scholarly journals Unidirectional Eph/ephrin signaling creates a cortical actomyosin differential to drive cell segregation

2016 ◽  
Vol 215 (2) ◽  
pp. 217-229 ◽  
Author(s):  
Audrey K. O’Neill ◽  
Abigail A. Kindberg ◽  
Terren K. Niethamer ◽  
Andrew R. Larson ◽  
Hsin-Yi Henry Ho ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Rho Kinase ◽  
Mouse Genetics ◽  
Tissue Formation ◽  
Cortical Actin ◽  
Mammalian Embryo ◽  
Actomyosin Contractility ◽  
Bidirectional Signaling ◽  
Fundamental Process ◽  
Cell Segregation

Cell segregation is the process by which cells self-organize to establish developmental boundaries, an essential step in tissue formation. Cell segregation is a common outcome of Eph/ephrin signaling, but the mechanisms remain unclear. In craniofrontonasal syndrome, X-linked mosaicism for ephrin-B1 expression has been hypothesized to lead to aberrant Eph/ephrin-mediated cell segregation. Here, we use mouse genetics to exploit mosaicism to study cell segregation in the mammalian embryo and integrate live-cell imaging to examine the underlying cellular and molecular mechanisms. Our data demonstrate that dramatic ephrin-B1–mediated cell segregation occurs in the early neuroepithelium. In contrast to the paradigm that repulsive bidirectional signaling drives cell segregation, unidirectional EphB kinase signaling leads to cell sorting by the Rho kinase–dependent generation of a cortical actin differential between ephrin-B1– and EphB-expressing cells. These results define mechanisms of Eph/ephrin-mediated cell segregation, implicating unidirectional regulation of cortical actomyosin contractility as a key effector of this fundamental process.

scholarly journals EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts

2021 ◽  
Vol 220 (6) ◽  
Author(s):  
Abigail A. Kindberg ◽  
Vasudha Srivastava ◽  
Jonathon M. Muncie ◽  
Valerie M. Weaver ◽  
Zev J. Gartner ◽  
...  
Keyword(s):  
Mouse Genetics ◽  
Self Organization ◽  
Cortical Tension ◽  
Force Microscopy ◽  
Tissue Organization ◽  
Angle Measurements ◽  
Actomyosin Contractility ◽  
Directed Cell Migration ◽  
The Stability ◽  
Cell Segregation

EPH/EPHRIN signaling is essential to many aspects of tissue self-organization and morphogenesis, but little is known about how EPH/EPHRIN signaling regulates cell mechanics during these processes. Here, we use a series of approaches to examine how EPH/EPHRIN signaling drives cellular self-organization. Contact angle measurements reveal that EPH/EPHRIN signaling decreases the stability of heterotypic cell:cell contacts through increased cortical actomyosin contractility. We find that EPH/EPHRIN-driven cell segregation depends on actomyosin contractility but occurs independently of directed cell migration and without changes in cell adhesion. Atomic force microscopy and live cell imaging of myosin localization support that EPH/EPHRIN signaling results in increased cortical tension. Interestingly, actomyosin contractility also nonautonomously drives increased EPHB2:EPHB2 homotypic contacts. Finally, we demonstrate that changes in tissue organization are driven by minimization of heterotypic contacts through actomyosin contractility in cell aggregates and by mouse genetics experiments. These data elucidate the biomechanical mechanisms driving EPH/EPHRIN-based cell segregation wherein differences in interfacial tension, regulated by actomyosin contractility, govern cellular self-organization.

scholarly journals RhoA is required for cortical retraction and rigidity during mitotic cell rounding

2003 ◽  
Vol 160 (2) ◽  
pp. 255-265 ◽  
Author(s):  
Amy Shaub Maddox ◽  
Keith Burridge
Keyword(s):  
Actin Cytoskeleton ◽  
Molecular Mechanisms ◽  
Shape Change ◽  
Rho Kinase ◽  
Mitotic Cell ◽  
Cortical Actin ◽  
Interphase Cell ◽  
Cell Rounding ◽  
Rhoa Activity ◽  
Cell Shape Change

