scholarly journals BNIP-2 retards breast cancer cell migration by coupling microtubule-mediated GEF-H1 and RhoA activation

2020 ◽  
Vol 6 (31) ◽  
pp. eaaz1534 ◽  
Author(s):  
Meng Pan ◽  
Ti Weng Chew ◽  
Darren Chen Pei Wong ◽  
Jingwei Xiao ◽  
Hui Ting Ong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Migration ◽  
Focal Adhesions ◽  
Fine Tuning ◽  
Nucleotide Exchange ◽  
Microtubule Depolymerization ◽  
Cell Contractility ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Guanosine Triphosphatase

Microtubules display dynamic turnover during cell migration, leading to cell contractility and focal adhesion maturation regulated by Rho guanosine triphosphatase activity. This interplay between microtubules and actomyosin is mediated by guanine nucleotide exchange factor (GEF)–H1 released after microtubule depolymerization or microtubule disconnection from focal adhesions. However, how GEF-H1 activates Rho upon microtubule disassembly remains elusive. Here, we found that BNIP-2, a BCH domain–containing protein that binds both RhoA and GEF-H1 and traffics with kinesin-1 on microtubules, is important for GEF-H1–driven RhoA activation upon microtubule disassembly. Depletion of BNIP-2 in MDA-MB-231 breast cancer cells decreases RhoA activity and promotes cell migration. Upon nocodazole-induced microtubule disassembly, the interaction between BNIP-2 and GEF-H1 increases, while knockdown of BNIP-2 reduces RhoA activation and cell rounding via uncoupling RhoA-GEF-H1 interaction. Together, these findings revealed that BNIP-2 couples microtubules and focal adhesions via scaffolding GEF-H1 and RhoA, fine-tuning RhoA activity and cell migration.

scholarly journals Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

2017 ◽  
Vol 43 (5) ◽  
pp. 1777-1789 ◽  
Author(s):  
Lei Zhang ◽  
Tianrong Ji ◽  
Qin Wang ◽  
Kexin Meng ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Protective Action ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Dependent Manner ◽  
Calcium Sensing Receptor ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Calcium Sensing

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+]i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists.

scholarly journals The Abl-related Gene Tyrosine Kinase Acts through p190RhoGAP to Inhibit Actomyosin Contractility and Regulate Focal Adhesion Dynamics upon Adhesion to Fibronectin

2007 ◽  
Vol 18 (10) ◽  
pp. 3860-3872 ◽  
Author(s):  
Justin G. Peacock ◽  
Ann L. Miller ◽  
William D. Bradley ◽  
Olga C. Rodriguez ◽  
Donna J. Webb ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Cell Periphery ◽  
Related Gene ◽  
Cell Contractility ◽  
Fibroblast Migration ◽  
Actomyosin Contractility

In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin–dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg−/− fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg−/− fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain–containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg−/− cells, the increased contractility of arg−/− cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions.

scholarly journals Novel Functions of Ect2 in Polar Lamellipodia Formation and Polarity Maintenance during “Contractile Ring-Independent” Cytokinesis in Adherent Cells

2008 ◽  
Vol 19 (1) ◽  
pp. 8-16 ◽  
Author(s):  
Masamitsu Kanada ◽  
Akira Nagasaki ◽  
Taro Q.P. Uyeda
Keyword(s):  
Mammalian Cells ◽  
Polar Regions ◽  
Nucleotide Exchange ◽  
Contractile Ring ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ht1080 Cells ◽  
Lamellipodia Formation

Some mammalian cells are able to divide via both the classic contractile ring-dependent method (cytokinesis A) and a contractile ring-independent, adhesion-dependent method (cytokinesis B). Cytokinesis A is triggered by RhoA, which, in HeLa cells, is activated by the guanine nucleotide-exchange factor Ect2 localized at the central spindle and equatorial cortex. Here, we show that in HT1080 cells undergoing cytokinesis A, Ect2 does not localize in the equatorial cortex, though RhoA accumulates there. Moreover, Ect2 depletion resulted in only modest multinucleation of HT1080 cells, enabling us to establish cell lines in which Ect2 was constitutively depleted. Thus, RhoA is activated via an Ect2-independent pathway during cytokinesis A in HT1080 cells. During cytokinesis B, Ect2-depleted cells showed narrower accumulation of RhoA at the equatorial cortex, accompanied by compromised pole-to-equator polarity, formation of ectopic lamellipodia in regions where RhoA normally would be distributed, and delayed formation of polar lamellipodia. Furthermore, C3 exoenzyme inhibited equatorial RhoA activation and polar lamellipodia formation. Conversely, expression of dominant active Ect2 in interphase HT1080 cells enhanced RhoA activity and suppressed lamellipodia formation. These results suggest that equatorial Ect2 locally suppresses lamellipodia formation via RhoA activation, which indirectly contributes to restricting lamellipodia formation to polar regions during cytokinesis B.

