Mason Final Dissertation Version 6.8.2011

Total Page:16

File Type:pdf, Size:1020Kb

Mason Final Dissertation Version 6.8.2011 Identification of a Novel Formin‑GAP Complex and Its Role in Macrophage Migration and Phagocytosis by Frank Marshall Mason, Jr. Department of Cell Biology Duke University Date:_______________________ Approved: ___________________________ Scott Soderling, Supervisor ___________________________ Vann Bennett, Chair ___________________________ John Klingensmith ___________________________ Terry Lechler ___________________________ Ann Marie Pendergast Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2011 i v ABSTRACT Identification of a Novel Formin‑GAP Complex and Its Role in Macrophage Migration and Phagocytosis by Frank Marshall Mason, Jr. Department of Cell Biology Duke University Date:_______________________ Approved: ___________________________ Scott Soderling, Supervisor ___________________________ Vann Bennett, Chair ___________________________ John Klingensmith ___________________________ Terry Lechler ___________________________ Ann Marie Pendergast An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2011 Copyright by Frank Marshall Mason, Jr. 2011 Abstract Essential and diverse biological processes such as cell division, morphogenesis and migration are regulated by a family of molecular switches called Rho GTPases. These proteins cycle between active, GTP‑bound and inactive, GDP‑bound states, and this cycle is regulated by families of proteins called Rho GEFs and GAPs. GAPs are proteins that stimulate the intrinsic GTPase activity of Rho‑family proteins, potentiating the active to inactive transition. GAPs target specific spatiotemporal pools of GTPases by responding to cellular cues and utilizing protein‑protein interactions. By dissecting these interactions and pathways, we can infer and then decipher the biological functions of these GAPs. This work focuses on the characterization of a novel Rho‑family GAP called srGAP2. In this study, we identify that srGAP2 is a Rac‑specific GAP that binds a Formin‑family member, Formin‑like 1 (FMNL1). FMNL1 is activated by Rac and polymerizes, bundles and severs actin filaments. srGAP2 specifically inhibits actin severing by active FMNL1, and the assembly of an srGAP2‑FMNL1 complex is regulated by Rac. Work on FMNL1 shows that it plays important roles in regulating phagocytosis and adhesion in macrophages. To learn more about srGAP2 and its role in regulating FMNL1, we studied macrophages isolated from an srGAP2 KO mouse we have recently generated. This work demonstrates that loss of srGAP2 decreases the iv ability for macrophages to invade through extracellular matrix but increases phagocytosis. These results suggest that these two processes might be coordinated in vivo by srGAP2 and that srGAP2 might be a critical regulator of the innate immune system. v Dedication To my wife and best friend, who makes me smile every day and loves me for the person I am. To my parents, who have always supported me. I could never fully express my gratitude. vi Contents Abstract .......................................................................................................................................... iv List of Figures ............................................................................................................................... xi List of Abreviations .................................................................................................................... xiii Acknowledgements .................................................................................................................... xv 1 Introduction ............................................................................................................................. 1 1.1 Rho‑Family GTPases and Their Regulation .............................................................. 1 1.1.1 Rho‑family GTPases ................................................................................................. 1 1.1.2 Guanine Nucleotide Exchange Factors ................................................................. 6 1.1.3 Rho GTPase Activating Proteins ............................................................................ 9 1.2 Actin and Actin Remodeling Proteins ...................................................................... 15 1.2.1 Actin ......................................................................................................................... 15 1.2.2 Arp2/3 and Arp2/3‑Activating Proteins .............................................................. 18 1.2.3 Formins .................................................................................................................... 21 1.2.4 WH2‑Containing Proteins ..................................................................................... 24 1.2.5 Actin Severing Proteins ......................................................................................... 