Methods in Cell Biology Sorting and Recycling Endosomes Volume 130 Series Editors Leslie Wilson Department of Molecular, Cellular and Developmental Biology University of California Santa Barbara, California Phong Tran University of Pennsylvania Philadelphia, USA & Institut Curie, Paris, France Methods in Cell Biology Sorting and Recycling Endosomes Volume 130 Edited by Wei Guo University of Pennsylvania, Biology Department, Philadelphia, PA, USA AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier Academic Press is an imprint of Elsevier 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 125 London Wall, London EC2Y 5AS, UK The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2015 Copyright © 2015 Elsevier Inc All Rights Reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein) Notices Knowledge and best practice in this field are constantly changing As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein ISBN: 978-0-12-802829-2 ISSN: 0091-679X For information on all Academic Press publications visit our website at http://store.elsevier.com Contributors Gerard Apodaca Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA Daniel D Billadeau Department of Biochemistry and Molecular Biology, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA Shanna L Bowman Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA William J Brown Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Steve Caplan Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Pei-Wen Chen Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Xiao-Wei Chen Institute of Molecular Medicine, Peking University, Beijing, China; PKU-THU Center for Life Sciences, Peking University, Beijing, China Nicholas D Condon Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia Paul de Figueiredo Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA; Department of Microbial Pathogenesis and Immunology, Norman Borlaug Center, Texas A&M University, College Station, TX, USA Ce´dric Delevoye Institut Curie, PSL Research University, Paris, France; CNRS UMR 144, Structure and Membrane Compartments, Paris, France Emmanuel Derivery Department of Biochemistry, Sciences II, University of Geneva, Geneva, Switzerland xiii xiv Contributors Julie G Donaldson Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Michael Robert Dores Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA, USA Stephen Doxsey Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA Dipannita Dutta Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Heike Foălsch Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA J Christopher Fromme Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Luciana Gallo Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA Alexis Gautreau Laboratoire de Biochimie, Unite´ Mixte de Recherche 7654, Centre National de la Recherche Scientifique, Ecole Polytechnique, Palaiseau Cedex, France Bruno Goud Institut Curie, PSL Research University, Paris, France; CNRS UMR 144, Molecular Mechanisms of Intracellular Transport, Paris, France Barth D Grant Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA Wei Guo Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Nicholas A Hamilton Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia; Research Computing Centre, The University of Queensland, Brisbane, QLD, Australia Contributors Heidi Hehnly Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA; Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY, USA; Department of Pharmacology, University of Washington, Seattle, WA, USA Victor W Hsu Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Boston, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA Hui-Fang Hung Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA Xiaoying Jian Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Danielle N Kalkofen Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Guangpu Li Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Jian Li Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Boston, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA Zhimin Liang Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Guodong Lu Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China Ruibai Luo Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA xv xvi Contributors M Caleb Marlin Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Naava Naslavsky Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Chao Nie Institute of Molecular Medicine, Peking University, Beijing, China Douglas G Osborne Department of Biochemistry and Molecular Biology, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA Andres E Perez Bay Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY, USA Christine A Phillips-Krawczak Department of Biochemistry and Molecular Biology, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA Itziar Pinilla-Macua Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Rytis