DOCSLIB.ORG

TLR4-Mediated Expulsion of Bacteria from Infected Bladder Epithelial Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
TLR4-Mediated Expulsion of Bacteria from Infected Bladder Epithelial Cells TLR4-mediated expulsion of bacteria from infected bladder epithelial cells Jeongmin Songa,1, Brian L. Bishopa,1, Guojie Lib, Richard Gradyc, Ann Stapletond,e, and Soman N. Abrahama,b,f,g,2 Departments of aMolecular Genetics and Microbiology, bPathology, and fImmunology, Duke University Medical Center, Durham, NC 27710; gProgram in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore 169547; and Departments of cUrology and dMedicine, and eDivision of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98195 Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved July 16, 2009 (received for review January 18, 2009) Uropathogenic Escherichia coli invade bladder epithelial cells bladder expansion. That E. coli are harbored in fusiform compart- (BECs) by direct entry into specialized cAMP regulated exocytic ments of infected BECs was confirmed by an immune microscopy, compartments. Remarkably, a significant number of these intra- which revealed bacteria encased in membranes closely associated cellular bacteria are subsequently expelled in a nonlytic and piece- with Rab27b, a marker of fusiform vesicles (9). meal fashion by infected BECs. Here, we report that expulsion of Surprisingly, a significant number of intracellular E. coli, har- intracellular E. coli by infected BECs is initiated by the pattern bored within exocytic compartments of cultured BECs, were sub- recognition receptor, Toll-like receptor (TLR)4, after activation by sequently returned into the extracellular medium in piecemeal LPS. Also, we reveal that caveolin-1, Rab27b, PKA, and MyRIP are fashion (9). Seemingly, BECs have the innate capacity to sense and components of the exocytic compartment, and that they form a expel infecting bacteria. Here, we sought to examine the specificity complex involved in the exocytosis of bacteria. This capacity of of this host cell response and elucidate the underlying mechanism. TLR4 to mediate the expulsion of intracellular bacteria from in- fected cells represents a previously unrecognized function for this Results innate immune receptor. Expulsion of E. coli from Infected Human BECs. Our earlier studies have suggested that infected BECs have the capacity to spontane- caveolin ͉ exocytosis ͉ signaling ͉ urinary tract infection ͉ ously exocytose UPEC (9). We sought to examine whether this uropathogenic Escherichia coli exocytic response of BECs was specific to E. coli, and if so, to identify the bacterial component(s) responsible. We infected the uring their lifetimes, 10–20% of American females will receive 5637 human BEC line with E. coli CI5, a previously described UTI Dmedical attention for a urinary tract infection (UTI), and Ϸ3% isolate (12), as well as ORN103(pSH2), a K12 strain of E. coli will experience more than one infection per year (1, 2). UTIs expressing type 1 fimbriae, the critical bacterial component respon- represent the second leading cause of physician visits in the U.S., sible for E. coli entry into BECs (10). To examine the specificity of costing the health care system over $2 billion per year (1, 2). the BEC response, we also used two other bacterial strains Salmo- Interestingly, when compared with other mucosal surfaces, the UT nella typhimurium SL1344 and Staphyloccus aureus 54 in this assay. is difficult to colonize. Much of the resistance of the UT is After1hofinfection with each of the 4 strains of bacteria, a attributable to the flushing actions of urine and to the imperme- standard gentamicin protection assay was undertaken, and after ability of the epithelial lining. Because of their specialized role in 24 h, intracellular bacterial numbers assessed again. Whereas the storing urine, the apical surface of superficial bladder epithelial numbers of intracellular S. typhimuium SL1344 and S. aureus 54 had cells (BECs) is lined by scalloped-shaped plaques comprising a markedly increased implying intracellular growth, the same could not be said of E. coli CI5 or E. coli ORN103(pSH2) (Fig. 1A). We tightly interlaced latticework of proteins called uroplakins (3). Ϸ These proteins are closely associated with a collection of lipids, found a 60% drop in intracellular E. coli numbers (Fig. 1A), E. coli sphingolipids, and cholesterol, often referred to as lipid rafts that suggesting active exocytosis by infected BECs, although we cannot rule out the possibility of intracellular bacterial killing. In cumulatively constitute a surface that is highly impregnable to any case, our findings suggest that this drop in intracellular numbers urine, solutes, and potential pathogens (3). of E. coli is a unique phenomenon, because significant increases in Uropathogenic Escherichia coli (UPEC) are uniquely successful intracellular S. typhimurium and S. aureus numbers were observed in overcoming the bladder defenses, accounting for Ͼ85% of