26 1.3 srGAP (WRP) Family of Rho GAPs .......................................................................... 28 1.3.1 F‑BAR and IF‑BAR domains ................................................................................. 30 1.3.2 SH3 domains ........................................................................................................... 32 1.3.3 srGAP1 ..................................................................................................................... 34 1.3.4 WRP .......................................................................................................................... 34 vii 1.3.5 srGAP2 ..................................................................................................................... 35 2 Materials and Methods ........................................................................................................ 37 2.1 Cell Culture .................................................................................................................. 37 2.2 Plasmids and Constructs ............................................................................................ 37 2.3 Antibodies for Staining and Immunoblots .............................................................. 38 2.4 Immunoprecipitations and Pulldowns .................................................................... 38 2.5 Immunostaining .......................................................................................................... 39 2.6 Yeast Two‑Hybrid ....................................................................................................... 40 2.7 Protein Production and Purification ......................................................................... 40 2.8 Production and Purification of Profilin .................................................................... 41 2.9 Actin Severing Assays ................................................................................................ 42 2.10 Preparation of NEM‑Treated Myosin ..................................................................... 44 2.11 Actin Filament Binding and Bundling Assays ...................................................... 45 2.12 Pyrene Actin Assays ................................................................................................. 46 2.13 GAP Assays and PAK Pulldowns ........................................................................... 48 2.14 Induction and Isolation of Peritoneal Macrophages ............................................ 49 2.15 2D Matrix Degradation by Macrophages .............................................................. 50 2.16 Preparation of Radiolabeled Protein for Peptide Array Overlay ....................... 53 2.17 Isolation of Bone Marrow Cells (Macrophages) ................................................... 53 2.18 3D‑Invasion of Bone Marrow Macrophages ......................................................... 55 2.19 Phagocytosis ............................................................................................................... 57 3 Bi‑modal regulation of a formin by srGAP2 ..................................................................... 59 viii 3.1 Introduction .................................................................................................................. 59 3.2 Results ........................................................................................................................... 61 3.2.1 srGAP2 binds Formin‑like 1, Formin‑like 3, and Afadin ................................. 61 3.2.2 srGAP2 regulates signaling between Rac and FMNL1 .................................... 66 3.2.3 Dynamic formation of the srGAP2/FMNL1 complex ....................................... 69 3.2.4 Both srGAP2 and FMNL1 co‑localize with F‑actin during Fc‑γ receptor phagocytosis ........................................................................................................................ 72 3.2.5 Direct regulation of FMNL1 actin severing, but not bundling or severing by the srGAP2 SH3 domain .................................................................................................... 73 3.3 Discussion ..................................................................................................................... 81 4 The Role of srGAP2 in Macrophages ................................................................................
Recommended publications
  • Formin Proteins in Normal Tissues and Cancer