Prekeris Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Manojkumar A Puthenveedu Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA Yaoyao Qi Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Paul A Randazzo Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA James B Reineke Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Contributors Brian C Richardson Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Enrique Rodriguez-Boulan Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY, USA Ryan Schreiner Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY, USA Anbing Shi Department of Medical Genetics, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; Institute for Brain Research, Huazhong University of Science and Technology, Wuhan, Hubei, China Amanda L Soohoo Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA Alexander Sorkin Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Jennifer L Stow Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia JoAnn Trejo Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA, USA Adam A Wall Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia Juanfei Wang Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Zonghua Wang Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China Bin Wu Department of Biology, University of Pennsylvania, Philadelphia, PA, USA xvii xviii Contributors Shuwei Xie Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Jeremy C Yeo Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia Dongmei Zhang Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China Yuting Zhao Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Jie Zhou Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China Volumes in Series Volume 127 (2015) Methods in Cilia & Flagella Edited by Renata Basto and Wallace F Marshall Volume 128 (2015) Building a Cell from its Component Parts Edited by Jennifer Ross and Wallace F Marshall Volume 129 (2015) Centrosome & Centriole Edited by Renata Basto and Karen Oegema 381 Index Note: Page numbers followed by “f” and “t” indicate figures and tables respectively A A280 protocol, 124 Abscission analyzing establishment, 25e26 data interpretation, 26e27 procedure, 26 asymmetric and symmetric models, 25f measuring delay, 24 data interpretation, 24 procedure, 24 mechanisms mediating, 21f ACAP1-based coat complex, 82e83 antibodies, 84 cargo binding, 85e87 cargo sequences recognizing, 90fe91f, 91 cells and reagents, 83e84 coat complex formation by ACAP1 and clathrin, 87e88, 89f endocytic recycling assays, 91e92 Glut4 recycling, 95, 96f integrin recycling, 94, 95f TfR recycling, 92, 93f plasmids and transfections, 84e85 ACAP1-based complex, 82 Actin dynamics, time-lapse analysis of, 27 analyzing actin dynamics, 29e30 and apical lumen formation in 3D cultures, 30e32 RAB11/FIP3-endosome transport analysis, 27e29 Actin filaments, 29 Additives, 116 DiR, 116 nickel, 116 Adenosine diphosphate ribosylation factor (ARF), 82e83 ADP ribosylation factor (Arf), 103 GEF activity measurement, 117e121 materials chemical reagents, 105e106 equipment, 104 software, 106 proteineArf1 interactions identification, 121e123 purification of required proteins, 106 protein buffers, 107t Sec7, 106e112 SMALL GTPases, 112e115 supporting protocol, 123e125 ADP-ribosylation factors (Arfs), 70 effects of, 70 Anion exchange, 110e112 Anti-early endosomal antigen-1 (anti-EEA1), 322 anti-EEA1, see Anti-early endosomal antigen-1 (anti-EEA1) Antibody/antibodies, 310, 322e323 internalization assays cell imaging, 133e134 cell plating, 132 internalization assays, 133 Anticellubrevin (Cbv), 84 AP-1B heterotetrameric clathrin adapter complex, 290 during endocytic recycling, biosynthetic delivery, or both, 299e303 instruments, 293 materials cell lines, 292 reagents and materials, 292e293 objectives and rationale, 291e292 Apical lumen formation in 3D cultures, 30 midbody-associated, 31f procedure, 30e32 Apical recycling endosome (ARE), 279e280 Apical sorting endosomes (ASE), 272e273 background and objective, 276 buffers composition, 276e277 experimental protocol, 277 apo-Tf loading with iron, 257e258 ARE, see Apical recycling endosome (ARE) ARF, see Adenosine diphosphate ribosylation factor (ARF) Arf, see ADP ribosylation factor (Arf) Arf G proteins in endosomal trafficking, 129f ARF-6-GFP, 83, 190 ARF6, 83 Arfs, see ADP-ribosylation factors (Arfs) ARH protein, see Autosomal recessive hypercholesterolemia protein (ARH protein) ASAP2, see Myc-tagged PAP ASE, see Apical sorting endosomes (ASE) Autosomal recessive hypercholesterolemia protein (ARH protein), 290 383 384 Index B B2AR, see Beta adrenergic receptor (B2AR) Baby hamster kidney cells (BHK cells), 37 Bacmid preparation, 107 Basolateral early endosomes (BEEs), 250 Basolateral sorting at steady state, see also AP-1B heterotetrameric clathrin adapter complex cargo proteins