all under the same incubation conditions (Fig. 1A). bladder infections. The singular success of UPEC in the UT is We next sought to establish that the drop in the number of ascribed primarily to its capacity to penetrate and harbor within the intracellular bacteria in E. coli infected BECs was attributable to superficial BECs (4). Several studies have reported that UPEC also expulsion of bacteria rather than loss of viability. For these and for assume distinct intracellular shapes and form ‘‘intracellular bacte- most of the subsequent studies, we used E. coli ORN103(pSH2), rial communities’’ (IBCs) in the cytoplasm of superficial epithelial because the genome of this strain is only a fraction of that found on cells of both rodent and human bladders (5–7). This ability of UPEC strains, making it infinitely easier to pinpoint the genetic UPEC to successfully breach the mucosal barrier and colonize these factor(s) responsible for reduced intracellular numbers. We in- cells represents a critical initiating event in the development of fected multiple cultures of BECs with E. coli ORN103(pSH2) for UTIs. 1 h followed by 30-min gentamicin treatment to eliminate extra- E. coli invasion of BECs has been reported to involve several components of lipid rafts such as caveolin-1, an integral membrane protein found in the inner leaflet of the lipid bilayer on many Author contributions: J.S., B.L.B., G.L., and S.N.A. designed research; J.S., B.L.B., and G.L. mammalian cells, and Rac1, a member of the Rho family of performed research; R.G. and A.S. contributed new reagents/analytic tools; J.S., B.L.B., G.L., GTPases (8). A remarkable aspect of E. coli entry into BECs came and S.N.A. analyzed data; and J.S., B.L.B., and S.N.A. wrote the paper. from the recent finding that, after entry, the bacteria were harbored The authors declare no conflict of interest. within specialized exocytic vesicles (9). These compartments (or This article is a PNAS Direct Submission. fusiform vesicles) in BECs perform an important physiologic 1J.S. and B.L.B. contributed equally to this work. function by providing the necessary membranes required for blad- 2To whom correspondence should be addressed. E-mail: [email protected]. der expansion. As urine volume increases, these vesicles fuse with This article contains supporting information online at www.pnas.org/cgi/content/full/ the apical bladder surface in a cAMP-dependent manner allowing 0900527106/DCSupplemental. 14966–14971 ͉ PNAS ͉ September 1, 2009 ͉ vol. 106 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900527106 Downloaded by guest on September 26, 2021 A B 450 *** 30000 400 0 hr 350 25000 24 hr Fig. 1. E. coli exocytosis from infected BECs. (A) The 300 5637 BECs were infected with various bacteria for 1 h, 20000 ** after which a standard gentamicin protection assay was 250 performed to quantify the number of intracellular bac- 15000 200 ** teria (0 h). A significant decrease in intracellular E. coli ORN103(pSH2) and E. coli CI5 was seen at 24 h after the 150 10000 addition of gentamicin (24 h). Unlike E. coli 100 ORN103(pSH2) and E. coli CI5, the numbers of intracel- 5000 lular S. typhimurium SL1344 and S. aureus 54 were 50 *** ** Viable bacteria (CFU/well) significantly increased at 24 h after initial infection and 0 Intracellular bacteria (% of 0 hour) 0 Intracellular Extracellular after gentamicin treatment. (B) Intracellular and extra- Escherichia coli Escherichia coli Salmonella Staphylococcus Intracellular ORN103(pSH2) CI5 typhimurium aureus E. coli 0 hr E. coli 4 hr E. coli 4 hr cellular E. coli ORN103(pSH2) numbers in BECs at 0 and 4 h after gentamicin treatment; 5637 BECs were in- fected with 1000 MOI E. coli ORN103(pSH2). The sum of C 150 D 3500 human primary BECs numbers of intracellular and extracellular E. coli at4h after gentamicin treatment was similar to intracellular 125 3000 bacteria numbers at 0 h, suggesting bacterial exocytosis from infected BECs. (C) Treatment of infected BECs with 2500 100 NH4Cl and Bafilomycin, which neutralize bactericidal activity within lysosomes, caused no change of numbers 2000 of intracellular bacteria, indicating the compartment 75 survival 1500 harboring E. coli did not possess bactericidal activity. (D) Quantitative E. coli exocytosis assays were per- 50 1000 formed by using human primary BECs and a clinical E. coli (% Control) isolate of UPEC strain E. coli CI5. Significant numbers of 25 500 intracellular E. coli CI5 were expelled from infected IMMUNOLOGY primary BECs at 4 h after the gentamicin treatment. Viable bacteria (CFU/well) 0 Bars represent the mean ϩ SEM. , P Ͻ 0.05; , P Ͻ 0.03; Control Bafilomycin NH Cl 0 * ** 4 Intracellular Intracellular Extracellular , P Ͻ 0.001, relative to Intracellular E. coli titers at 0 h (1μ M) (10mM) *** E. coli 0 hr E. coli 4 hr E. coli 4 hr in A, B, and D. cellular bacteria. The intracellular bacterial numbers at this time (0 protein), and cytokeratin, all of which are hallmarks of terminal h) were assessed in one of the cultures.
