell-ECM adhesion, and microtubules as regulators of the viscoelastic response of myofibrils. Here we probed the viscoelastic response of myofibrils using laser-assisted dissection. As a proof-of-concept, we found actomyosin contractility was required to endow myofibrils with their viscoelastic response, with blebbistatin treatment resulting in decreased myofibril tension and viscous damping. Focal adhesion kinase (FAK) is a key regulator of cell-ECM adhesion, microtubule stability, and myofibril assembly. We found inhibition of FAK signaling altered the viscoelastic properties of myofibrils. Specifically, inhibition of FAK resulted in increased viscous damping of myofibril retraction following laser ablation. This damping was not associated with acute changes in the electrophysiological properties of cardiac myocytes. These results implicate FAK as a regulator of mechanical properties of myofibrils.

Abstract 4205: The role of non-muscle myosin IIA in HER2+ breast cancers

Abstract HER2 is amplified in about 20% of breast cancers. HER3 plays a critical role in HER2 mediated tumorigenesis. It is now clear that inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using the irreversible pan HER inhibitor neratinib. We immunoaffinity-purified HER3 from HER2+ BT474 cells treated ± neratinib. Following immunoprecipitation using a HER3 antibody, binding partners were released under reducing conditions. Mass spectrometry experiments identified non-muscle myosin IIA (NMMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To confirm the presence of NMMIIA, we performed immunoprecipitation experiments in BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes the protein nonmuscle myosin of class II, isoform A (NMMIIA). NMMIIA localizes to actin stress fibers and has been implicated in regulation of cell contractility and stress fiber organization To further investigate the role of NMMIIA, we used blebbistatin, a myosin IIa inhibitor, in growth assays using BT474 and MDA-MB-453 breast cancer cells in vitro. The combination of blebbistatin and neratinib significantly decreased the proliferation in both cell lines compared to neratinib or blebbistatin treatment alone. Additionally, we knocked out myosin IIa in BT474 cells using myosin IIa specific siRNA to examine the effect of myosin IIa silencing on HER3 and Myosin IIa protein levels in the presence or absence of neratinib treatment. Results indicated that in BT474 cells knockdown of NMMIIA resulted in both decreased HER3 and Myosin IIa protein expression and that neratinib treatment rescued this effect. We next examined long term overall survival of primary breast cancer patients who have high and low levels of MYH9 gene expression from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients who express low levels of MYH9. Currently, further experiments are ongoing to determine the mechanism of action of the Myosin IIA/HER3 complex, which could be an adaptive response to HER2 inhibitors in HER2+ breast cancers and to evaluate the role of myosin IIa on proliferation, migration, and invasion of HER2+ breast cancer cells. In conclusion, we have identified that neratinib treatment results in increased NMMIIA bound to HER3. Further studies to determine if NMMIIA could play a role in invasion of HER2+ breast cancer cells and/or resistance to HER2 inhibitors are ongoing. Citation Format: Samar M. Alanazi, Rosalin Mishra, Long Yuan, Hima Patel, Joan Garrett. The role of non-muscle myosin IIA in HER2+ breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4205.

Traction Forces Control Cell-Edge Dynamics and Mediate Distance Sensitivity during Cell Polarization.

Traction forces are generated by cellular actin-myosin system and transmitted to the environment through adhesions. They are believed to drive cell motion, shape changes, and extracellular matrix remodeling [1-3]. However, most of the traction force analysis has been performed on stationary cells, investigating forces at the level of individual focal adhesions or linking them to static cell parameters, such as area and edge curvature [4-10]. It is not well understood how traction forces are related to shape changes and motion, e.g., forces were reported to either increase or drop prior to cell retraction [11-15]. Here, we analyze the dynamics of traction forces during the protrusion-retraction cycle of polarizing fish epidermal keratocytes and find that forces fluctuate together with the cycle, increasing during protrusion and reaching maximum at the beginning of retraction. We relate force dynamics to the recently discovered phenomenological rule [16] that governs cell-edge behavior during keratocyte polarization: both traction forces and probability of switch from protrusion to retraction increase with the distance from the cell center. Diminishing forces with cell contractility inhibitor leads to decreased edge fluctuations and abnormal polarization, although externally applied force can induce protrusion-retraction switch. These results suggest that forces mediate distance sensitivity of the edge dynamics and organize cell-edge behavior, leading to spontaneous polarization. Actin flow rate did not exhibit the same distance dependence as traction stress, arguing against its role in organizing edge dynamics. Finally, using a simple model of actin-myosin network, we show that force-distance relationship might be an emergent feature of such networks.