immunoprecipitation alternative approaches, 298 background and objective, 296 considerations, 297 expected results, 297e298 flow of experiment, 296e297 LLC-PK1 cells, protein sorting analysis in, 295f alternative approaches, 295 background and objective, 293 considerations, 294 expected results, 294e295 flow of experiment, 293e294 Basolateral sorting endosomes (BSE), 272e273, 278 Basolateral TfReholo-Tf complex, 250 Batch nickel affinity purification, 110e111 BBC, see Bovine brain cytoplasm (BBC) BEEs, see Basolateral early endosomes (BEEs) BEL, see Bromoenol lactone (BEL) Beta adrenergic receptor (B2AR), 334 BFA, see Brefeldin A (BFA) BHK cells, see Baby hamster kidney cells (BHK cells) BHK-21 cells transfection, 40 Biochemical-based assay, 283e285, see also Microscopy-based trafficking assays Bioluminescence resonance energy transfer technology (BRET technology), 60, 61f analyzing conformational change induced by ERK1/2, 64e65 ERK2 purification, 65 materials, 65 quantification of Rabin8 conformational change, 66 autoinhibitory conformation of Rabin8, 62f Bovine brain cytoplasm (BBC), 163 Bovine serum albumin (BSA), 160, 254, 278, 292, 322, 350 Bradford semiquantitative analysis, 123e124 Brefeldin A (BFA), 163 stimulation of endosome membrane tubules formation, 167e168 BRET technology, see Bioluminescence resonance energy transfer technology (BRET technology) Bromoenol lactone (BEL), 175 BSA, see Bovine serum albumin (BSA) BSE, see Basolateral sorting endosomes (BSE) C C-terminal regions, 88 Caenorhabditis elegans (C elegans), 182 general mechanisms of membrane traffic in, 182 materials and instruments reagents, 184 and software, 184 methods endocytic tracer analysis in intestinal epithelia, 186e188 endosomal dynamics, 194e195 living intact animals, morphometric analysis of endosomal markers in, 188e191 transmembrane recycling cargo assays, 191e194 yolk protein YP170 uptake assay, 184e186 MIG-14, 192 objectives and rationale, 183 polarized epithelia, 182e183 Caenorhabditis Genetics Center (CGC), 190e191 Canine holo-Tf, reagents to iodinate, 254 Cargo binding by ACAP1, 85, 86f ARF6 GAPs, 86e87 cytoplasmic domains of cargoes, 85e86 HeLa cells, 85 pulldown studies, 87 Cargo movement through RE, experimental considerations, 11 flow of experiment, 9e10 results, 11e14 VAMP3 recruitment, 13f Cargo proteins immunoprecipitation alternative approaches, 298 background and objective, 296 considerations, 297 expected results, 297e298 flow of experiment, 296e297 Cbv, see Anticellubrevin (Cbv) CDE, see Clathrin-dependent endocytosis (CDE) Cell imaging, 133e134 Cell plating, 132, 204 Cell-free, endosome membrane tubules in vitro formation, 163 equipment, 164 microfuge tubes, 165 negative-stained endosomes, 166f reaction, 165 Index reagents, 163e164 solutions and reagents, 164 Centrosome isolation and identification, 53 at interphase, methods to, 53e55 reconstituting GST-Rab11, methods for, 56 staining isolated centrosomes, methods for, 55 methods to isolate centrosome at interphase, 53e55 CGC, see Caenorhabditis Genetics Center (CGC) CHC, see Clathrin heavy chain (CHC) CI-M6PR, 201e203 antibody labeling to monitor retrograde trafficking, 204e205 CIE, see Clathrin-independent endocytosis (CIE) Clathrin heavy chain (CHC), 84 Clathrin-dependent endocytosis (CDE), 140e141 Clathrin-independent endocytosis (CIE), 128, 140e141 Clathrin-mediated endocytosis (CME), 128 Cloning, reagents for, 37 CM, see Complete medium (CM) CME, see Clathrin-mediated endocytosis (CME) Coat protein II complex (COPII complex), 82e83 Coat proteins, 82 Colloidal gold particles, 160 Colloidal gold-loaded endosomes, 161 equipment, 161 procedure, 161e163 reagents, 161 Column volumes (CV), 110 Common recycling endosome (CRE), 250 Complete medium (CM), 38 Confocal fluorescence microscopy localization by, 42 materials and reagents for, 38 Confocal immunofluorescence microscopy assay reagents, 323 Confocal microscopy, 168e169, 335 procedure, 169 Fiji macro script, 173e174 Sholl analysis, 172e173 step-by-step endosome membrane tubule “tracing”, 171e173 semiautomated endosome membrane tubule segmentation, 170f Constitutively active ERK2 (ERK2-CA), 65 Coomassie gel analysis, 124e125 COPII complex, see Coat protein II complex (COPII complex) CRE, see Common recycling endosome (CRE) CRETfR, see TfR-rich perinuclear common recycling endosomes (CRETfR) Culture MDCK cells, reagents to, 256 Curve comparison, 120 extraction, 120 fitting, 120 CV, see Column volumes (CV) Cytokinesis, 20 methods for analyzing proteins regulating, 22 multinucleation analysis, 22e24 time-lapse analysis of endosome and actin dynamics during, 27 analyzing actin dynamics, 29e30 and apical lumen formation in 3D cultures, 30e32 RAB11/FIP3-endosome transport analysis, 27e29 Cytoskeleton, 216e217 D Data interpretation, 23e24, 26e27 DEAE cellulose, see Diethylaminoethyl cellulose (DEAE