    FORMIN PROTEINS IN NORMAL TISSUES AND CANCER Maria Gardberg TURUN YLIOPISTON JULKAISUJA – ANNALES UNIVERSITATIS TURKUENSIS Sarja - ser. D osa - tom. 1163 | Medica - Odontologica | Turku 2015 University of Turku Faculty of Medicine Institute of Biomedicine Department of Pathology National Graduate School of Clinical Investigation (CLIGS) Doctoral Programme of Clinical Investigation (CLIDP) University of Turku and Turku University Hospital Supervised by Professor Olli Carpén Department of Pathology Turku University Hospital and University of Turku, Turku, Finland Reviewed by Docent Maria Vartiainen Professor Veli-Pekka Lehto Institute of Biotechnology Department of Pathology University of Helsinki, Helsinki, Finland University of Helsinki, Helsinki, Finland Opponent Professor Robert Grosse Institute of Pharmacology University of Marburg, Marburg, Germany The originality of this thesis has been checked in accordance with the University of Turku quality assurance system using the Turnitin OriginalityCheck service. ISBN 978-951-29-6044-6 (PRINT) ISBN 978-951-29-6045-3 (PDF) ISSN 0355-9483 Painosalama Oy – Turku, Finland 2015 To my family 4 Abstract ABSTRACT Maria Gardberg Formin proteins in normal tissues and cancer Department of Pathology, University of Turku, Turku, Finland (2015) The actin cytoskeleton is a dynamic structure that determines cell shape. Actin turnover is mandatory for migration in normal and malignant cells. In epithelial cancers invasion is frequently accompanied by epithelial to mesenchymal transition (EMT). In EMT, cancer cells acquire a migratory phenotype through transcriptional reprogramming. EMT requires substantial re-organization of actin. During the past decade, new actin regulating proteins have been discovered. Among these are members of the formin family. To study formin expression in tissues and cells, antibodies for detection of formin proteins FMNL1 (Formin-like protein 1), FMNL2 (Formin-like protein 2) and FHOD1 (Formin homology 2 domain containing protein 1) were used.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Bundling of Cytoskeletal Actin by the Formin FMNL1 Contributes to Celladhesion and Migration
    Bundling of cytoskeletal actin by the formin FMNL1 contributes to celladhesion and migration Item Type Dissertation Authors Miller, Eric Rights Attribution-NonCommercial-NoDerivatives 4.0 International Download date 27/09/2021 05:11:17 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/20.500.12648/1760 Bundling of cytoskeletal actin by the formin FMNL1 contributes to cell adhesion and migration Eric W. Miller A Dissertation in the Department of Cell and Developmental Biology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies of State University of New York, Upstate Medical University Approved ______________________ Dr. Scott D. Blystone Date______________________ i Table of Contents Title Page-------------------------------------------------------------------------------------------------------i Table of Contents-------------------------------------------------------------------------------------------ii List of Tables and Figures------------------------------------------------------------------------------vi Abbreviations----------------------------------------------------------------------------------------------viii Acknowledgements--------------------------------------------------------------------------------------xiii Thesis Abstract-------------------------------------------------------------------------------------------xvi Chapter 1: General Introduction-----------------------------------------------------------------------1
    [Show full text]
  • Formin Mdia1, a Downstream Molecule of FMNL1, Regulates Profilin1 for Actin Assembly and Spindle Organization During Mouse Oocyte Meiosis☆
    Biochimica et Biophysica Acta 1853 (2015) 317–327 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr Formin mDia1, a downstream molecule of FMNL1, regulates Profilin1 for actin assembly and spindle organization during mouse oocyte meiosis☆ Yu Zhang a,FeiWanga,Ying-JieNiua,Hong-LinLiua,RongRuib, Xiang-Shun Cui c, Nam-Hyung Kim c, Shao-Chen Sun a,⁎ a College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China b College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China c Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea article info abstract Article history: Mammalian diaphanous1 (mDia1) is a homologue of Drosophila diaphanous and belongs to the Formin- Received 26 August 2014 homology family of proteins that catalyze actin nucleation and polymerization. Although Formin family proteins, Received in revised form 6 October 2014 such as Drosophila diaphanous, have been shown to be essential for cytokinesis, whether and how mDia1 Accepted 4 November 2014 functions during meiosis remain uncertain. In this study, we explored possible roles and the signaling pathway Available online 15 November 2014 involved for mDia1 using a mouse oocyte model. mDia1 depletion reduced polar body extrusion, which may have been due to reduced cortical actin assembly. mDia1 and Profilin1 had similar localization patterns in Keywords: fi mDia1 mouse oocytes and mDia1 knockdown resulted in reduced Pro lin1 expression. Depleting FMNL1, another Profilin1 Formin family member, resulted in reduced mDia1 expression, while RhoA inhibition did not alter mDia1 expres- Actin sion, which indicated that there was a FMNL1-mDia1-Profilin1 signaling pathway in mouse oocytes.
