DOCSLIB.ORG

A Rhog-Mediated Signaling Pathway That Modulates Invadopodia Dynamics in Breast Cancer Cells Silvia M

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Rhog-Mediated Signaling Pathway That Modulates Invadopodia Dynamics in Breast Cancer Cells Silvia M © 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 1064-1077 doi:10.1242/jcs.195552 RESEARCH ARTICLE A RhoG-mediated signaling pathway that modulates invadopodia dynamics in breast cancer cells Silvia M. Goicoechea, Ashtyn Zinn, Sahezeel S. Awadia, Kyle Snyder and Rafael Garcia-Mata* ABSTRACT micropinocytosis, bacterial uptake, phagocytosis and leukocyte One of the hallmarks of cancer is the ability of tumor cells to invade trans-endothelial migration (deBakker et al., 2004; Ellerbroek et al., surrounding tissues and metastasize. During metastasis, cancer cells 2004; Jackson et al., 2015; Katoh et al., 2006, 2000; van Buul et al., degrade the extracellular matrix, which acts as a physical barrier, by 2007). Recent studies have revealed that RhoG plays a role in tumor developing specialized actin-rich membrane protrusion structures cell invasion and may contribute to the formation of invadopodia called invadopodia. The formation of invadopodia is regulated by Rho (Hiramoto-Yamaki et al., 2010; Kwiatkowska et al., 2012). GTPases, a family of proteins that regulates the actin cytoskeleton. Invadopodia are actin-rich adhesive structures that form in the Here, we describe a novel role for RhoG in the regulation of ventral surface of cancer cells and allow them to degrade the invadopodia disassembly in human breast cancer cells. Our results extracellular matrix (ECM) (Gimona et al., 2008). Formation of show that RhoG and Rac1 have independent and opposite roles invadopodia involves a series of steps that include the disassembly in the regulation of invadopodia dynamics. We also show that SGEF of focal adhesions and stress fibers, and the relocalization of several (also known as ARHGEF26) is the exchange factor responsible of their components into the newly formed invadopodia (Hoshino for the activation of RhoG during invadopodia disassembly. When et al., 2012; Oikawa et al., 2008). Invadopodia assembly starts with the expression of either RhoG or SGEF is silenced, invadopodia the formation of actin- and cortactin-rich puncta, followed by the are more stable and have a longer lifetime than in control cells. recruitment of adhesion proteins, such as vinculin and paxillin, and Our findings also demonstrate that RhoG and SGEF modulate the proteinases that allow ECM degradation (Hoshino et al., 2013). phosphorylation of paxillin, which plays a key role during invadopodia Even though many of the molecular components required for disassembly. In summary, we have identified a novel signaling invadopodia formation have been identified, the signaling pathways pathway involving SGEF, RhoG and paxillin phosphorylation, which that regulate these events are still poorly understood (Linder et al., functions in the regulation of invadopodia disassembly in breast 2011). Invadopodia formation is controlled by the integrated cancer cells. activity of several GTPases, including Cdc42, RhoA, RhoC and Rac1 (Spuul et al., 2014). Cdc42 promotes invadopodia formation KEY WORDS: RhoG, Invadopodia, SGEF, Guanine-nucleotide in almost every system tested (Ayala et al., 2009; Di Martino et al., exchange factors, Src, Paxillin, Rac1 2014; Moreau et al., 2006, 2003; Nakahara et al., 2003; Tatin et al., 2006). However, generalized conclusions cannot be drawn from INTRODUCTION studies of other Rho GTPases, since their activities can both inhibit Rho GTPases control many aspects of cell behavior ranging from or promote invadopodia formation depending on the experimental the regulation of cytoskeletal organization, cell motility and cell conditions or cell type used (Spuul et al., 2014). polarity, to nuclear gene expression and control of cell growth In this study, we describe a novel function for RhoG in the (Hodge and Ridley, 2016). Rho proteins cycle between an active regulation of invadopodia dynamics in human breast cancer cells. (GTP-bound) and an inactive (GDP-bound) state. The activation of Our findings describe a signaling pathway involving SGEF and Rho proteins involves the exchange of GDP for