Load more

Recommended publications
  • Abl Family Tyrosine Kinases Govern Igg Extravasation in the Skin in a Murine Pemphigus Model
    ARTICLE https://doi.org/10.1038/s41467-019-12232-3 OPEN Abl family tyrosine kinases govern IgG extravasation in the skin in a murine pemphigus model Sachiko Ono1, Gyohei Egawa1, Takashi Nomura1, Akihiko Kitoh1, Teruki Dainichi 1, Atsushi Otsuka1, Saeko Nakajima1, Masayuki Amagai2, Fumi Matsumoto3, Mami Yamamoto 3, Yoshiaki Kubota4, Toshiyuki Takai5, Tetsuya Honda1 & Kenji Kabashima 1,6 1234567890():,; The pathway of homeostatic IgG extravasation is not fully understood, in spite of its importance for the maintenance of host immunity, the management of autoantibody- mediated disorders, and the use of antibody-based biologics. Here we show in a murine model of pemphigus, a prototypic cutaneous autoantibody-mediated disorder, that blood- circulating IgG extravasates into the skin in a time- and dose-dependent manner under homeostatic conditions. This IgG extravasation is unaffected by depletion of Fcγ receptors, but is largely attenuated by specific ablation of dynamin-dependent endocytic vesicle for- mation in blood endothelial cells (BECs). Among dynamin-dependent endocytic vesicles, IgG co-localizes well with caveolae in cultured BECs. An Abl family tyrosine kinase inhibitor imatinib, which reduces caveolae-mediated endocytosis, impairs IgG extravasation in the skin and attenuates the murine pemphigus manifestations. Our study highlights the kinetics of IgG extravasation in vivo, which might be a clue to understand the pathological mechanism of autoantibody-mediated autoimmune disorders. 1 Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan. 2 Department of Dermatology, Keio University Graduate School of Medicine, Tokyo, Japan. 3 Research Unit/Immunology & Inflammation, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Japan. 4 Department of Anatomy, Keio University School of Medicine, Tokyo, Japan.
    [Show full text]
  • The Mir-199–Dynamin Regulatory Axis Controls Receptor-Mediated Endocytosis Juan F
    © 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233 RESEARCH ARTICLE The miR-199–dynamin regulatory axis controls receptor-mediated endocytosis Juan F. Aranda1,2, Alberto Canfrán-Duque1,2, Leigh Goedeke1,2, Yajaira Suárez1,2 and Carlos Fernández-Hernando1,2,* ABSTRACT mechanism for the selective uptake of essential nutrients such as Small non-coding RNAs (microRNAs) are important regulators of low-density lipoprotein (LDL), through the LDL receptor (LDLR) gene expression that modulate many physiological processes; (Brown and Goldstein, 1986), or iron, through transferrin receptor however, their role in regulating intracellular transport remains (TfR) (Harding et al., 1983). Thus, factors that affect RME have a largely unknown. Intriguingly, we found that the dynamin (DNM) direct effect on these receptors, and, in the case of LDLR, to regulate genes, a GTPase family of proteins responsible for endocytosis in intracellular cholesterol levels. In both the LDLR and TfR eukaryotic cells, encode the conserved miR-199a and miR-199b internalization processes, clathrin plays a key role during the family of miRNAs within their intronic sequences. Here, we formation of coated vesicles (Moore et al., 1987). Once vesicles are demonstrate that miR-199a and miR-199b regulate endocytic internalized, their passage through a broad endosomal compartment transport by controlling the expression of important mediators of system is required; first they are rapidly transported into early endocytosis such as clathrin heavy chain (CLTC), Rab5A, low- endosomes, where Rab5A is a key regulator (Nielsen et al., 1999), density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1).