    [Show full text]
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts
    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
    [Show full text]
  • Rho Activation of Mdia Formins Is Modulated by an Interaction with Inverted Formin 2 (INF2)
    Rho activation of mDia formins is modulated by an interaction with inverted formin 2 (INF2) Hua Suna,b,c, Johannes S. Schlondorffb,c, Elizabeth J. Brownc,d, Henry N. Higgse, and Martin R. Pollakb,c,1 aNephrology Division, Department of Medicine, Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China; bNephrology Division, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215; cDepartment of Medicine, Harvard Medical School, Boston, MA 02115; dDivision of Nephrology, Department of Medicine, Children’s Hospital, Boston, MA 02115; and eDepartment of Biochemistry, Dartmouth Medical School, Hanover, NH 03755 Edited by Christine E. Seidman, Harvard Medical School, Boston, MA, and approved December 30, 2010 (received for review November 12, 2010) Inverted formin 2 (INF2) encodes a member of the diaphanous glomerular slit diaphragm (11–13). The importance of the actin subfamily of formin proteins. Mutations in INF2 cause human cytoskeleton in maintaining the glomerular filtration barrier is kidney disease characterized by focal and segmental glomerulo- supported by the fact that mutations in α-actinin-4, an actin cross- sclerosis. Disease-causing mutations occur only in the diaphanous linking protein, cause a similar form of autosomal-dominant FSGS inhibitory domain (DID), suggesting specific roles for this domain in (14). Numerous lines of evidence support the notion that podo- the pathogenesis of disease. In a yeast two-hybrid screen, we cytes are highly sensitive to perturbations in their actin cytoskel- identified the diaphanous autoregulatory domains (DADs) of the eton (15). Consistent with this, FSGS-associated mutant forms of mammalian diaphanous-related formins (mDias) mDia1, mDia2, INF2 induce distinct patterns of actin polymerization in cultured and mDia 3 as INF2_DID-interacting partners.
    [Show full text]
  • Integrated Characterisation of Cancer Genes Identifies Key Molecular Biomarkers in Stomach Adenocarcinoma Haifeng Wang,1 Liyijing Shen,2 Yaoqing Li ‍ ,1 Jieqing Lv1
    Original research J Clin Pathol: first published as 10.1136/jclinpath-2019-206400 on 7 February 2020. Downloaded from Integrated characterisation of cancer genes identifies key molecular biomarkers in stomach adenocarcinoma Haifeng Wang,1 Liyijing Shen,2 Yaoqing Li ,1 Jieqing Lv1 ► Additional material is ABSTRact is essential for the early diagnosis and prognosis of published online only. To view Aims Gastric cancer is one of the leading causes patients with GC. please visit the journal online In recent years, numerous next generation (http:// dx. doi. org/ 10. 1136/ for cancer mortality. Recent studies have defined the jclinpath- 2019- 206400). landscape of genomic alterations of gastric cancer and sequencing studies have characterised the genomics their association with clinical outcomes. However, the basis and found many actionable genetic drivers 1 Department of Gastrointestinal pathogenesis of gastric cancer has not been completely in GC. CDH1, RhoA and ARID1A mutations are Surgery, Shaoxing People’s characterised. a common set of genetic variations related to the Hospital, Shaoxing Hospital of 3–6 Zhejiang University, Shaoxing, Methods Driver genes were detected by five diffuse subtype of GCs from various regions. Zhejiang Province, China computational tools, MutSigCV, OncodriveCLUST, TP53, TGFβR2, ARID1A, CDH1, SYNE1 and 2 Department of radiology, OncodriveFM, dendrix and edriver, using mutation data TMPRSS2 were recurrently mutated genes in 49 4 Shaoxing People’s Hospital, of stomach adenocarcinoma (STAD) from the cancer late stage GC tumours. The Cancer Genome Altas Shaoxing Hospital of Zhejiang (TCGA) project classified GC tumours into four University, Shaoxing, Zhejiang genome altas database, followed by an integrative Province, China investigation.