Mitotic cell rounding is the process of cell shape change in which a flat interphase cell becomes spherical at the onset of mitosis. Rearrangement of the actin cytoskeleton, de-adhesion, and an increase in cortical rigidity accompany mitotic cell rounding. The molecular mechanisms that contribute to this process have not been defined. We show that RhoA is required for cortical retraction but not de-adhesion during mitotic cell rounding. The mitotic increase in cortical rigidity also requires RhoA, suggesting that increases in cortical rigidity and cortical retraction are linked processes. Rho-kinase is also required for mitotic cortical retraction and rigidity, indicating that the effects of RhoA on cell rounding are mediated through this effector. Consistent with a role for RhoA during mitotic entry, RhoA activity is elevated in rounded, preanaphase mitotic cells. The activity of the RhoA inhibitor p190RhoGAP is decreased due to its serine/threonine phosphorylation at this time. Cumulatively, these results suggest that the mitotic increase in RhoA activity leads to rearrangements of the cortical actin cytoskeleton that promote cortical rigidity, resulting in mitotic cell rounding.

Abstract 126: High Efficiency Reprogramming of Fibroblasts Into Cardiomyocytes Requires Suppression of Pro-fibrotic Signaling

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Kunhua Song ◽  
Yuanbiao Zhao ◽  
Pilar Londono ◽  
Emily Sharpe ◽  
Joshua R Clair ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
High Efficiency ◽  
Transforming Growth Factor ◽  
Cardiac Fibroblasts ◽  
Rho Kinase ◽  
Transforming Growth Factor Β ◽  
Direct Reprogramming ◽  
Cardiac Gene Expression ◽  
Cardiac Gene ◽  
Kinetics Of

The mammalian heart is composed of ~30% cardiomyocytes which have limited capacity to regenerate and ~70% non-cardiomyocytes including endothelial cells and cardiac fibroblasts. Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic transcription factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. Despite extensive efforts, the efficiency of direct reprogramming of embryonic or adult fibroblasts into cardiomyocytes has yet to exceed 20%, or 0.1% respectively, leading many in the field to question the clinical translatability of this method. Here, we demonstrate that pro-fibrotic signaling events governed by transforming growth factor-β (TGF-β) and Rho kinase (ROCK) are concomitantly activated in GHMT-expressing fibroblasts, leading to potent suppression of cardiac reprogramming ( Figure 1 ). Remarkably, pharmacological inhibition of TGF-β, or ROCK leads to conversion of ≥ 60% of fibroblasts into highly functional cardiomyocytes, displaying global cardiac gene expression, spontaneous contractility, action potentials and calcium transients. Furthermore, inhibition of TGF-β, or ROCK dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than two weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts, and should enhance efforts to generate cardiomyocytes for clinical applications.

scholarly journals Identification of Aortic Proteins Involved in Arterial Stiffness in Spontaneously Hypertensive Rats Treated With Perindopril:A Proteomic Approach

2021 ◽  
Vol 12 ◽  
Author(s):  
Danyelle S. Miotto ◽  
Aline Dionizio ◽  
André M. Jacomini ◽  
Anderson S. Zago ◽  
Marília Afonso Rabelo Buzalaf ◽  
...  
Keyword(s):  
Arterial Stiffness ◽  
Spontaneously Hypertensive Rats ◽  
Wistar Rats ◽  
Molecular Mechanisms ◽  
Rho Kinase ◽  
Vascular Wall ◽  
Hypertensive Rats ◽  
Spontaneously Hypertensive ◽  
Cytoskeleton Organization ◽  
Proteomic Approach