cellulose) DEPC, see Diethylpyrocarbonate (DEPC) Desferoxamine B, see Iron chelator desferrioxamine DEX, see Dexamethasone (DEX) Dexamethasone (DEX), 312 Diethylaminoethyl cellulose (DEAE cellulose), 106 Diethylpyrocarbonate (DEPC), 37 Dimethyl sulfoxide (DMSO), 239, 256, 284, 292 DiR die, 116 Discontinuous sucrose gradient flotation, 122 3,3’-Dithiobis(sulfosuccinimidylpropionate) (DTSSP), 292 Divalent metal transporter (DMT1), 250 DMEM, see Dulbecco’s modified eagle medium (DMEM) DMSO, see Dimethyl sulfoxide (DMSO) DMT1, see Divalent metal transporter (DMT1) Dominant negative forms of G proteins, 130e131 DTSSP, see 3,3’-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) Dulbecco’s minimum essential medium, see Dulbecco’s modified eagle medium (DMEM) Dulbecco’s modified eagle medium (DMEM), 84, 209, 322, 335, 350 385 386 Index Dulbecco’s modified essential medium, see Dulbecco’s modified eagle medium (DMEM) Dynamin inhibitor, 352 considerations, 356 experimental protocol, 352e353 results, 353e354 variation, 353 Dyngo-4a, 349e350 on EGFR endocytosis, 353, 355f E Early endosomes maturation, biochemical analysis of, 245 Early/sorting endosome, 140e141 EDTA, see Ethylenediamminetetraacetate (EDTA) EGF, see Epidermal growth factor (EGF) EGF-Rhodamine (EGF-Rh), 351e352 EGF:EGFR complex recycling using 125I-EGF considerations, 366 experimental protocol, 364e365 results, 365 theory, 363e364 variation, 365 EGFR, see Epidermal growth factor receptor (EGFR) EHD proteins, see Eps15 homology domain proteins (EHD proteins) Electron microscopy (EM), 239e240 EM, see Electron microscopy (EM) Endocytic compartment, 70 Endocytic pathway, 141f, 216, 236, see also Rab GTPasesekinesin interactions Endocytic recycling, 308 assays, 91e92 Glut4 recycling, 95, 96f integrin recycling, 94, 95f TfR recycling, 92, 93f buffers, 143 cells, 142 equipment, 142 materials, 142 MHC I antibody-receptor complex recycling quantification, 152e154 qualitative assessment, 143e149 quantitative assessment, 149e152 reagents, 143 Endocytic recycling compartment (ERC), 140e141 Endocytic tracers, 182e183 analysis in intestinal epithelia, 186e188 Endocytosis, 128, 352 Endoplasmic reticulum (ER), 36 Endosomal compartments, 128 in epithelial cells, 272, 273f, 275 Endosomal dynamics experimental strategies and considerations, 194 principles, 194 time-lapse imaging, 194e195 Endosomal sorting complexes required for transport (ESCRT), 148e149, 320e321 complexes, 20 ESCRT-III complex, 20 Endosomal trafficking materials and instruments, 131e132 methods, 132 antibody internalization assays, 132e134 expected results, 136e137 expression of wild type and mutant Rab and Arf proteins and effects, 134 flow of experiment, 134e136 objectives and rationale, 130e131 Rab and Arf G proteins, 129f Endosome dynamics, time-lapse analysis of, 27 analyzing actin dynamics, 29e30 and apical lumen formation in 3D cultures, 30e32 RAB11/FIP3-endosome transport analysis, 27e29 Endosome tubulation imaging, 218, see also Wash microdomains live imaging considerations, 220e222 inducing by WASH inhibition, 221f microscope, 219 procedure cell plating and Tf uptake, 220 cell transfection, 219 imaging and results, 220 reagents, 218e219 Endosome(s), 128, see also Liposome(s); Recycling endosome (RE) membrane tubule formation, 158e160 from Golgi complex, 160 PLA2 enzymes, 174e178 in vitro, 160e165 in vivo, 167e174 membrane tubule tracing, 171e172 visualization of sorting and recycling in, 341e342, 343f considerations, 342e343 flow of experiments, 342 results, 343 Index Ensemble measurement of surface levels, 337, 337f considerations, 338 flow of experiments, 338 results, 339 Epidermal growth factor (EGF), 142, 158e160, 239e240, 348e349 Epidermal growth factor receptor (EGFR), 239e240, 272, 348e349 cell culture, 351 degradation, 362f dynamin inhibitor, 352e356 EGF:EGFR complex, 349e350 recycling using 125I-EGF, 363e366 fluorescence microscopy, 352 imaging EGFR endocytosis in living cells, 352e356 instruments, 351 internalization rates measurement, 356 considerations, 358e359 experimental protocol, 356e357 results, 357e358 variation, 357 ligand-induced EGFR degradation, 361e363 PQ effect, 363e366 reagents, 350e351 SDS-page, 351e352 trafficking, 349 ubiquitination, 360f considerations, 361 experimental protocol, 359e360 results, 360e361 theory, 359 western blot, 351e352 Epithelial cells, 248 Epithelial tissues, 248 Eps15 homology domain proteins (EHD proteins), 141e142 ER, see Endoplasmic reticulum (ER) ERC, see Endocytic recycling compartment (ERC) ERK1/2, BRET assay to analyzing conformational change, 64e65 ERK2 purification, 65 materials, 65 quantification of Rabin8 conformational change, 66 ERK2-CA, see Constitutively active ERK2 (ERK2-CA) ERK2-KD, see Kinase-dead ERK2 (ERK2-KD) ESCRT, see Endosomal sorting complexes required for transport (ESCRT) Ethanol (EtOH), 241e242 