    [Show full text]
  • Statistical and Bioinformatic Analysis of Hemimethylation Patterns in Non-Small Cell Lung Cancer Shuying Sun1* , Austin Zane2, Carolyn Fulton3 and Jasmine Philipoom4
    Sun et al. BMC Cancer (2021) 21:268 https://doi.org/10.1186/s12885-021-07990-7 RESEARCH ARTICLE Open Access Statistical and bioinformatic analysis of hemimethylation patterns in non-small cell lung cancer Shuying Sun1* , Austin Zane2, Carolyn Fulton3 and Jasmine Philipoom4 Abstract Background: DNA methylation is an epigenetic event involving the addition of a methyl-group to a cytosine- guanine base pair (i.e., CpG site). It is associated with different cancers. Our research focuses on studying non-small cell lung cancer hemimethylation, which refers to methylation occurring on only one of the two DNA strands. Many studies often assume that methylation occurs on both DNA strands at a CpG site. However, recent publications show the existence of hemimethylation and its significant impact. Therefore, it is important to identify cancer hemimethylation patterns. Methods: In this paper, we use the Wilcoxon signed rank test to identify hemimethylated CpG sites based on publicly available non-small cell lung cancer methylation sequencing data. We then identify two types of hemimethylated CpG clusters, regular and polarity clusters, and genes with large numbers of hemimethylated sites. Highly hemimethylated genes are then studied for their biological interactions using available bioinformatics tools. Results: In this paper, we have conducted the first-ever investigation of hemimethylation in lung cancer. Our results show that hemimethylation does exist in lung cells either as singletons or clusters. Most clusters contain only two or three CpG sites. Polarity clusters are much shorter than regular clusters and appear less frequently. The majority of clusters found in tumor samples have no overlap with clusters found in normal samples, and vice versa.
    [Show full text]
  • Multiple Formin Proteins Participate in Glioblastoma Migration Vanina D
    Heuser et al. BMC Cancer (2020) 20:710 https://doi.org/10.1186/s12885-020-07211-7 RESEARCH ARTICLE Open Access Multiple formin proteins participate in glioblastoma migration Vanina D. Heuser1,2, Aida Kiviniemi3, Laura Lehtinen1,2, Sune Munthe4, Bjarne Winther Kristensen5, Jussi P. Posti6,7, Jussi O. T. Sipilä8,9,10, Ville Vuorinen11, Olli Carpén2,12 and Maria Gardberg1,2* Abstract Background: The prognosis of glioblastoma remains poor, related to its diffuse spread within the brain. There is an ongoing search for molecular regulators of this particularly invasive behavior. One approach is to look for actin regulating proteins that might be targeted by future anti-cancer therapy. The formin family of proteins orchestrates rearrangement of the actin cytoskeleton in multiple cellular processes. Recently, the formin proteins mDia1 and mDia2 were shown to be expressed in glioblastoma in vitro, and their function could be modified by small molecule agonists. This finding implies that the formins could be future therapeutic targets in glioblastoma. Methods: In cell studies, we investigated the changes in expression of the 15 human formins in primary glioblastoma cells and commercially available glioblastoma cell lines during differentiation from spheroids to migrating cells using transcriptomic analysis and qRT-PCR. siRNA mediated knockdown of selected formins was performed to investigate whether their expression affects glioblastoma migration. Using immunohistochemistry, we studied the expression of two formins, FHOD1 and INF2, in tissue samples from 93 IDH-wildtype glioblastomas. Associated clinicopathological parameters and follow-up data were utilized to test whether formin expression correlates with survival or has prognostic value. Results: We found that multiple formins were upregulated during migration.
    [Show full text]
  • Gene Section Review
    Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL INIST-CNRS Gene Section Review FMNL1 (formin like 1) methyltransferase) Matheus Rodrigues Lopes, Patricia Favaro Hematology and Hemotherapy Center, University of Campinas - UNICAMP, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas São Paulo (MRL, PF), MRL Presently at Federal University of Vale do São Francisco at Paulo Afonso, Bahia; Department of Biological Sciences, Federal University of São Paulo (PF), Diadema, São Paulo, Brazil. [email protected], [email protected] Published in Atlas Database: October 2016 Online updated version : http://AtlasGeneticsOncology.org/Genes/FMNL1ID46313ch17q21.html Printable original version : http://documents.irevues.inist.fr/bitstream/handle/2042/68240/10-2016-FMNL1ID46313ch17q21.pdf DOI: 10.4267/2042/68240 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2017 Atlas of Genetics and Cytogenetics in Oncology and Haematology from the Human Cancer Genome Project (Dias Neto Abstract et al., 2000). The entire FMNL1 gene is located in Formin-like 1 (FMNL1) is a member of the Formin chromosome 17 (17q21) and has a size of protein family, which are regulators of actin and approximately 25.8 Kb (start: 45221444 and end: microtubule cytoskeletal dynamics. FMNL1 belongs 45247320 bp; orientation: Plus strand) and contains to the subfamily of formins knows as Diaphanous- 17 exons. The FMNL1 cDNA contains 3973 bp and related formins (DRF) and is involved in processes is located in chromosome 17 (17q21). such as phagocytosis, cell adhesion, podosome dynamics, cell migration, cytokinesis, and polarity Protein control. It has been suggested that spatial and temporal regulation of FMNL1 is controlled by Description small Rho GTPases.