GTP, which is RhoG that regulates invadopodia disassembly independently of catalyzed by specific guanine-nucleotide-exchange factors (GEFs). Rac1 through regulation of paxillin phosphorylation. Once activated, Rho GTPases interact with a wide variety of downstream effectors to modulate their activity and/or localization. RESULTS The hydrolysis of GTP to GDP, a reaction that is stimulated by RhoG is a negative regulator of invadopodia formation GTPase-activating proteins (GAPs), inactivates the GTPases and We have previously shown that the breast cancer cell line SUM159 terminates the signal. With more than 80 Rho GEFs, 70 Rho GAPs forms invadopodia when treated with phorbol esters such phorbol and over 100 effectors, cells regulate the activity of Rho proteins 12,13-dibutyrate (PDBu) and 12-O-tetradecanoylphorbol-13- through multiple pathways, thus acting as key nodes for signal acetate (PMA), and that the formation of these structures integration and dissemination (Bustelo et al., 2007; Rossman et al., correlates with their metastatic potential (Goicoechea et al., 2009). 2005; Tcherkezian and Lamarche-Vane, 2007). To determine the requirement of RhoG in invadopodia formation, RhoG, a Rho protein related to Rac, has been associated we generated stable SUM159 cell lines in which RhoG expression with cell migration, neurite outgrowth, microtubule dynamics, was silenced using lentivirally encoded shRNA. We used two different RhoG-specific shRNAs (shRNA#1 and shRNA#4) to rule Department of Biological Sciences, University of Toledo, Toledo, OH 43606, USA. out off-target effects. A stable cell line expressing a non-targeting shRNA was used as control (CTRL). Fig. 1A shows that RhoG *Author for correspondence ([email protected]) silencing was efficient, especially for shRNA #4. SUM159 cells are S.M.G., 0000-0003-1107-389X; R.G.-M., 0000-0002-7116-4411 able to form invadopodia spontaneously, so we first looked at the effect of RhoG depletion in untreated cells. To identify invadopodia Received 22 July 2016; Accepted 14 January 2017 structures, we stained the cells with the invadopodia markers Journal of Cell Science 1064 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1064-1077 doi:10.1242/jcs.195552 Fig. 1. RhoG negatively regulates invadopodia formation in SUM159 cells. (A) Cell lysates from SUM159 cells stably expressing non-targeting (CTRL) or RhoG-specific shRNAs (shRNA#1 and shRNA #4) were analyzed by western blotting and probed for RhoG and Rac1, and for tubulin as a loading control. (B) Quantification of cortactin- and actin-containing invadopodia in CTRL and RhoG KD cells. Results are expressed as the percentage of cells with invadopodia. Data are mean±s.e.m. for at least three independent experiments. Cells were either untreated, or treated with PDBu or PMA. (C) CTRL or RhoG KD SUM159 cells were treated with PDBu for 30 min and stained with anti-cortactin antibody (red), Alexa-Fluor-488–phalloidin (green) and Hoechst 33342 (blue). Arrowheads indicate representative invadopodia. (D) Cell lysates from CTRL, RhoG KD and RhoG KD cells expressing shRNA resistant Myc– RhoG (rescue) were immunoblotted with anti-RhoG and -Myc antibodies. Tubulin was used as a loading control. (E) Quantification of cortactin- and actin-containing invadopodia in CTRL, RhoG KD and rescue cells. Data are mean±s.e.m. of three independent experiments. (F) Cell lysates from CTRL, Myc–RhoG wt (RhoG wt) or Myc–RhoG Q61L (RhoG Q61L) cells were immunoblotted with anti-RhoG and -Myc antibodies. Tubulin was used as a loading control. (G) SUM159 cells transiently transfected with Myc- tagged wild-type (RhoG wt) or constitutively active RhoG (RhoG Q61L) were treated with PDBu for 30 min and stained with anti-cortactin antibody (red), anti-Myc antibody (green), Alexa-Fluor-647–phalloidin (magenta) and Hoechst 33342 (blue). Arrowheads indicate transfected cells and arrows indicate representative invadopodia. (H) Invadopodia were quantified in CTRL (non-transfected cells), and RhoG wt and RhoG Q61L transfected cells. Results are representative of three independent experiments in which at least 100 cells per experiment were counted. Data are mean±s.e.m. (error bars). Scale bars: 10 µm. *P<0.05; **P<0.01; ***P<0.001; ns, not significant. cortactin and actin. We found