Ethylenediaminetetraacetic acid, see Ethylenediamminetetraacetate (EDTA) Ethylenediamminetetraacetate (EDTA), 87, 256 Evi5 centrosome-targeted protein, 49 Exocyst complex, 309 directing outbound trafficking, 309 Glut4 outbound trafficking, 310 RalA cellular activity, 309 Extrusion, 116e117 F FACS, see Fluorescence-activated cell sorting (FACS) Fast recycling route, 242 FBS, see Fetal bovine serum (FBS) FCS, see Fetal calf serum (FCS) Fetal bovine serum (FBS), 37, 84, 167, 240, 256, 292, 312, 335, 350 Fetal calf serum (FCS), 84 Fiji macro script, 173e174 Fix cells, 135 Fluorescence intensity analysis, 330 Fluorescence microscopy, 352 imaging EGFR endocytosis in living cells by, 352e356 Fluorescence-activated cell sorting (FACS), 208 stripping buffer, 208e209 Fluorescent protein (FP), 237 Fluorescent TF uptake, labeling tubular endocytic compartments by procedure, 167e168 reagents and supplies, 167 FP, see Fluorescent protein (FP) Fractional conversion calculation, 76e78 Functional assays, 239e240 biochemical analysis of early endosomes maturation, 245 kinesin depletion, 240e241 slow Tf recycling route analysis, 242e244 ultrastructural localization of internalized Tf, 241e242 G G protein-coupled receptor (GPCR), 320e321, 334 sorting at MVEs, 321 cell plating and transfection, 323e324 GFP, 321e322 IEM, 321 instrumentation, 323 387 388 Index G protein-coupled receptor (GPCR) (Continued) proteinase-protection assay, 324e326 by Rab5-Q79L expression, 327e330 reagents, 322e323 visualizing and quantitating GPCR recycling, 334 cell culture, plating, and transfection, 336 confocal microscopy, 335 endocytosis, 335 ensemble measurement of surface levels, 337e339 equipment, 336 live cell imaging conditions, 336 quantifying recycling at single-event resolution, 339e341 reagents, 335e336 visualization of sorting and recycling in endosome, 341e343 G proteins, see GTP-binding proteins (G proteins) GAPs, see GTPase-activating proteins (GAPs) GDP, see Guanosine diphosphate (GDP) GEFs, see Guanine nucleotide exchange factors (GEFs) GFP, see Green fluorescent protein (GFP) GGA, see Golgi-localized, gamma-adaptin (GGA) Glucose transporter type (Glut4), 83, 88 detection of cell surface, 315e317 recycling, 95, 96f 3T3-L1 preadipocytes maintenance and differentiation, 312 buffers, 310 detection of cell surface Glut4, 315e317 exocyst complex, 309 GST-RAL binding domain preparation, 311e312 instruments, 311 plasmids and cell lines, 311 pulldown assay, 312e314 reagents, 310 RNAi-mediated depletion of RalA, 314e315 Glutathione-S-transferase (GST), 84 Gold stabilized with bovine serum albumin (Gold-BSA particles), 160 Gold-BSA particles, see Gold stabilized with bovine serum albumin (Gold-BSA particles) Golgi-localized, gamma-adaptin (GGA), 82 GPCR, see G protein-coupled receptor (GPCR) Green fluorescent protein (GFP), 48e49, 84, 130, 134, 182, 321e322 GFP-RAB-10 fusion protein, 190 GFP-RAB-11.1 marker, 190 GFP-VAMP3 mirrors, 16 GFP::RME-1 fusion protein, 188e190 GST, see Glutathione-S-transferase (GST) GST-RAL binding domain preparation, 311e312 GTP-binding proteins (G proteins), 128 GTPase-activating proteins (GAPs), 70, 82e83, 130 background, 74 exchanging GXP for [a32P]GTP, 75 materials, 74e75 preparing large unilamellar vesicles, 75 results and analysis, 76e78 GTPases, see Guanosine triphosphatases (GTPases) Guanine nucleotide exchange factors (GEFs), 36, 60, 103, 130 activity measurement native tryptophan fluorescence of Arf1, 117e120 small GTPase crosstalk variation, 121 Guanosine diphosphate (GDP), 103 Guanosine triphosphatases (GTPases), 82e83, 103, 272 H HaloTag protein, 61 Hanks balanced salt solution (HBSS), 276 HBSS, see Hanks balanced salt solution (HBSS) HEK cells, see Human embryonic kidney cells (HEK cells) HeLa cells, 84, 203, 238e239, 243e245, 323e324 Holo-Tf iodination, 258e259 Horseradish peroxidase (HRP), 51e53, 251 Horseradish peroxide, see Horseradish peroxidase (HRP) HRP, see Horseradish peroxidase (HRP) hTAC, 192 hTfR, see human transferrin receptor (hTfR) Human embryonic kidney cells (HEK cells), 335 human transferrin receptor (hTfR), 192 Humidity chamber, 145f 125 I-EGF, 349e350 125 I-EGF-loaded cells, 364e365 EGF:EGFR complex recycling using, 363e366 EGFR internalization rates measurement using, 356 considerations, 358e359 experimental protocol, 356e357 results, 357e358 variation, 357 internalization, 358f recycling, 364f 125 I-Tf recycling, reagents to, 256 Index I IC, see Intermediate compartment (IC) ICB, see Intracellular bridge (ICB) ICl method, see Iodine monochloride method (ICl method) IEM, see Immune-electron microscopy (IEM) IF, see Immunofluorescence (IF) ILVs, see Intraluminal vesicles (ILVs) Immune-electron microscopy (IEM), 321 Immunoblotting antibodies, 322 Immunofluorescence (IF), 205, 237 antibodies, 323 confocal microscopy, 321e322 In vitro endosome membrane tubule