    [Show full text]
  • Statistical and Bioinformatic Analysis of Hemimethylation Patterns in Lung Cancer
    Statistical and Bioinformatic Analysis of Hemimethylation Patterns in Lung Cancer Shuying Sun ( [email protected] ) Texas State University San Marcos https://orcid.org/0000-0003-3974-6996 Austin Zane Texas A&M University College Station Carolyn Fulton Schreiner University Jasmine Philipoom Case Western Reserve University Research article Keywords: Methylation, Hemimethylation, Lung Cancer, Bioinformatics, Epigenetics Posted Date: March 20th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-17794/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on March 12th, 2021. See the published version at https://doi.org/10.1186/s12885-021-07990-7. Page 1/28 Abstract Background: DNA methylation is an epigenetic event involving the addition of a methyl-group to a cytosine-guanine base pair (i.e., CpG site). It is associated with different cancers. Our research focuses on studying lung cancer hemimethylation, which refers to methylation occurring on only one of the two DNA strands. Many studies often assume that methylation occurs on both DNA strands at a CpG site. However, recent publications show the existence of hemimethylation and its impact. It is important to identify cancer hemimethylation patterns. Methods: In this paper, we use the Wilcoxon signed rank test to identify hemimethylated CpG sites based on publicly available lung cancer methylation sequencing data. We then identify two types of hemimethylated CpG clusters, regular and polarity clusters, and genes with large numbers of hemimethylated sites. Highly hemimethylated genes are then studied for their biological interactions using available bioinformatics tools.
    [Show full text]
  • FMNL1 Sirna (H): Sc-62325
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . FMNL1 siRNA (h): sc-62325 BACKGROUND STORAGE AND RESUSPENSION Formin-like protein 1 (FMNL1, formin-related protein, Frl) is a 1,094 amino acid Store lyophilized siRNA duplex at -20° C with desiccant. Stable for at least protein encoded by the mouse gene Fmnl1. FMNL1 belongs to the formin one year from the date of shipment. Once resuspended, store at -20° C, homology family and has one DAD (diaphanous autoregulatory domain), one avoid contact with RNAses and repeated freeze thaw cycles. FH2 (formin homology 2) domain and one GBD/FH3 (Rho GTPase-binding/ Resuspend lyophilized siRNA duplex in 330 µl of the RNAse-free water formin homology 3) domain. Formins are a conserved class of proteins pro vided. Resuspension of the siRNA duplex in 330 µl of RNAse-free water expressed in all eukaryotes, with known roles in generating cellular Actin- makes a 10 µM solution in a 10 µM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM based structures. FMNL1 is believed to play a role in the control of cell motility EDTA buffered solution. and survival of macrophages. FMNL1 has been found to interact with Rac 1, PFN1 and PFN2 and can block apoptotic cell death and inhibit cell adhesion APPLICATIONS and migration. FMNL1 is located in the cytoplasm and is highly expressed in the spleen, lymph nodes and bone marrow cells. FMNL1 siRNA (h) is recommended for the inhibition of FMNL1 expression in human cells. REFERENCES SUPPORT REAGENTS 1. Yayoshi-Yamamoto, S., et al. 2000.
    [Show full text]