    [Show full text]
  • Palmitoylation of Caveolin-1 and Its Importance for Structural and Functional Plasticity
    Palmitoylation of Caveolin-1 and its importance for structural and functional plasticity A DISSERTATION SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Katherine R. Tonn IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Paul G. Mermelstein November 2018 © Katherine R. Tonn Eisinger 2018 Acknowledgments I would like to thank my adviser, Dr. Paul Mermelstein, for his mentorship, training, support, and scientific flexibility. Infinite thanks to past and present members of the Mermelstein and Meisel Labs, especially Dr. Brittni Peterson, Dr. Kelsey Moore, Dr. Laura Been, Dr. Luis Martinez, Dr. Valerie Hedges, and Dr. John Meitzen, for being a source of scientific inspiration and friendship. I could not have asked for a better friend and lab colleague than Dr. Kellie Gross, whose input has kept me sane and made me a better scientist. Thanks also to stellar undergrads Kerry Trotter, Julia Dworsky, and Sam Swanson for their help and energy. Thanks to the many people who collaborated or helped in some way on the experiments presented in this thesis, including Dr. Mark Thomas, Dr. Lorene Lanier, Dr. Mark Dell’Acqua, Dr. Kevin Woolfrey, Dr. Brian Head, Dr. Jing Tong, and Dr. John Meitzen. In particular, I am extremely grateful for the collaboration of Dr. Lorene Lanier, whose enthusiasm helped reinvigorate me and this project. Finally, thanks to the members of my thesis committee, Drs. Harry Orr, Timothy Ebner, Anna Lee, and Robert Meisel, for their encouragement and guidance. i Dedication For Mom, my first and most important example of what it is to be smart, versatile, and kind.
    [Show full text]
  • Role of Stromal Caveolin-1 (CAV1) Levels in Breast Cancer Angiogenesis
    Universidad Autónoma de Madrid Programa de Doctorado en Biociencias Moleculares Role of stromal Caveolin-1 (CAV1) levels in breast cancer angiogenesis Alberto Díez Sánchez Madrid, 2018 0 1 Departamento de Bioquímica Facultad de Medicina Universidad Autónoma de Madrid Role of stromal Caveolin-1 (CAV1) levels in breast cancer angiogenesis Doctorando: Alberto Díez Sánchez, Licenciado en Biotecnología Director: Miguel Ángel del Pozo Barriuso, MD, PhD. Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) Madrid, 2018 1 2 CERTIFICADO DEL DIRECTOR DE TESIS El doctor Miguel Ángel del Pozo Barriuso CERTIFICA que el doctorando Alberto Díez Sánchez ha desarrollado y concluido su trabajo de tesis doctoral “Role of stromal Caveolin-1 (CAV1) levels in breast cancer angiogenesis” bajo su supervisión, en el Centro Nacional de Investigaciones Cardiovasculares (CNIC). Y para que así conste lo firma en Madrid, a 10 de Julio de 2018, Fdo. Dr. Miguel Ángel del Pozo Barriuso Centro Nacional de Investigaciones Cardiovasculares (CNIC) 3 4 ACKNOWLEDGMENTS It is said that scientific knowledge is built on top of the shoulder of giants, in more practical terms, I consider all these people below my personal giants. First ones I encountered, were my parents and grandparents, everything I have achieved has been done on top of their previous efforts, to them I dedicate my most sincere gratitude for teaching this once lazy kid the value of effort. Next, I have to thank all those high-school teachers and university professors that during my education have been able to spark in me the sense of amazement derived from understanding how nature works.