Blebbistatin Inhibits Neomycin-Induced Apoptosis in Hair Cell-Like HEI-OC-1 Cells and in Cochlear Hair Cells

Aging, noise, and ototoxic drug-induced hair cell (HC) loss are the major causes of sensorineural hearing loss. Aminoglycoside antibiotics are commonly used in the clinic, but these often have ototoxic side effects due to the accumulation of oxygen-free radicals and the subsequent induction of HC apoptosis. Blebbistatin is a myosin II inhibitor that regulates microtubule assembly and myosin–actin interactions, and most research has focused on its ability to modulate cardiac or urinary bladder contractility. By regulating the cytoskeletal structure and reducing the accumulation of reactive oxygen species (ROS), blebbistatin can prevent apoptosis in many different types of cells. However, there are no reports on the effect of blebbistatin in HC apoptosis. In this study, we found that the presence of blebbistatin significantly inhibited neomycin-induced apoptosis in HC-like HEI-OC-1 cells. We also found that blebbistatin treatment significantly increased the mitochondrial membrane potential (MMP), decreased ROS accumulation, and inhibited pro-apoptotic gene expression in both HC-like HEI-OC-1 cells and explant-cultured cochlear HCs after neomycin exposure. Meanwhile, blebbistatin can protect the synaptic connections between HCs and cochlear spiral ganglion neurons. This study showed that blebbistatin could maintain mitochondrial function and reduce the ROS level and thus could maintain the viability of HCs after neomycin exposure and the neural function in the inner ear, suggesting that blebbistatin has potential clinic application in protecting against ototoxic drug-induced HC loss.

Effects of Mavacamten and Blebbistatin on the Small-Angle X-ray Scattering Structure of Human β-cardiac Myosin

Cardiac myosin is a motor protein that converts chemical energy from ATP hydrolysis to mechanical work that keeps the heart beating. Full-length myosin and even the smaller sub-fragment, heavy meromyosin (HMM) are large and flexible molecules and have not been possible to crystallize and therefore has been unavailable for structural studies. These properties make myosin a good candidate for small-angle X-ray scattering (SAXS) studies. Here we use size exclusion chromatography coupled with SAXS to study structural changes of human β-cardiac 2-hep and 25-hep HMM constructs in relaxing conditions treated with two different myosin inhibitors: blebbistatin and mavacamten. Under resting conditions, the 25-hep HMM had a radius of gyration (Rg) of ∼ 118 Å (Guinier fit) to ∼ 137 Å (GNOM fit) and the maximum dimension of the molecule (Dmax) of 526 Å. The Rg of 25-hep HMM decreased by about 5-10% with either blebbistatin or mavacamten treatment. The Dmax of 25-hep HMM decreased ∼ 20 Å by blebbistatin treatment while it decreased ∼ 80 Å by mavacamten treatment. There were only moderate structural changes (< 2%) for the 2-hep HMM with either blebbistatin or mavacamten treatment. Our results indicate that most of the HMM molecules in relaxing conditions maintain an open configuration and that the myosin S2 region is essential for HMM to adopt the folded back closed configuration in presence of mavacamten and blebbistatin. Supported, in part, by NIH P41 GM103622.

Targeting non-muscle myosin II promotes corneal endothelial migration through regulating lamellipodial dynamics.

Corneal endothelial cell (CEC) dysfunction causes corneal edema that may lead to blindness. In addition to corneal transplantation, simple descemetorhexis has been proposed to treat centrally located disease with adequate peripheral cell reserve, but promoting the centripetal migration of CECs is pivotal to this strategy. Here, we show that targeting non-muscle myosin II (NMII) activity by Y27632, a ROCK inhibitor, or blebbistatin, a selective NMII inhibitor, promotes directional migration of CECs and accelerates in vitro wound healing. The lamellipodial protrusion persistence is increased, and actin retrograde flow is decreased after NMII inhibition. Counteracting lamellipodial protrusion by actin-related protein 2/3 (ARP2/3) inhibitor abolishes this migration-promoting effect. Although both Y27632 and blebbistatin accelerate wound healing, cell junctional integrity and barrier function are better preserved after blebbistatin treatment, leading to more rapid corneal deturgescence in rabbit corneal endothelial wounding model. Our findings indicate that NMII is a promising therapeutic target in the treatment of CEC dysfunction. KEY MESSAGES: NMII inhibition promotes directional migration and wound healing of CECs in vitro. Lamellipodial protrusion persistence is increased after NMII inhibition. Selective NMII inhibitor preserves junctional integrity better than ROCK inhibitor. Selective NMII inhibitor accelerates corneal deturgescence after wounding in vivo.

Mesenchymal actomyosin contractility is required for androgen-driven urethral masculinization in mice

The morphogenesis of mammalian embryonic external genitalia (eExG) shows dynamic differences between males and females. In genotypic males, eExG are masculinized in response to androgen signaling. Disruption of this process can give rise to multiple male reproductive organ defects. Currently, mechanisms of androgen-driven sexually dimorphic organogenesis are still unclear. We show here that mesenchymal-derived actomyosin contractility, by MYH10, is essential for the masculinization of mouse eExG. MYH10 is expressed prominently in the bilateral mesenchyme of male eExG. Androgen induces MYH10 protein expression and actomyosin contractility in the bilateral mesenchyme. Inhibition of actomyosin contractility through blebbistatin treatment and mesenchymal genetic deletion induced defective urethral masculinization with reduced mesenchymal condensation. We also suggest that actomyosin contractility regulates androgen-dependent mesenchymal directional cell migration to form the condensation in the bilateral mesenchyme leading to changes in urethral plate shape to accomplish urethral masculinization. Thus, mesenchymal-derived actomyosin contractility is indispensable for androgen-driven urethral masculinization.