formation, 160 BBC, 163 cell-free, 163e165 colloidal gold particles preparation, 160 colloidal gold-loaded endosomes preparation, 161e163 In vivo endosome membrane tubule formation, 167 confocal microscopy, 168e174 labeling tubular endocytic compartments, 167e168 quantitative image analysis, 168e174 Insect cell transfection, 107e109 Insulin responsive glucose transporter, see Glucose transporter type (Glut4) Insulin-stimulated Glut4 trafficking, 308e309 Integrin recycling analysis, 94, 95f, 208, see also Retrograde trafficking analysis experimental analysis, 210 experimental strategy equipment, 208 FACS stripping buffer, 208e209 reagents, 208 software, 208 methods antibody labeling of cells, 209 cell plating, RNAi transfection, and harvesting of cells, 209 controls, 210 recycling and acid-stripping of antibodylabeled cells, 209e210 Intermediate compartment (IC), 36 Internalization assays, 133, 135 process, 140e141 of receptor, 252 Internalized cargo proteins trafficking, 281e282 Intracellular bridge (ICB), 20 Intraluminal vesicles (ILVs), 320e321 Iodine monochloride method (ICl method), 251 IPTG, see Isopropyl b-D-galactopyranoside (IPTG) IRE, see Iron response/regulatory element (IRE) Iron chelator desferrioxamine, 252 Iron load canine apo-Tf, reagents to, 253e254 Iron response/regulatory element (IRE), 248e250 Isobutyl-1-methylzanthine (MIX), 312 Isopropyl b-D-galactopyranoside (IPTG), 105 J JNK-interacting protein (JIP4), 130 K Kinase-dead ERK2 (ERK2-KD), 65 Kinesin, 236 depletion, 240e241 live cell imaging on cells coexpressing, 238f microtubule cytoskeleton, 239 material, 239 methods, 239 results, 239 Rab GTPasesekinesin interactions, screen for, 236 methods, 236e237 results, 237 spatiotemporal distribution, 237 material, 237 results, 238 L Labeling surface receptors, 251e252 Large unilamellar vesicles (LUVs), 74 Last eukaryotic common ancestor (LECA), 37 Late endosomes (LE), 272 Late-endosomal/lysosomal pathway (LE/Lys pathway), 140e141 LDLR, see Low-density lipoprotein receptor (LDLR) LE, see Late endosomes (LE) LE/Lys pathway, see Late-endosomal/lysosomal pathway (LE/Lys pathway) LECA, see Last eukaryotic common ancestor (LECA) Ligand-induced EGFR degradation considerations, 363 experimental protocol, 361e362 results, 362e363 theory, 361 Lilly Laboratories cell porcine kidney model cell line (LLC-PK1 model cell line), 292 389 390 Index Lipid mixes, 116 Liposome(s), see also ADP ribosylation factor (Arf); Endosome(s) binding, 122 composition, 115 additives, 116 flotation, 122e123, 123f analysis, 122e123 discontinuous sucrose gradient flotation, 122 nucleotide exchange, 122 quantification of recovery, 122 production lipid mixes, 116 solubilization and extrusion, 116e117 size, 116 Live cell imaging, Living intact animals, morphometric analysis of endosomal markers in ARF-6-GFP, 190 broadspectrum autofluorescence, 191 experimental strategies and considerations, 190 GFP emission, 191 GFP-RAB-10 fusion protein, 190 GFP-RAB-11.1 marker, 190 GFP::RME-1 fusion protein, 188e190 intestinal cell, 191 organelle markers, 190e191 principles, 188 LLC-PK1 cells alternative approaches, 295 background and objective, 293 considerations, 294 expected results, 294e295 flow of experiment, 293e294 protein sorting analysis, 295f LLC-PK1 model cell line, see Lilly Laboratories cell porcine kidney model cell line (LLC-PK1 model cell line) Low-density lipoprotein receptor (LDLR), 272, 294 LROs, see Lysosome-related organelles (LROs) LUVs, see Large unilamellar vesicles (LUVs) Lysate clearing, 109e111 Lysis clearing, 109e111 Lysosome-related organelles (LROs), 191 M mAb, see Monoclonal antibody (mAb) Macrophages, 2e3, see also Recycling endosomes (REs) localization of recycling endosome-associated Rab GTPases, 7f recycling endosome cargo and trafficking in, 12f MadineDarby canine kidney cell lines (MDCK cell lines), 248, 292, see also Polarized MDCK cells Magnaporthe oryzae (M oryzae), 37, see also Rabs recycling Major histocompatibility complex class I protein (MHC I protein), 133, 149e151, 158e160 antibody-receptor complex recycling quantification, 152e154 MDCK cell lines, see MadineDarby canine kidney cell lines (MDCK cell lines) Mean intensity fluorescence (MIF), 283 MEM, see Minimum essential medium (MEM) Membrane receptors, 295 trafficking events, 342e343 MHC I protein, see Major histocompatibility complex class I protein (MHC I protein) MICAL-L1, see Molecule interacting with CasLlike1 (MICAL-L1) Microscopy-based apical transcytosis assay, 282e283 Microscopy-based protocols, 275 ARE, 279e280 ASE, 276e277 BSE, 278 LE/Lys, 280e281 ligands and endosomal markers, 276t TfR-rich perinuclear common recycling endosomes, 279 Microscopy-based trafficking assays, 281 internalized cargo proteins trafficking, 281e282 microscopy-based apical transcytosis assay, 282e283 Microtubule dynamics analysis during cytokinesis, 