that silencing RhoG induced a depletion in cells treated with phorbol esters. In PDBu-treated cells, significant increase in the number of spontaneous invadopodia the number of cells with invadopodia increased significantly in when compared to CTRL cells, from 7% in CTRL cells to 14% and RhoG-knockdown (KD) cells, from 35% in CTRL cells to 64% 23% in RhoG shRNA#1 and shRNA#4 cells, respectively (Fig. 1B, and 81% in RhoG shRNA#1 and shRNA#4 cells respectively untreated; Fig. S1A). We next looked at the effect of RhoG (Fig. 1B,C). Similarly, the percentage of cells with invadopodia Journal of Cell Science 1065 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1064-1077 doi:10.1242/jcs.195552 increased from 31% in CTRL cells to 79% of cells in PMA-treated SUM159 cells, using specific shRNAs. We tested two independent RhoG KD cells (Fig. 1B; Fig. S1B). Based on these results, and shRNAs for each of the GEFs and compared them to cells unless otherwise indicated, we used shRNA#4 for the rest of these expressing a non-targeting shRNA (CTRL). The KD efficiency was studies (referred to as RhoG KD). We next performed a rescue verified either by quantitative real-time PCR (qRT-PCR; Fig. 3A; experiment
Load more

Recommended publications
  • Dock3 Protects Myelin in the Cuprizone Model for Demyelination
    Citation: Cell Death and Disease (2014) 5, e1395; doi:10.1038/cddis.2014.357 OPEN & 2014 Macmillan Publishers Limited All rights reserved 2041-4889/14 www.nature.com/cddis Dock3 protects myelin in the cuprizone model for demyelination K Namekata1, A Kimura1, C Harada1, H Yoshida2, Y Matsumoto1 and T Harada*,1,2 Dedicator of cytokinesis 3 (Dock3) belongs to an atypical family of the guanine nucleotide exchange factors. It is predominantly expressed in the neural tissues and causes cellular morphological changes by activating the small GTPase Rac1. We previously reported that Dock3 overexpression protects retinal ganglion cells from excitotoxic cell death. Oligodendrocytes are the myelinating cells of axons in the central nervous system and these cells are damaged in demyelinating disorders including multiple sclerosis (MS) and optic neuritis. In this study, we examined if Dock3 is expressed in oligodendrocytes and if increasing Dock3 signals can suppress demyelination in a cuprizone-induced demyelination model, an animal model of MS. We demonstrate that Dock3 is expressed in oligodendrocytes and Dock3 overexpression protects myelin in the corpus callosum following cuprizone treatment. Furthermore, we show that cuprizone demyelinates optic nerves and the extent of demyelination is ameliorated in mice overexpressing Dock3. Cuprizone treatment impairs visual function, which was demonstrated by multifocal electroretinograms, an established non-invasive method, and Dock3 overexpression prevented this effect. In mice overexpressing Dock3, Erk activation is increased, suggesting this may at least partly explain the observed protective effects. Our findings suggest that Dock3 may be a therapeutic target for demyelinating disorders including optic neuritis. Cell Death and Disease (2014) 5, e1395; doi:10.1038/cddis.2014.357; published online 28 August 2014 Dedicator of cytokinesis 3 (Dock3), an atypical member of the will restore the visual function.
    [Show full text]
  • Structure of the Dock2âˆ'elmo1 Complex Provides Insights Into
    ARTICLE https://doi.org/10.1038/s41467-020-17271-9 OPEN Structure of the DOCK2−ELMO1 complex provides insights into regulation of the auto-inhibited state Leifu Chang1,7, Jing Yang1, Chang Hwa Jo 2, Andreas Boland 1,8, Ziguo Zhang 1, Stephen H. McLaughlin 1, Afnan Abu-Thuraia 3, Ryan C. Killoran2, Matthew J. Smith 2,4,9, Jean-Francois Côté 3,5,6,9 & ✉ David Barford 1 DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange 1234567890():,; factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2−ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG−ELMO2RBD complex. The binary DOCK2−ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto- inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF. 1 MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK. 2 Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec H3T 1J4, Canada. 3 Montreal Institute of Clinical Research (IRCM), Montréal, QC H2W 1R7, Canada.