scholarly journals RhoA GTPase inhibition organizes contraction during epithelial morphogenesis

2016 ◽  
Vol 214 (5) ◽  
pp. 603-617 ◽  
Author(s):  
Frank M. Mason ◽  
Shicong Xie ◽  
Claudia G. Vasquez ◽  
Michael Tworoger ◽  
Adam C. Martin
Keyword(s):  
Cell Shape ◽  
Shape Change ◽  
Critical Role ◽  
Central Position ◽  
Nucleotide Exchange ◽  
Apical Constriction ◽  
Cell Contractility ◽  
Rhoa Activity ◽  
Cell Shape Change ◽  
Rhoa Gtpase

During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding.

scholarly journals p190RhoGAP is the convergence point of adhesion signals from α5β1 integrin and syndecan-4

2008 ◽  
Vol 181 (6) ◽  
pp. 1013-1026 ◽  
Author(s):  
Mark D. Bass ◽  
Mark R. Morgan ◽  
Kirsty A. Roach ◽  
Jeffrey Settleman ◽  
Andrew B. Goryachev ◽  
...  
Keyword(s):  
Adhesion Formation ◽  
Focal Adhesions ◽  
Dependent Manner ◽  
Rhoa Activity ◽  
Focal Adhesion Formation ◽  
Convergence Point ◽  
Individual Contributions ◽  
Guanosine Triphosphatase ◽  
Extracellular Matrix Receptors ◽  
Α5β1 Integrin

The fibronectin receptors α5β1 integrin and syndecan-4 cocluster in focal adhesions and coordinate cell migration by making individual contributions to the suppression of RhoA activity during matrix engagement. p190Rho–guanosine triphosphatase–activating protein (GAP) is known to inhibit RhoA during the early stages of cell spreading in an Src-dependent manner. This paper dissects the mechanisms of p190RhoGAP regulation and distinguishes the contributions of α5β1 integrin and syndecan-4. Matrix-induced tyrosine phosphorylation of p190RhoGAP is stimulated solely by engagement of α5β1 integrin and is independent of syndecan-4. Parallel engagement of syndecan-4 causes redistribution of the tyrosine-phosphorylated pool of p190RhoGAP between membrane and cytosolic fractions by a mechanism that requires direct activation of protein kinase C α by syndecan-4. Activation of both pathways is necessary for the efficient regulation of RhoA and, as a consequence, focal adhesion formation. Accordingly, we identify p190RhoGAP as the convergence point for adhesive signals mediated by α5β1 integrin and syndecan-4. This molecular mechanism explains the cooperation between extracellular matrix receptors during cell adhesion.

scholarly journals PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

2008 ◽  
Vol 180 (1) ◽  
pp. 187-203 ◽  
Author(s):  
Yangmi Lim ◽  
Ssang-Taek Lim ◽  
Alok Tomar ◽  
Margaret Gardel ◽  
Joie A. Bernard-Trifilo ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cellular Contact

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

scholarly journals Nitric oxide may regulate focal adhesion turnover and cell migration in MDA-MB-231 breast cancer cells by modulating early endosome trafficking

2021 ◽  
Vol 9 (2) ◽  
pp. 60-72
Author(s):  
Dhurgham Al-Fahad ◽  
Bandar Fahad Alharbi ◽  
Clementino Ibeas Bih ◽  
Philip Richard Dash
Keyword(s):  
Breast Cancer ◽  
Nitric Oxide ◽  
Cell Migration ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Focal Adhesions ◽  
Early Endosome ◽  
No Synthase ◽  
Endosome Trafficking ◽  
In Cells

Abstract Cell migration is an essential process for wound healing, metastasis and inflammation. Focal adhesions (FA) are local regions of plasma membrane consisting of multiprotein complexes providing adhesive contact between the cell and the extracellular matrix (ECM). FA turnover regulates different signalling pathways implicated in various cellular responses (e.g. cell migration). Endocytosis, specifically the dynamin and clathrin pathways, is known to regulate cell migration by modulating FA dynamics. In this study, we investigated whether NO activity regulates cell migration, FA dynamics and early endosome trafficking in MDA-MB-231 cells. The assessment of cell migration showed a slowing down of cell migration and an increased duration of FA turnover in cells treated with inhibitors of NO synthase (NOS) such as L-NAME or 1400W. In addition, these treatments were found to exhibit no effect on transferrin and dextran uptake mediated by endocytosis and micropinocytosis, respectively. The number of early endosome antigen 1 (EEA1)-positive endosomes was reduced while their sizes were found to increase in cells treated with L-NAME or 1400W. In contrast, these inhibitors did not affect the number nor the size of Rab5-positive endosomes. Furthermore, we demonstrated that EEA1, endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) were colocalised. Using the biotin switch assay followed by western blot, we showed that early endosome proteins such as APPL1, EEA1, Rab5 were found to be S-nitrosylated. These results were further supported by the sequence analysis performed with the GPS-SNO algorithm which predicted the S-nitrosylation of these endosomal proteins. Taken together, our findings suggest that NO might be involved in cell migration and FA turnover through early endosome trafficking in MDA-MB-231 cells. Running title: Nitric oxide in MDA-MB-231 breast cancer cells

scholarly journals Novel Association of Vav2 and Nek3 Modulates Signaling through the Human Prolactin Receptor