Arterial stiffness, frequently associated with hypertension, is associated with disorganization of the vascular wall and has been recognized as an independent predictor of all-cause mortality. The identification of the molecular mechanisms involved in aortic stiffness would be an emerging target for hypertension therapeutic intervention. This study evaluated the effects of perindopril on pulse wave velocity (PWV) and on the differentially expressed proteins in aorta of spontaneously hypertensive rats (SHR), using a proteomic approach. SHR and Wistar rats were treated with perindopril (SHRP) or water (SHRc and Wistar rats) for 8 weeks. At the end, SHRC presented higher systolic blood pressure (SBP, +70%) and PWV (+31%) compared with Wistar rats. SHRP had higher values of nitrite concentration and lower PWV compared with SHRC. From 21 upregulated proteins in the aortic wall from SHRC, most of them were involved with the actin cytoskeleton organization, like Tropomyosin and Cofilin-1. After perindopril treatment, there was an upregulation of the GDP dissociation inhibitors (GDIs), which normally inhibits the RhoA/Rho-kinase/cofilin-1 pathway and may contribute to decreased arterial stiffening. In conclusion, the results of the present study revealed that treatment with perindopril reduced SBP and PWV in SHR. In addition, the proteomic analysis in aorta suggested, for the first time, that the RhoA/Rho-kinase/Cofilin-1 pathway may be inhibited by perindopril-induced upregulation of GDIs or increases in NO bioavailability in SHR. Therefore, we may propose that activation of GDIs or inhibition of RhoA/Rho-kinase pathway could be a possible strategy to treat arterial stiffness.

scholarly journals Effects of Tolvaptan on Oxidative Stress in ADPKD: A Molecular Biological Approach

2022 ◽  
Vol 11 (2) ◽  
pp. 402
Author(s):  
Matteo Rigato ◽  
Gianni Carraro ◽  
Irene Cirella ◽  
Silvia Dian ◽  
Valentina Di Di Vico ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Renal Deterioration ◽  
Deterioration Rate ◽  
Function Loss ◽  
State Marker ◽  
Molecular Biological Approach

Autosomal dominant polycystic disease (ADPKD) is the most frequent monogenic kidney disease. It causes progressive renal failure, endothelial dysfunction, and hypertension, all of which are strictly linked to oxidative stress (OxSt). Treatment with tolvaptan is known to slow the renal deterioration rate, but not all the molecular mechanisms involved in this effect are well-established. We evaluated the OxSt state in untreated ADPKD patients compared to that in tolvaptan-treated ADPKD patients and healthy subjects. OxSt was assessed in nine patients for each group in terms of mononuclear cell p22phox protein expression, NADPH oxidase key subunit, MYPT-1 phosphorylation state, marker of Rho kinase activity (Western blot) and heme oxygenase (HO)-1, induced and protective against OxSt (ELISA). p22phox protein expression was higher in untreated ADPKD patients compared to treated patients and controls: 1.42 ± 0.11 vs. 0.86 ± 0.15 d.u., p = 0.015, vs. 0.53 ± 0.11 d.u., p < 0.001, respectively. The same was observed for phosphorylated MYPT-1: 0.96 ± 0.28 vs. 0.68 ± 0.09 d.u., p = 0.013 and vs. 0.47 ± 0.13 d.u., p < 0.001, respectively, while the HO-1 expression of untreated patients was significantly lower compared to that of treated patients and controls: 5.33 ± 3.34 vs. 2.08 ± 0.79 ng/mL, p = 0.012, vs. 1.97 ± 1.22 ng/mL, p = 0.012, respectively. Tolvaptan-treated ADPKD patients have reduced OxSt levels compared to untreated patients. This effect may contribute to the slowing of renal function loss observed with tolvaptan treatment.

scholarly journals Dia- and Rok-dependent enrichment of capping proteins in a cortical region

2021 ◽  
Author(s):  
Anja Schmidt ◽  
Long Li ◽  
Zhiyi Lv ◽  
Shuling Yan ◽  
Jörg Großhans
Keyword(s):  
Myosin Ii ◽  
Rho Kinase ◽  
Cortical Region ◽  
Protein Enrichment ◽  
Cortical Actin ◽  
Capping Protein ◽  
Capping Proteins ◽  
Muscle Myosin ◽  
Drosophila Embryos

Rho signaling with its major targets the formin Dia, Rho kinase (Rok) and non-muscle myosin II control turnover, amount and contractility of actomyosin. Much less investigated has been a potential function for the distribution of F-actin plus and minus ends. In syncytial Drosophila embryos Rho1 signaling is high between actin caps, i. e. the cortical intercap region. Capping protein binds to free plus ends of F-actin to prevent elongation of the filament. Capping protein has served as a marker to visualize the distribution of F-actin plus ends in cells and in vitro. Here, we probed the distribution of plus ends with capping protein in syncytial Drosophila embryos. We found that Capping proteins are specifically enriched in the intercap region similar to Dia and MyoII but distinct from overall F-actin. The intercap enrichment of Capping protein was impaired in dia mutants and embryos, in which Rok and MyoII activation was inhibited. Our observations reveal that Dia and Rok/MyoII control Capping protein enrichment and support a model that Dia and Rok/MyoII control the organization of cortical actin cytoskeleton downstream of Rho1 signaling.