    [Show full text]
  • Active Rac1 Detection Kit 2012 09/20
    Active Rac1 Detection Kit 1 Kit Orders n 877-616-CELL (2355) (30 immunoprecipitations) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com rev. 09/24/20 #8815 For Research Use Only. Not For Use In Diagnostic Procedures. Species Cross-Reactivity: H, M Components Ship As: 11894S Item # Kit Quantity Storage Temp Description: The Active Rac1 Detection Kit provides all GTPgS 11521 1 X 50 µl –80°C reagents necessary for measuring activation of Rac1 GTPase in the cell. GST-PAK1-PBD fusion protein is used to bind GDP 11522 1 X 50 µl –80°C the activated form of GTP-bound Rac1, which can then be Components Ship As: 11894S Item # Kit Quantity Storage Temp immunoprecipitated with glutathione resin. Rac1 activation levels are then determined by western blot using a Rac1 GST-Human PAK1-PBD 8659 1 X 600 µg –20°C Mouse mAb. Rac1 Mouse mAb 8631 1 X 50 µl –20°C Specificity/Sensitivity: Active Rac1 Detection Kit detects Components Ship As: 11860S Item # Kit Quantity Storage Temp endogenous levels of GTP-bound (active) Rac1 as shown in Lysis/Binding/Wash Buffer 11524 1 X 100 mL 4°C Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect Glutathione Resin 11523 1 X 3 ml 4°C homologous proteins from other species. SDS Sample Buffer 11525 1 X 1.5 ml 4°C Background: The Ras superfamily of small GTP-binding Spin Cup and Collection Tubes 11526 1 X 30 vial RT proteins (G proteins) comprise a large class of proteins (over 150 members) that can be classified into at least five families based on their sequence and functional similarities: 1 2 3 4 Figure 1.
    [Show full text]
  • Rac1 Accumulates in the Nucleus During the G2 Phase of the Cell
    Published Online: 28 April, 2008 | Supp Info: http://doi.org/10.1083/jcb.200801047 JCB: ARTICLE Downloaded from jcb.rupress.org on May 9, 2018 Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division David Michaelson , 2,5 Wasif Abidi , 2,5 Daniele Guardavaccaro , 4,5 Mo Zhou, 3,5 Ian Ahearn , 3,5 Michele Pagano , 4,5 and Mark R. Philips 1,2,3,5 1 Department of Medicine, 2 Department of Cell Biology, 3 Department of Pharmacology, 4 Department of Pathology, and the 5 New York University Cancer Institute, New York University School of Medicine, New York, NY 10016 ac1 regulates a wide variety of cellular processes. zation of cells stably expressing low levels of GFP-Rac1, The polybasic region of the Rac1 C terminus func- and time-lapse microscopy of asynchronous cells revealed Rtions both as a plasma membrane – targeting motif Rac1 accumulation in the nucleus in late G2 and exclu- and a nuclear localization sequence (NLS). We show that sion in early G1. Although constitutively active Rac1 re- a triproline N-terminal to the polybasic region contributes stricted to the cytoplasm inhibited cell division, activated to the NLS, which is cryptic in the sense that it is strongly Rac1 expressed constitutively in the nucleus increased the inhibited by geranylgeranylation of the adjacent cysteine. mitotic rate. These results show that Rac1 cycles in and out Subcellular fractionation demonstrated endogenous Rac1 of the nucleus during the cell cycle and thereby plays a in the nucleus and Triton X-114 partition revealed that this role in promoting cell division.
    [Show full text]
  • The Rac Gtpase in Cancer: from Old Concepts to New Paradigms Marcelo G
    Published OnlineFirst August 14, 2017; DOI: 10.1158/0008-5472.CAN-17-1456 Cancer Review Research The Rac GTPase in Cancer: From Old Concepts to New Paradigms Marcelo G. Kazanietz1 and Maria J. Caloca2 Abstract Rho family GTPases are critical regulators of cellular func- mislocalization of Rac signaling components. The unexpected tions that play important roles in cancer progression. Aberrant pro-oncogenic functions of Rac GTPase-activating proteins also activity of Rho small G-proteins, particularly Rac1 and their challenged the dogma that these negative Rac regulators solely regulators, is a hallmark of cancer and contributes to the act as tumor suppressors. The potential contribution of Rac tumorigenic and metastatic phenotypes of cancer cells. This hyperactivation to resistance to anticancer agents, including review examines the multiple mechanisms leading to Rac1 targeted therapies, as well as to the suppression of antitumor hyperactivation, particularly focusing on emerging paradigms immune response, highlights the critical need to develop ther- that involve gain-of-function mutations in Rac and guanine apeutic strategies to target the Rac pathway in a clinical setting. nucleotide exchange factors, defects in Rac1 degradation, and Cancer Res; 77(20); 5445–51. Ó2017 AACR. Introduction directed toward targeting Rho-regulated pathways for battling cancer. Exactly 25 years ago, two seminal papers by Alan Hall and Nearly all Rho GTPases act as molecular switches that cycle colleagues illuminated us with one of the most influential dis- between GDP-bound (inactive) and GTP-bound (active) forms. coveries in cancer signaling: the association of Ras-related small Activation is promoted by guanine nucleotide exchange factors GTPases of the Rho family with actin cytoskeleton reorganization (GEF) responsible for GDP dissociation, a process that normally (1, 2).