2005 ◽  
Vol 19 (4) ◽  
pp. 939-949 ◽  
Author(s):  
Sommer L. Miller ◽  
Jamie E. DeMaria ◽  
David O. Freier ◽  
Angela M. Riegel ◽  
Charles V. Clevenger
Keyword(s):  
Breast Cancer ◽  
Signaling Pathways ◽  
Prolactin Receptor ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Signal Transducer ◽  
Nucleotide Exchange ◽  
T47d Cells ◽  
Related Family ◽  
Guanosine Triphosphatase

Abstract Prolactin (PRL) receptor activation contributes to the progression and motility of human breast cancer. This event activates multimeric signaling pathways, including the activation of the Vav family of guanine nucleotide exchange factors. To detect novel proteins interacting with Vav, yeast two-hybrid analysis was performed and demonstrated an interaction between the serine/threonine NIMA (never in mitosis A)-related family kinase p56Nek3 and Vav1. The PRL-dependent interaction of Nek3 with Vav1 and Vav2 was confirmed by coimmunoprecipitation analysis. PRL stimulation of T47D cells induced Nek3 kinase activity and the interaction of Vav2/Nek3 with the PRL receptor. Increased Nek3 levels up-regulated Vav2 serine and tyrosine phosphorylation, whereas knockdown of Nek3 resulted in a reduction of Vav2 phosphorylation. Activation of guanosine triphosphatase Rac-1 in Chinese hamster ovary transfectants required both Nek3 and Vav2 and was inhibited by the overexpression of a kinase inactivating Nek3 mutant. However, overexpression of either Nek3 or kinase-inactive Nek3 had no effect on Vav2-potentiated signal transducer and activator of transcription 5-mediated gene expression. Overexpression of kinase inactive Nek3 in T47D cells led to a 50% increase in apoptosis vs. controls. These data suggest that the PRL-mediated activation of Nek3 contributes differentially to Vav2 signaling pathways involving Rac1 and signal transducer and activator of transcription 5 and implicates Nek3 during PRL-mediated actions in breast cancer.

scholarly journals Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed

2008 ◽  
Vol 183 (6) ◽  
pp. 999-1005 ◽  
Author(s):  
Margaret L. Gardel ◽  
Benedikt Sabass ◽  
Lin Ji ◽  
Gaudenz Danuser ◽  
Ulrich S. Schwarz ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesions ◽  
Traction Force ◽  
Flow Speed ◽  
Retrograde Flow ◽  
Filamentous Actin ◽  
Rho Family ◽  
Guanosine Triphosphatase ◽  
Protein Density ◽  
Traction Stress

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8–10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

scholarly journals Leptin induces cell migration and invasion in a FAK-Src-dependent manner in breast cancer cells

2019 ◽  
Vol 8 (11) ◽  
pp. 1539-1552 ◽  
Author(s):  
Juan Carlos Juárez-Cruz ◽  
Miriam Daniela Zuñiga-Eulogio ◽  
Monserrat Olea-Flores ◽  
Eduardo Castañeda-Saucedo ◽  
Miguel Ángel Mendoza-Catalán ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Migration ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Breast Cancer Cells ◽  
Focal Adhesions ◽  
Migration And Invasion ◽  
Breast Cancer Cell Lines ◽  
Dependent Manner

Breast cancer is the most common invasive neoplasia, and the second leading cause of the cancer deaths in women worldwide. Mammary tumorigenesis is severely linked to obesity, one potential connection is leptin. Leptin is a hormone secreted by adipocytes, which contributes to the progression of breast cancer. Cell migration, metalloproteases secretion, and invasion are cellular processes associated with various stages of metastasis. These processes are regulated by the kinases FAK and Src. In this study, we utilized the breast cancer cell lines MCF7 and MDA-MB-231 to determine the effect of leptin on FAK and Src kinases activation, cell migration, metalloprotease secretion, and invasion. We found that leptin activates FAK and Src and induces the localization of FAK to the focal adhesions. Interestingly, leptin promotes the activation of FAK through a Src- and STAT3-dependent canonical pathway. Specific inhibitors of FAK, Src and STAT3 showed that the effect exerted by leptin in cell migration in breast cancer cells is dependent on these proteins. Moreover, we established that leptin promotes the secretion of the extracellular matrix remodelers, MMP-2 and MMP-9 and invasion in a FAK and Src-dependent manner. Our findings strongly suggest that leptin promotes the development of a more aggressive invasive phenotype in mammary cancer cells.

Sign in / Sign up

Export Citation Format

Share Document