    [Show full text]
  • Novel Missense Mutation in the Caveolin-3 Gene in a Belgian Family
    1349 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.2003.028217 on 16 August 2004. Downloaded from SHORT REPORT Novel missense mutation in the caveolin-3 gene in a Belgian family with rippling muscle disease P Y K Van den Bergh, J M Ge´rard, J A Elosegi, M U Manto, C Kubisch, B G H Schoser ............................................................................................................................... J Neurol Neurosurg Psychiatry 2004;75:1349–1351. doi: 10.1136/jnnp.2003.028217 too small’’). The patient was diagnosed as having fibromyal- Rippling muscle disease (RMD) is a rare muscle disorder gia, which led to secondary depression and loss of her job. characterised by muscle stiffness, exercise induced myalgia, Muscle weakness and pigmenturia were absent. The medical and cramp-like sensations. It is genetically heterogeneous history was remarkable for mild hypothyroidism, peptic and can be acquired, but most cases show autosomal oesophagitis, and tobacco related asthmatiform bronchitis dominant inheritance due to mutations in the caveolin-3 with emphysema. At age 38, she had a neurological work-up. (CAV3) gene. We report a novel heterozygous missense The cranial nerves, muscle bulk, muscle strength, and deep mutation in CAV3 in a Belgian family with autosomal tendon reflexes were normal, and plantar responses were dominant RMD. flexor. Tapping with the reflex hammer or with the finger on A 40 year old woman complained of fatigue, exercise the muscles provoked an immediate, short lasting, forceful induced muscle pain, and muscle cramps since the age of 35. contraction and/or painful local mounding lasting for several Neurological examination revealed percussion induced seconds. PIRCs were most pronounced in the sternocleido- rapid muscle contractions (PIRCs) and localised muscle mastoid, deltoid, biceps, brachioradialis, finger and wrist mounding on percussion; muscle rippling was not observed.
    [Show full text]
  • Snapshot: Caveolae, Caveolins, and Cavins Nicholas Ariotti and Robert G
    704 Cell SnapShot: Caveolae, Caveolins, and Cavins Nicholas Ariotti and Robert G. Parton 154 Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD 4072, Australia , August 1,2013©2013 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2013.07.009 ORGAN/ COMPONENTS DISEASE AND TISSUE-SPECIFIC CAVEOLAR COMPLEXES TISSUE CELLULAR PROCESS Rippling muscle disease Caveolin3 Striated muscle fiber Limb girdle muscular dystrophy Muscle Cavin1 skeletal and Cardiomyopathy Cavin4 cardiac Mechanoprotection Cav1 (heart) T-tubule formation Caveolin1 Lipodystrophy Caveolin2 Lipotoxicity Adipose Cavin1 Fatty acid regulation tissue Cavin2 Insulin signaling Cavin3 Mechanoprotection Atherosclerosis Caveolin1 Inammation Caveolin2 Lung Pulmonary hypertension and other Cavin1 Adipocyte Pulmonary brosis endothelia Cavin2 Mechanosensation Cavin3 Signaling Fatty liver Hepatocellular carcinoma Caveolin1 LIPID DROPLET Liver Lipid metabolism Caveolin2 Carbohydrate metabolism Liver regeneration Caveolin1 Autism Brain Cavin1 Schizophrenia Cavin3 LOW EXPRESSION HIGH EXPRESSION Capillary GENERAL CELLULAR CONTEXT ENDOCYTOSIS MECHANOPROTECTION SIGNALING See online version for legend and references. and legend for version online See Membrane stretch LUMEN Cavin complex Caveola EHD2 eNOS Ca2+ inactive Rab5 Dynamin2 EARLY ENDOSOME Intracellular targets MVB/ Caveolin 1 Caveolin 2 Caveolin 3 Dynamin2 late endosome eNOS Cavin1 Cavin2 Cavin3 Cavin4 eNOS EHD2 active SnapShot: Caveolae, Caveolins, and Cavins Nicholas Ariotti and Robert G. Parton Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD 4072, Australia Caveolae, submicroscopic bulb-shaped plasma membrane pits, are an abundant feature of many mammalian cells (Parton and del Pozo, 2013). Caveolae and the major proteins of caveolae, caveolins (Rothberg et al., 1992), and cavins (Hill et al., 2008), are linked to a number of human diseases such as muscular dystrophy, cardiomyopathy, and lipodys- trophy (see Glossary) (Bruno et al., 1993; Fernández et al., 2006; Hayashi et al., 2009).