    [Show full text]
  • The Atypical Guanine-Nucleotide Exchange Factor, Dock7, Negatively Regulates Schwann Cell Differentiation and Myelination
    The Journal of Neuroscience, August 31, 2011 • 31(35):12579–12592 • 12579 Cellular/Molecular The Atypical Guanine-Nucleotide Exchange Factor, Dock7, Negatively Regulates Schwann Cell Differentiation and Myelination Junji Yamauchi,1,3,5 Yuki Miyamoto,1 Hajime Hamasaki,1,3 Atsushi Sanbe,1 Shinji Kusakawa,1 Akane Nakamura,2 Hideki Tsumura,2 Masahiro Maeda,4 Noriko Nemoto,6 Katsumasa Kawahara,5 Tomohiro Torii,1 and Akito Tanoue1 1Department of Pharmacology and 2Laboratory Animal Resource Facility, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan, 3Department of Biological Sciences, Tokyo Institute of Technology, Midori, Yokohama 226-8501, Japan, 4IBL, Ltd., Fujioka, Gumma 375-0005, Japan, and 5Department of Physiology and 6Bioimaging Research Center, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan In development of the peripheral nervous system, Schwann cells proliferate, migrate, and ultimately differentiate to form myelin sheath. In all of the myelination stages, Schwann cells continuously undergo morphological changes; however, little is known about their underlying molecular mechanisms. We previously cloned the dock7 gene encoding the atypical Rho family guanine-nucleotide exchange factor (GEF) and reported the positive role of Dock7, the target Rho GTPases Rac/Cdc42, and the downstream c-Jun N-terminal kinase in Schwann cell migration (Yamauchi et al., 2008). We investigated the role of Dock7 in Schwann cell differentiation and myelination. Knockdown of Dock7 by the specific small interfering (si)RNA in primary Schwann cells promotes dibutyryl cAMP-induced morpholog- ical differentiation, indicating the negative role of Dock7 in Schwann cell differentiation. It also results in a shorter duration of activation of Rac/Cdc42 and JNK, which is the negative regulator of myelination, and the earlier activation of Rho and Rho-kinase, which is the positive regulator of myelination.
    [Show full text]
  • Related Malignant Phenotypes in the Nf1-Deficient MPNST
    Published OnlineFirst February 19, 2013; DOI: 10.1158/1541-7786.MCR-12-0593 Molecular Cancer Genomics Research RAS/MEK–Independent Gene Expression Reveals BMP2- Related Malignant Phenotypes in the Nf1-Deficient MPNST Daochun Sun1, Ramsi Haddad2,3, Janice M. Kraniak2, Steven D. Horne1, and Michael A. Tainsky1,2 Abstract Malignant peripheral nerve sheath tumor (MPNST) is a type of soft tissue sarcoma that occurs in carriers of germline mutations in Nf1 gene as well as sporadically. Neurofibromin, encoded by the Nf1 gene, functions as a GTPase-activating protein (GAP) whose mutation leads to activation of wt-RAS and mitogen-activated protein kinase (MAPK) signaling in neurofibromatosis type I (NF1) patients' tumors. However, therapeutic targeting of RAS and MAPK have had limited success in this disease. In this study, we modulated NRAS, mitogen-activated protein/extracellular signal–regulated kinase (MEK)1/2, and neurofibromin levels in MPNST cells and determined gene expression changes to evaluate the regulation of signaling pathways in MPNST cells. Gene expression changes due to neurofibromin modulation but independent of NRAS and MEK1/2 regulation in MPNST cells indicated bone morphogenetic protein 2 (Bmp2) signaling as a key pathway. The BMP2-SMAD1/5/8 pathway was activated in NF1-associated MPNST cells and inhibition of BMP2 signaling by LDN-193189 or short hairpin RNA (shRNA) to BMP2 decreased the motility and invasion of NF1-associated MPNST cells. The pathway-specific gene changes provide a greater understanding of the complex role of neurofibromin in MPNST pathology and novel targets for drug discovery. Mol Cancer Res; 11(6); 616–27.