Abstract 533: Crucial Role of Rho-Kinase in Pressure Overload--Induced Right Ventricular Hypertrophy and Dysfunction in Mice: A Possible Novel Therapeutic Target of Right Ventricular Failure

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Shohei Ikeda ◽  
Kimio Satoh ◽  
Nobuhiro Kikuchi ◽  
Satoshi Miyata ◽  
Kota Suzuki ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Right Ventricular ◽  
Therapeutic Target ◽  
Crucial Role ◽  
Molecular Mechanisms ◽  
Rho Kinase ◽  
Pressure Overload ◽  
Dominant Negative ◽  
Term Survival ◽  
Novel Therapeutic

Rationale: Right ventricular (RV) failure is the leading cause of death in various cardiopulmonary diseases, including pulmonary hypertension. It is generally considered that the RV is vulnerable to pressure-overload as compared with the left ventricle (LV). However, as compared with LV failure, the molecular mechanisms of RV failure are poorly understood. Objective: We aimed to identify molecular therapeutic targets for RV failure in a mouse model of pressure-overload. Methods and Results: To induce pressure-overload to respective ventricles, we performed pulmonary artery constriction (PAC) or transverse aortic constriction (TAC) in mice. We first performed microarray analysis and found that the molecules related to RhoA/Rho-kinase and integrin pathways were significantly up-regulated in the RV with PAC compared with the LV with TAC. Then, we examined the responses of both ventricles to chronic pressure-overload in vivo. We demonstrated that compared with TAC, PAC caused greater extents of mortality, Rho-kinase expression (especially ROCK2 isoform) and oxidative stress in pressure-overloaded RV, reflecting the weakness of the RV in response to pressure-overload. Additionally, mechanical stretch of RV cardiomyocytes from rats immediately up-regulated ROCK2 expression (not ROCK1), suggesting the specific importance of ROCK2 in stretch-induced responses of RV tissues. Furthermore, mice with myocardial-specific overexpression of dominant-negative Rho-kinase (DN-RhoK) showed resistance to pressure-overload-induced hypertrophy and dysfunction associated with reduced oxidative stress. Finally, DN-RhoK mice showed a significantly improved long-term survival in both PAC and TAC as compared with littermate controls. Conclusions: These results indicate that the Rho-kinase pathway plays a crucial role in RV hypertrophy and dysfunction, suggesting that the pathway is a novel therapeutic target of RV failure in humans.

scholarly journals Distinct roles for paxillin and Hic-5 in regulating breast cancer cell morphology, invasion, and metastasis

2011 ◽  
Vol 22 (3) ◽  
pp. 327-341 ◽  
Author(s):  
Nicholas O. Deakin ◽  
Christopher E. Turner
Keyword(s):  
Breast Cancer ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Cell Morphology ◽  
Molecular Mechanisms ◽  
Adhesion Formation ◽  
Three Dimensional ◽  
Rho Kinase ◽  
Adaptor Protein ◽  
Metastatic Tumor

Individual metastatic tumor cells exhibit two interconvertible modes of cell motility during tissue invasion that are classified as either mesenchymal or amoeboid. The molecular mechanisms by which invasive breast cancer cells regulate this migratory plasticity have yet to be fully elucidated. Herein we show that the focal adhesion adaptor protein, paxillin, and the closely related Hic-5 have distinct and unique roles in the regulation of breast cancer cell lung metastasis by modulating cell morphology and cell invasion through three-dimensional extracellular matrices (3D ECMs). Cells depleted of paxillin by RNA interference displayed a highly elongated mesenchymal morphology, whereas Hic-5 knockdown induced an amoeboid phenotype with both cell populations exhibiting reduced plasticity, migration persistence, and velocity through 3D ECM environments. In evaluating associated signaling pathways, we determined that Rac1 activity was increased in cells devoid of paxillin whereas Hic-5 silencing resulted in elevated RhoA activity and associated Rho kinase–induced nonmuscle myosin II activity. Hic-5 was essential for adhesion formation in 3D ECMs, and analysis of adhesion dynamics and lifetime identified paxillin as a key regulator of 3D adhesion assembly, stabilization, and disassembly.