    [Show full text]
  • Comprehensive Analysis of Lncrna‑Associated Cerna Network Reveals the Novel Potential of Lncrna, Mirna and Mrna Biomarkers in Human Rectosigmoid Junction Cancer
    ONCOLOGY LETTERS 21: 144, 2021 Comprehensive analysis of lncRNA‑associated ceRNA network reveals the novel potential of lncRNA, miRNA and mRNA biomarkers in human rectosigmoid junction cancer QIANSHI ZHANG1*, ZHEN FENG1*, SHASHA SHI2, YU ZHANG3 and SHUANGYI REN1 Departments of 1Gastrointestinal Surgery, 2Ultrasound and 3Clinical Laboratory, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116023, P.R. China Received March 11, 2020; Accepted November 20, 2020 DOI: 10.3892/ol.2020.12405 Abstract. Although accumulating evidence has confirmed gene 20) and three mRNAs (sodium‑ and chloride‑dependent the potential biological functions of long non‑coding RNAs taurine transporter, fibroblast growth factor 13 and tubulin (lncRNAs) as competitive endogenous RNAs (ceRNAs) in polyglutamylase TTLL7) were significantly associated with colorectal tumorigenesis and progression, few studies have OS (P<0.05). Additionally, two lncRNAs (KCNQ1OT1 focused on rectosigmoid junction cancer. In the present and MIR17HG) interacted with most of the DEmiRNAs. study, a comprehensive analysis was conducted to explore Notably, two top‑ranked miRNAs (hsa‑miR‑374a‑5p and lncRNA‑mediated ceRNA implications and their potential hsa‑miR‑374b‑5p) associated networks were identified to be value for prognosis. lncRNA, microRNA (miR/miRNA) markedly associated with the pathogenesis. Furthermore, four and mRNA expression profiles were downloaded from DEmRNAs (caveolin‑1, MET, filamin‑A and AKT3) were The Cancer Genome Atlas database. Subsequently, a enriched in the Kyoto Encylopedia of Gene and Genomes lncRNA‑miRNA‑mRNA regulatory network was constructed pathway analysis, as well as being included in the ceRNA to evaluate the functions of these differentially expressed genes network. In summary, the present results revealed that a on overall survival (OS) for rectosigmoid junction cancer.
    [Show full text]
  • Caveolar Endocytosis of Simian Virus 40 Reveals a New Two-Step Vesicular- Transport Pathway to the ER
    articles Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular- transport pathway to the ER Lucas Pelkmans*, Jürgen Kartenbeck† and Ari Helenius*‡ *Institute of Biochemistry, Swiss Federal Institute of Technology, Universitaetstrasse 16, CH-8092 Zürich, Switzerland †German Cancer Research Center (DKFZ) Heidelberg, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany ‡e-mail: [email protected] Simian virus 40 (SV40) is unusual among animal viruses in that it enters cells through caveolae, and the internalized virus accumulates in a smooth endoplasmic reticulum (ER) compartment. Using video-enhanced, dual-colour, live fluorescence microscopy, we show the uptake of individual virus particles in CV-1 cells. After associating with cave- olae, SV40 leaves the plasma membrane in small, caveolin-1-containing vesicles. It then enters larger, peripheral organelles with a non-acidic pH. Although rich in caveolin-1, these organelles do not contain markers for endo- somes, lysosomes, ER or Golgi, nor do they acquire ligands of clathrin-coated vesicle endocytosis. After several hours in these organelles, SV40 is sorted into tubular, caveolin-free membrane vesicles that move rapidly along microtubules, and is deposited in perinuclear, syntaxin 17-positive, smooth ER organelles. The microtubule-disrupt- ing agent nocodazole inhibits formation and transport of these tubular carriers, and blocks viral infection. Our results demonstrate the existence of a two-step transport pathway from plasma-membrane caveolae, through an intermediate organelle (termed the caveosome), to the ER. This pathway bypasses endosomes and the Golgi com- plex, and is part of the productive infectious route used by SV40. any animal viruses take advantage of receptor-mediated mutants of caveolin-3 localize to intracellular vesicles that are dis- endocytosis to enter their host cells.