Micro-scaled topographies direct differentiation of human epidermal stem cells

Human epidermal stem cells initiate terminal differentiation when spreading is restricted on ECM-coated micropatterned islands, soft hydrogels or hydrogel-nanoparticle composites with high nanoparticle spacing. The effect of substrate topography, however, is incompletely understood. To explore this, primary human keratinocytes enriched for stem cells were seeded on a topographical library with over 2000 different topographies in the micrometre range. Twenty-four hours later the proportion of cells expressing the differentiation marker transglutaminase-1 was determined by high content imaging. As predicted, topographies that prevented spreading promoted differentiation. However, we also identified topographies that supported differentiation of highly spread cells. Topographies supporting differentiation of spread cells were more irregular than those supporting differentiation of round cells. Low topography coverage promoted differentiation of spread cells, whereas high coverage promoted differentiation of round cells. Based on these observations we fabricated a topography in 6-well plate format that supported differentiation of spread cells, enabling us to examine cell responses at higher resolution. We found that differentiated spread cells did not assemble significant numbers of hemidesmosomes, focal adhesions, adherens junctions, desmosomes or tight junctions. They did, however, organise the actin cytoskeleton in response to the topographies. Rho kinase inhibition and blebbistatin treatment blocked the differentiation of spread cells, whereas SRF inhibition did not. These observations suggest a potential role for actin polymerization and actomyosin contraction in the topography-induced differentiation of spread cells. Statement of SignificanceThe epidermis is the outer covering of the skin. It is formed by layers of cells called keratinocytes. The basal cell layer contains stem cells, which divide to replace cells in the outermost layers that are lost through a process known as differentiation. In this manuscript we have developed surfaces that promote the differentiation of epidermal stem cells in order to understand the signals that control differentiation. The experimental tools we have developed have the potential to help us to devise new treatments that control diseases such as psoriasis and eczema in which epidermal stem cell proliferation and differentiation are disturbed.

Inverse tissue mechanics of cell monolayer expansion.

Living tissues undergo deformation during morphogenesis. In this process, cells generate mechanical forces that drive the coordinated cell motion and shape changes. Recent advances in experimental and theoretical techniques have enabled in situ measurement of the mechanical forces, but the characterization of mechanical properties that determine how these forces quantitatively affect tissue deformation remains challenging, and this represents a major obstacle for the complete understanding of morphogenesis. Here, we proposed a non-invasive reverse-engineering approach for the estimation of the mechanical properties, by combining tissue mechanics modeling and statistical machine learning. Our strategy is to model the tissue as a continuum mechanical system and to use passive observations of spontaneous tissue deformation and force fields to statistically estimate the model parameters. This method was applied to the analysis of the collective migration of Madin-Darby canine kidney cells, and the tissue flow and force were simultaneously observed by the phase contrast imaging and traction force microscopy. We found that our monolayer elastic model, whose elastic moduli were reverse-engineered, enabled a long-term forecast of the traction force fields when given the tissue flow fields, indicating that the elasticity contributes to the evolution of the tissue stress. Furthermore, we investigated the tissues in which myosin was inhibited by blebbistatin treatment, and observed a several-fold reduction in the elastic moduli. The obtained results validate our framework, which paves the way to the estimation of mechanical properties of living tissues during morphogenesis.

Blebbistatin induces chondrogenesis of single mesenchymal cells via PI3K/PDK1/mTOR/p70S6K pathway

AbstractRearrangement of the actin cytoskeleton plays an inductive role in chondrogenic differentiation. Our previous study showed that blebbistatin, an inhibitor of myosin II, removes actin stress fibres and induces chondrogenesis of mesenchymal cells in monolayer cultures. In the present study, we investigated signalling pathways implicated in the induction of chondrogenesis by dissolving actin stress fibres after blebbistatin treatment. Blebbistatin increased the activity of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K with LY294002 blocked blebbistatin-induced chondrogenesis without affecting blebbistatin-induced reorganization of actin filaments. Blebbistatin also upregulated the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), and inhibition of PDK1 with GSK2334470 suppressed blebbistatin-induced chondrogenesis, indicating that removal of actin stress fibres by blebbistatin induced chondrogenesis by activating PI3K/PDK1. Although inhibition of Akt activity by Akt inhibitor IV blocked blebbistatin-induced chondrogenesis, phosphorylation of Akt was not affected by blebbistatin. Blebbistatin increased the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and p70 ribosomal protein S6 kinase (p70S6K). Inhibition of mTOR with rapamycin almost completely abolished the phosphorylation of p70S6K. Inhibition of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) with pp242 diminished phosphorylation of Akt at Ser473, whereas inhibition of mTORC1 with rapamycin did not. However, blebbistatin did not affect the phosphorylation of mTOR at Ser2481. Taken together, the present results suggest that blebbistatin induces chondrogenesis by activating the PI3K/PDK1/mTOR/p70S6K pathway. Our data also indicate that Akt activity is essential for chondrogenesis but is regulated by mTORC2, which is independent of blebbistatin treatment.