    [Show full text]
  • Involvement of the Rho/Rac Family Member Rhog in Caveolar Endocytosis
    Oncogene (2006) 25, 2961–2973 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Involvement of the Rho/Rac family member RhoG in caveolar endocytosis RM Prieto-Sa´ nchez1, IM Berenjeno1 and XR Bustelo Centro de Investigacio´n del Ca´ncer, Instituto de Biologı´a Molecular y Celular del Ca´ncer (IBMCC), and Red Tema´tica Cooperativa de Centros de Ca´ncer, CSIC-University of Salamanca, Campus Unamuno, Salamanca, Spain We show here that the GTPase RhoG is involved in transduction, cell polarity, and intercellular communi- caveolar trafficking. Wild-type RhoG moves sequentially cations (Gruenberg, 2001; Conner and Schmid, 2003; Di to the plasma membrane, intracellular vesicles, and the Guglielmo et al., 2003; Felberbaum-Corti et al., 2003; Golgi apparatus along markers of this endocytic pathway. Gonzalez-Gaitan, 2003). The best-known endocytic Such translocation is associated with changes in RhoG pathway is that composed of clathrin-coated vesicles GDP/GTP levels and is highly dependent on lipid raft (Zerial and McBride, 2001; Conner and Schmid, 2003). integrity and on the function of the GTPase dynamin2. In In this case, we have information regarding the addition, the constitutively active RhoGQ61L mutant is internalization, sorting, recycling, fusion, and delivery preferentially located in endocytic vesicles that can be events that participate in this type of endocytosis (Zerial decorated with markers of the caveola-derived endocytic and McBride, 2001; Conner and Schmid, 2003). This pathway. RhoGQ61L, but not the analogous Rac1 mutant pathway has also been molecularly dissected, resulting protein, affects caveola internalization and the subsequent in the characterization of a large number of regulatory delivery of endocytic vesicles to the Golgi apparatus.
    [Show full text]
  • Role of Rac1-Pak Pathway in Aggressive B-Cell Lymphoma
    University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-4-2019 Role of Rac1-Pak pathway in aggressive b-cell lymphoma Tian Tian University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Hemic and Lymphatic Diseases Commons, and the Neoplasms Commons Recommended Citation Tian, Tian, "Role of Rac1-Pak pathway in aggressive b-cell lymphoma" (2019). Theses & Dissertations. 344. https://digitalcommons.unmc.edu/etd/344 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. i Role of Rac1-PAK Pathway in Aggressive B-cell Lymphomas By Tian Tian A DISSERTATION Presented to the Faculty of The University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Pathology & Microbiology Graduate program Under the Supervision of Professor Kai Fu University of Nebraska Medical Center, Omaha, Nebraska Dec, 2018 Supervisory Committee: Ying Yan, Ph.D. John S Davis, Ph.D. Timothy C Greiner, M.D. Javeed Iqbal, Ph.D. ii Role of Rac1-PAK pathway in Aggressive B-cell Lymphomas Tian Tian University of Nebraska Medical Center, 2018 Advisor: Kai Fu, M.D. Ph.D. Aggressive B-cell lymphomas are diverse group of neoplasms that arise at different stages of B-cell development and by various mechanisms of neoplastic transformation. Aggressive B-cell lymphomas include many types, subtypes and variants of diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), mantle cell lymphoma (MCL) and B lymphoblastic lymphoma.