    [Show full text]
  • Starting a Molecular Systems View of Endocytosis
    ANRV356-CB24-20 ARI 3 September 2008 19:11 ANNUAL Protein Kinases: Starting REVIEWS Further Click here for quick links to Annual Reviews content online, a Molecular Systems View including: • Other articles in this volume of Endocytosis • Top cited articles • Top downloaded articles • Our comprehensive search Prisca Liberali, Pauli Ram¨ o,¨ and Lucas Pelkmans Institute of Molecular Systems Biology, ETH Zurich, CH-8093 Zurich, Switzerland; email: [email protected] Annu. Rev. Cell Dev. Biol. 2008. 24:501–23 Key Words First published online as a Review in Advance on membrane trafficking, phosphorylation, signal transduction, July 3, 2008 complexity, nonlinear systems, genetical physics The Annual Review of Cell and Developmental Biology is online at cellbio.annualreviews.org Abstract This article’s doi: The field of endocytosis is in strong need of formal biophysical model- 10.1146/annurev.cellbio.041008.145637 ing and mathematical analysis. At the same time, endocytosis must be Copyright c 2008 by Annual Reviews. much better integrated into cellular physiology to understand the for- by Universitat Zurich- Hauptbibliothek Irchel on 04/05/13. For personal use only. All rights reserved mer’s complex behavior in such a wide range of phenotypic variations. Annu. Rev. Cell Dev. Biol. 2008.24:501-523. Downloaded from www.annualreviews.org 1081-0706/08/1110-0501$20.00 Furthermore, the concept that endocytosis provides the space-time for signal transduction can now be experimentally addressed. In this review, we discuss these principles and argue for a systematic and top-down ap- proach to study the endocytic membrane system. We provide a summary of published observations on protein kinases regulating endocytic ma- chinery components and discuss global unbiased approaches to further map out kinase regulatory networks.
    [Show full text]
  • Modulation of the Caveolin-3 Localization to Caveolae and STAT3 to Mitochondria by Catecholamine-Induced Cardiac Hypertrophy in H9c2 Cardiomyoblasts
    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 41, No. 4, 226-235, April 2009 Modulation of the caveolin-3 localization to caveolae and STAT3 to mitochondria by catecholamine-induced cardiac hypertrophy in H9c2 cardiomyoblasts Kyuho Jeong*, Hayeong Kwon*, amine-induced cardiac hypertrophy. Chanhee Min and Yunbae Pak1 Keywords: cardiomegaly; caveolae; caveolin-3; cell Department of Biochemistry nucleus; heart; isoproterenol; mitochondria; phenyl- Division of Applied Life Science (BK21), PMBBRC ephrine; STAT3 transcription factor Environmental Biotechnology National Core Research Center Gyeongsang National University Jinju 660-701, Korea Introduction 1Corresponding author: Tel, 82-55-751-5961; Fax, 82-55-759-9363; E-mail, [email protected] Hypertension is major risk factors for cardiac da- mage, ischemia, myocardial infarction, and conge- *These authors contributed equally to this work. stive heart failure (Zampaglione et al., 1996). In DOI 10.3858/emm.2009.41.4.025 response to increased demands for cardiac work caused by various pathologic stresses, heart adapts Accepted 20 November 2008 through compensatory hypertrophy of myocytes. Abbreviations: Akt, protein kinase B; CsA, cyclosporin A; GPCR, Thus, cardiac hypertrophy is recognized in many G protein-coupled receptor; ISO, isoproterenol; PE, phenylephrine; cardiovascular diseases, such as hypertension, RTK, receptor tyrosine kinase; STAT3, signal transducers and acti- vascular disease, and myocardial infarction, and is vator of transcription 3 an independent risk factor for cardiac morbidity and mortality. Hypertrophic stimuli induce an in- crease in cell size in the absence of cell division through Ca2+ signaling and activation of PKC, Abstract MAPK and PKB/ Akt (Watanabe et al., 2001; Dorn and Force, 2005), and are accompanied by We investigated the effect of phenylephrine (PE)- and increased protein synthesis with reprogramming of isoproterenol (ISO)-induced cardiac hypertrophy on gene expression (Takeo et al., 2000).