Genetic Basis For Hematopoietic Stem Cell Generation In The Mammalian Embryo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3667-3667
Author(s):  
Xin Gao ◽  
Kirby D Johnson ◽  
Yuan-I Chang ◽  
Meghan E Boyer ◽  
Colin N Dewey ◽  
...  
Keyword(s):  
Stem Cell ◽  
Knockout Mice ◽  
Molecular Mechanisms ◽  
Single Cells ◽  
Genetic Network ◽  
Dorsal Aorta ◽  
Hemogenic Endothelium ◽  
Mammalian Embryo ◽  
Hematopoietic Stem ◽  
Hematopoietic System

Abstract The generation of hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. Many questions remain unanswered regarding the molecular program in hemogenic endothelium that promotes the budding of hematopoietic cell clusters containing HSCs. Previously, we described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13-14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia (MDS)/leukemia. In contrast to Gata2 knockout mice, which die around E10.5 because of anemia, the prolonged embryonic development of +9.5 site knockout mice provides a unique model system to investigate the potential roles for GATA-2 in HSC production, migration and function, and more specifically, the requirement for the +9.5 element to regulate Gata2 expression during these processes. Using an ex vivo system involving culturing intact AGM, or AGM dissociated into single cells and then reaggregated into an organoid, we demonstrated that the +9.5 deletion reduced Gata2 expression in uncultured AGM (1.4 fold, p<0.05), cultured intact AGM (4 fold, p<0.001) and cultured AGM reaggregates (3.4 fold, p<0.001). The importance of the +9.5 element for Gata2 expression in the AGM suggested that it might control the function of hemogenic endothelium and/or the HSC progeny. The homozygous +9.5 mutation resulted in a complete loss of progenitors and long-term repopulating HSCs in the AGM, as determined by quantitative colony assays and competitive transplantation assays, respectively. To determine whether the ablation of HSC repopulating activity in the +9.5-/- mutant AGM reflects a +9.5 element requirement for HSC genesis from hemogenic endothelium, we used a whole-mount three-dimensional embryo immunostaining assay to visualize HSC genesis in +9.5+/+ and +9.5-/- AGMs. Imaging of E10.5 embryos revealed CD31+c-Kit+ hematopoietic clusters in +9.5+/+ dorsal aorta, while clusters were absent from the +9.5-/- embryos. The absence of hematopoietic clusters in the +9.5-/- dorsal aorta, and the ablation of HSC repopulating activity, demonstrated that the +9.5 element is required for hemogenic endothelium to generate HSCs in the AGM. In principle, the +9.5-dependent genetic network should reveal clues regarding the molecular mechanisms underlying the defective HSC generation in +9.5-/- AGMs. We conducted RNA-seq to define +9.5+/+ and +9.5-/- AGM explant transcriptomes, and this genomic analysis indicated that the +9.5 element instigates a stem cell-regulatory genetic network consisting of genes encoding established regulators of hemogenic endothelium and HSCs, and genes not implicated previously in hematopoiesis. We investigated whether the +9.5 element contributes to the transcriptome of AGM endothelium. Quantitative RT-PCR analysis revealed a similar impact of +9.5 deletion on representative genes in the fraction enriched in endothelial cells (CD31+c-Kit-) from the AGM. These studies establish a new model whereby a composite cis-regulatory element induces Gata2 expression and instigates a complex genetic network in the AGM, which controls the transition of hemogenic endothelium to HSCs in the AGM. Studies are ongoing to establish the genetic network in hemogenic endothelium that mediates the development of the adult hematopoietic system and the applicability of the respective mechanisms to distinct biological and pathological contexts. Disclosures: No relevant conflicts of interest to declare.

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