    [Show full text]
  • Rac1 Promotes Cell Motility by Controlling Cell Mechanics in Human Glioblastoma
    cancers Article Rac1 Promotes Cell Motility by Controlling Cell Mechanics in Human Glioblastoma Jing Xu 1,2, Nicola Galvanetto 1, Jihua Nie 1,3, Yili Yang 2,* and Vincent Torre 1,2,3,4,* 1 International School for Advanced Studies (SISSA), 34136 Trieste, Italy; [email protected] (J.X.); [email protected] (N.G.); [email protected] (J.N.) 2 Joint Laboratory of Biophysics and Translational Medicine, Suzhou Institute of Systems Medicine (ISM)- International School for Advanced Studies (SISSA), Suzhou 215123, China 3 School of Radiation Medicine and Protection, State Key Laboratory of Radiation Medicine and Protection Medical College of Soochow University, Suzhou 215123, China 4 Cixi Institute of Biomedical Engineering, Ningbo Institute of Materials Technology and Engineering Chinese Academy of Sciences, Ningbo 315201, China * Correspondence: [email protected] (Y.Y.);[email protected] (V.T.) Received: 21 May 2020; Accepted: 2 June 2020; Published: 23 June 2020 Abstract: The failure of existing therapies in treating human glioblastoma (GBM) mostly is due to the ability of GBM to infiltrate into healthy regions of the brain; however, the relationship between cell motility and cell mechanics is not well understood. Here, we used atomic force microscopy (AFM), live-cell imaging, and biochemical tools to study the connection between motility and mechanics in human GBM cells. It was found thatRac1 inactivation by genomic silencing and inhibition with EHT 1864 reduced cell motility, inhibited cell ruffles, and disrupted the dynamics of cytoskeleton organization and cell adhesion. These changes were correlated with abnormal localization of myosin IIa and a rapid suppression of the phosphorylation of Erk1/2.
    [Show full text]
  • A Rac/Cdc42 Exchange Factor Complex Promotes Formation of Lateral filopodia and Blood Vessel Lumen Morphogenesis
    ARTICLE Received 1 Oct 2014 | Accepted 26 Apr 2015 | Published 1 Jul 2015 DOI: 10.1038/ncomms8286 OPEN A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis Sabu Abraham1,w,*, Margherita Scarcia2,w,*, Richard D. Bagshaw3,w,*, Kathryn McMahon2,w, Gary Grant2, Tracey Harvey2,w, Maggie Yeo1, Filomena O.G. Esteves2, Helene H. Thygesen2,w, Pamela F. Jones4, Valerie Speirs2, Andrew M. Hanby2, Peter J. Selby2, Mihaela Lorger2, T. Neil Dear4,w, Tony Pawson3,z, Christopher J. Marshall1 & Georgia Mavria2 During angiogenesis, Rho-GTPases influence endothelial cell migration and cell–cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell–cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis. 1 Institute of Cancer Research, Division of Cancer Biology, 237 Fulham Road, London SW3 6JB, UK.
    [Show full text]
  • Anti-CRK Monoclonal Antibody, Clone 4H22D2 (DCABH-1226) This Product Is for Research Use Only and Is Not Intended for Diagnostic Use
    Anti-CRK monoclonal antibody, clone 4H22D2 (DCABH-1226) This product is for research use only and is not intended for diagnostic use. PRODUCT INFORMATION Product Overview Mouse monoclonal to Crk p38 Antigen Description The Crk-I and Crk-II forms differ in their biological activities. Crk-II has less transforming activity than Crk-I. Crk-II mediates attachment-induced MAPK8 activation, membrane ruffling and cell motility in a Rac-dependent manner. Involved in phagocytosis of apoptotic cells and cell motility via its interaction with DOCK1 and DOCK4. Immunogen Purified recombinant fragment of Human Crk p38 expressed in E. Coli. Isotype IgG2b Source/Host Mouse Species Reactivity Mouse, Human Clone 4H22D2 Purity Ascites Conjugate Unconjugated Applications WB, ELISA, IHC-P, Flow Cyt, ICC/IF Positive Control Recombinant Crk p38 protein; Crk p38-hIgGFc transfected HEK293 cell lysate; Human rectum cancer and bladder cancer tissues; MCF7 and 3T3 L1 cells. Format Liquid Size 100 μl Buffer Preservative: 0.03% Sodium azide; Constituent: 99% Ascites Preservative 0.03% Sodium Azide Storage Store at 4°C or at -20°C for long term storage. GENE INFORMATION 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 1 © Creative Diagnostics All Rights Reserved Gene Name CRK v-crk sarcoma virus CT10 oncogene homolog (avian) [ Homo sapiens ] Official Symbol CRK Synonyms CRK; v-crk sarcoma virus CT10 oncogene homolog (avian); v crk avian sarcoma virus CT10 oncogene homolog; adapter molecule crk; proto-oncogene
    [Show full text]