    [Show full text]
  • Caveolin-1 Expression in Schwann Cells
    GLIA 27:39–52 (1999) Caveolin-1 Expression in Schwann Cells DANIEL D. MIKOL,* HOYLOND L. HONG, HSIN-LIN CHENG, AND EVA L. FELDMAN Department of Neurology, University of Michigan, Ann Arbor, Michigan KEY WORDS caveolae; cholesterol; differentiation; myelin; nerve ABSTRACT Caveolae are non-clathrin-coated invaginations of the plasma mem- brane, which are present in most cell types. An integral component of caveolae is the caveolin family of related proteins, which not only forms the structural framework of caveolae, but also likely subserves its functional roles, including regulation of signal transduction and cellular transport, in particular, cholesterol trafficking. Although caveolae have been identified ultrastructurally in the peripheral nervous system (PNS), caveolin expression has not previously been studied. To date, three caveolin genes have been reported. Here, we show for the first time that caveolin-1 is expressed by Schwann cells (SC) as well as several SC-derived cell lines. Caveolin-1 is enriched in the buoyant, detergent-insoluble membranes of rat sciatic nerve (SN) and SC, a hallmark of the caveolar compartment. Caveolin-1 exists as both soluble and insoluble forms in rat SN and SC, and localizes to SC cytoplasm and abaxonal myelin. SC caveolin-1 decreases after axotomy, when SC revert to a premyelinating phenotype. We speculate that caveolin-1 may regulate signal transduction and/or cholesterol transport in myelinating SC. GLIA 27:39–52, 1999. ௠ 1999 Wiley-Liss, Inc. INTRODUCTION A number of functions are proposed for caveolae: (1) the uptake of small molecules such as folate into cells, a Caveolae are plasma membrane microdomains that process known as potocytosis (Anderson et al., 1992); can invaginate to form 50–100 nm vesicles, and are (2) the transcytosis of molecules such as low density enriched in cholesterol, glycosphingolipids, a variety of lipoprotein across cells (Simionescu, 1983); (3) polar- signaling molecules, and the caveolins, a family of ized trafficking of proteins, especially in epithelial cells 18–24 kD proteins.
    [Show full text]
  • Caveolin-1 Expression and Cavin Stability Regulate Caveolae Dynamics in Adipocyte Lipid Store Fluctuation
    4032 Diabetes Volume 63, December 2014 Nolwenn Briand,1 Cécilia Prado,1 Guillaume Mabilleau,2 Françoise Lasnier,1 Xavier Le Lièpvre,1 Jeffrey D. Covington,3 Eric Ravussin,3 Soazig Le Lay,4 and Isabelle Dugail5 Caveolin-1 Expression and Cavin Stability Regulate Caveolae Dynamics in Adipocyte Lipid Store Fluctuation Diabetes 2014;63:4032–4044 | DOI: 10.2337/db13-1961 Adipocytes specialized in the storage of energy as fat Caveolae are small flask-shaped invaginations of the plasma are among the most caveolae-enriched cell types. Loss membrane (1) that are found with remarkable abundance in of caveolae produces lipodystrophic diabetes in humans, endothelial cells, myotubes, and adipocytes. They are consid- which cannot be reversed by endothelial rescue of ered a subset of the so-called lipid raft domains and segregate caveolin expression in mice, indicating major impor- a number of membrane-related processes (2). An accepted tance of adipocyte caveolae. However, how caveolae paradigm is that caveolae formation is primarily driven by participate in fat cell functions is poorly understood. the assembly of a cytoplasmic coat consisting of oligomeric We investigated dynamic conditions of lipid store caveolins (3), a protein family with 3 highly-related members fluctuations and demonstrate reciprocal regulation of (caveolin-1 through -3). Invalidation of individual caveolin caveolae density and fat cell lipid droplet storage. We genes led to the generation of mice models lacking caveolae METABOLISM identified caveolin-1 expression as a crucial step in in all cell types or in a tissue-restricted manner (4). Caveolin- adipose cell lines and in mice to raise the density of 1–null mice, which also lack caveolin-2, suffer from severe caveolae, to increase adipocyte ability to accommodate vascular dysfunction and pulmonary defects (5,6) and de- larger lipid droplets, and to promote cell expansion velop lipodystrophy (7), a metabolic phenotype that cannot by increased glucose utilization.
    [Show full text]