29e30 Midbody, 29 Middle loop regions, 88 MIF, see Mean intensity fluorescence (MIF) MIG-14-GFP, 193e194 MIG-14, 192 Minimum essential medium (MEM), 161, 256, 292 MIX, see Isobutyl-1-methylzanthine (MIX) Molecular switches, 309 Molecule interacting with CasL-like1 (MICAL-L1), 141e142 Monoclonal antibody (mAb), 38 MOR, see mu opioid receptor (MOR) MoRab11, MoRab8, and MoRab1 cloning Index amplifying cDNAs, 39e40 total RNA extraction, 38e39 colocalization in BHK cells, 43f expression and intracellular localization BHK-21 cells transfection, 40 localization by confocal fluorescence microscopy, 42 protein expression immunoblot analysis, 40e42 mu opioid receptor (MOR), 334 Multinucleation analysis, 22e23 data interpretation, 23e24 procedure, 23 Multivesicular bodies (MVB), 200 Multivesicular endosomes (MVEs), 320e321, see also G protein-coupled receptor (GPCR) MVB, see Multivesicular bodies (MVB) MVEs, see Multivesicular endosomes (MVEs) Myc-tagged PAP, 84 Myc7-Glut4-eGFP reporter, cell surface Glut4 detection with, 315e317 Myristoylated Arf1 (myrArf1), 73 expression, 72e73 GAP assay, 75e76 background, 74 exchanging GXP for [a32P]GTP, 75 materials, 74e75 preparing large unilamellar vesicles, 75 results and analysis, 76e78 preparation background, 71e72 buffers, 72 materials, 72 purification, 73e74, 112e114 protein expression, 113 protein purification, 113e114 Rab GTPases, 115 N N-terminal regions, 88 NanoLuc, 61 Native tryptophan fluorescence of Arf1, 117 data analysis curve comparison, 120 curve extraction, 120 curve fitting, 120 data collection notes, 117e119 protein fluorescence calibration, 119 protocol, 119 before starting, 119 nucleotide exchange kinetics calculation, 118f working stocks, 117 Neuron-glia cell adhesion molecule (Ng-CAM), 301 Nickel, 116 Nocodazole (Noco), 239, 251e252 Nucleotide exchange, 122 O Object-based colocalization method, 226 Open reading frame (ORF), 62 Optical axis integration scan function (OAI scan function), Optical density (OD), 72e73 P PAF, see Platelet-activating factor (PAF) PAR1, see Protease-activated receptor (PAR1) Paraformaldehyde (PFA), 38, 203, 292, 323 pBI-Tet vector amplifying cDNAs of MoRab11, MoRab8, and MoRab1, 39e40 eGFP-labeled MoRabs bidirectional expression vector, 41f PBS, see Phosphate buffered saline (PBS) PC, see Phosphatidylcholine (PC) PE, see Phosphatidylethanolamine (PE) PEI, see Polyethylenimine (PEI) Personal Deltavision, PFA, see Paraformaldehyde (PFA) Phagosomes, RE delivery to, 14 experimental considerations, 15 flow of experiment, 15 results, 16 Phenylmethylsulfonyl fluoride (PMSF), 63, 105, 323 Phosphate buffered saline (PBS), 23, 26, 37, 53, 85e86, 132, 203, 277, 296, 322, 350 Phosphate-buffered salt, see Phosphate buffered saline (PBS) Phosphatidylcholine (PC), 74 Phosphatidylethanolamine (PE), 74 Phosphatidylinositiol 3,4,5-trisphosphate (PIP3), 74 Phosphatidylinositol (PI), 74 Phosphatidylinositol 4,5-bisphosphate (PIP2), 74 Phosphatidylserine (PS), 74 Phosphoinositides (PIPs), 128 Phospholipase A2 (PLA2), 158e160 antagonists, 175 inhibitors use in in vitro assays, 178 reversible inhibitors, 175e177 391 392 Index Phospholipase A2 (PLA2) (Continued) treatment of living cells, 175 endocytic organelles and steps, 159f in endosome membrane tubule formation, 174 inhibitors, 176te177t Phosphotungstic acid (PTA), 164, 350 PI, see Phosphatidylinositol (PI) pIgR, see Polymeric IgA receptor (pIgR) PIP2, see Phosphatidylinositol 4,5-bisphosphate (PIP2) PIP3, see Phosphatidylinositiol 3,4,5trisphosphate (PIP3) PIPs, see Phosphoinositides (PIPs) PLA2, see Phospholipase A2 (PLA2) Plasma membrane (PM), 140, 272 Plasmids, 37, 84e85 Platelet-activating factor (PAF), 174 PM, see Plasma membrane (PM) PMSF, see Phenylmethylsulfonyl fluoride (PMSF) PNS, see Postnuclear supernatant (PNS) Polarized epithelial cells microscopy-based protocols for endosomal labeling, 275 ARE, 279e280 ASE, 276e277 BSE, 278 LE/Lys, 280e281 ligands and endosomal markers, 276t TfR-rich perinuclear common recycling endosomes, 279 microscopy-based trafficking assays, 281 internalized cargo proteins trafficking, 281e282 microscopy-based apical transcytosis assay, 282e283 Polarized MDCK cells culture, 255f endocytic pathways in, 249f materials and instruments, 253 culture MDCK cells, reagents to, 256 equipment, 257 125 I-Tf recycling, reagents to, 256 iodinate canine holo-Tf, reagents to, 254 iron load canine apo-Tf, reagents to, 253e254 tricholoracetic acid precipitation, reagents to, 256e257 methods culturing MDCK cells on 12-MM transwells, 259e260 experimental strategy to receptor recycling measurement, 260e266 iodinating holo-Tf, 258e259 loading apo-Tf with iron, 257e258 objectives and rationale, 250e251 internalization of receptor, 252 labeling surface receptors, 251e252 receptor recycling measurement, 252e253 Polyethylenimine (PEI), 322 Polymeric IgA receptor (pIgR), 272e273 Postnuclear supernatant (PNS), 161 PQ, see Primaquine (PQ) Primaquine (PQ), 349e350 effect, 363e366 Protease-activated receptor (PAR1), 320e321 Protein buffers, 107t concentration quantification A280, 124 Bradford semiquantitative analysis, 123e124 Coomassie gel analysis, 124e125 quick analysis of elution fractions, 123 expression, 110e111 approximately 4e5 days beforehand, 109 day 1, 109 day 3, 109 immunoblot analysis, 40e42 one day beforehand, 109 reagents for protein expression and analysis, 37e38 fluorescence calibration, 119 purification, 108f, 109 anion exchange, 110 batch nickel affinity purification, 110 lysis and lysate clearing, 109e110 ProteineArf1 interactions identification liposome flotation, 122e123, 123f analysis, 122e123 discontinuous sucrose gradient flotation, 122 liposome binding, 122 nucleotide exchange, 122 quantification of recovery, 122 notes, 121 Proteinase K protection assay reagents, 323 Proteinase-protection assay, 321, 324 considerations, 326 proteinase-protection endosomal sorting assay, 325f results, 326 workflow of experiment, 324e326 PS, see Phosphatidylserine (PS) PTA, see Phosphotungstic acid (PTA) Pulldown assay, 312e314 Index Q Qualitative assessment of endocytic recycling experimental strategies, 143e144 humidity chamber, 145f imaging, 147e148 immunfluorescence, 144e147 postimage processing, 148 remarks, 148e149 Tf and EGF treatment, 144 Quantitative assessment of endocytic recycling experimental strategies, 149e151 flow cytometry analysis of fluorescently labeled Tf, 151e152 remarks, 152 Quantitative image analysis, 168e169 Quick analysis of elution fractions, 123 R Rab GTPases, 36, 115, 236 live cell imaging on cells coexpressing, 238f microtubule cytoskeleton in, 239 material, 239 methods, 239 results, 239 screen for Rab GTPasesekinesin interactions, 236 methods, 236e237 results, 237 spatiotemporal distribution, 237 material, 237 results, 238 Rab proteins, 60, 128e129, 141 in endosomal trafficking, 129f Rab11-effector proteins, 22 RAB11/FIP3-endosome transport analysis, 27e28 during cytokinesis, 28f data interpretation, 29 procedure, 28e29 Rab5 protein, 128e129 Rab5-Q79L expression, GPCR sorting quantification at MVEs by, 327 considerations and approaches, 328e329 fluorescence intensity analysis, 330 quench and wash buffers, 328t results, 330 workflow of experiment, 327e328 Rab5Q79L, 222e223 Rab8 protein, 36 Rabin8, 60 autoinhibitory conformation, 61 BRET assay setup, 64 detecting Rabin8 conformational change, 64 BRET technology, 61fe62f materials, 62e63 NanoLuc-HaloTag fusion proteins expression and purification, 63 Rabs protein, 128e129 Rabs protein, 128e129 Rabs protein, 128e129 Rabs 11 protein, 128e129 Rabs 22 protein, 128e129 Rabs 35 protein, 128e129 Rabs recycling, 37 equipment, 38 materials and reagents for confocal fluorescence microscopy, 38 MoRab11, MoRab8, and MoRab1 cloning, 38e40 expression and intracellular localization, 40e42 plasmid construction, 37 RE subcompartments by, 5e8 reagents for cloning, 37 reagents for protein expression and analysis, 37e38 Radioactive pulse-chase experiments, see also Polarized MDCK cells alternative approaches, 302e303 background and objective, 299 considerations, 301 expected results, 301 flow of experiment, 299e301 RalA cellular activity, 309 preparing GST-RAL binding domain, 311e312 pulldown assay using immobilized GST-RBD to determining, 312e314 RNAi-mediated depletion of RalA, 314e315 Ras-MEK-ERK signal pathway, 64e65 RAW264 macrophages, RE, see Recycling endosome (RE) Receptor recycling measurement, 252e253 background and objective, 260 flow of experiment considerations, 264e266 data analysis, 263e264 labeling cohort of TfR with 125I-Tf, and ensuring internalization, 260e261 perform recycling assay, 261e263 removal of cell-surface bound 125I-Tf, 261, 262f results, 266 TCA precipitation, 263 Reconstituting GST-Rab11, methods for, 56 Recycling, 248 recycling/transcytosis assay, 284e285 393 394 Index Recycling endosome (RE), 2e3, 20, 48e49, see also Endosome(s) cargo movement, experimental considerations, 11 flow of experiment, 9e10 results, 11e14 VAMP3 recruitment, 13f cell culture and transfection, centrosome isolation and identification, 53 centrosomes isolation at interphase, methods to, 53e55 reconstituting GST-Rab11, methods for, 56 staining isolated centrosomes, methods for, 55 centrosome localization by electron microscopy, 51e53 delivery to phagosomes, 14 experimental considerations, 15 flow of experiment, 15 results, 16 instruments, live cell imaging, 50 in interphase cells, 50e51 in mitotic cells, 51 live cell imaging, in macrophages, RE-associated proteins and lipids, reagents, SNARE protein family, subcompartments by Rabs, 5e6 flow of experiment, results, 6e8 Tfn-HRP-loaded endosomes, 52f Region of interest (ROI), 10 Retinal pigment epithelium (RPE), 274e275 Retrograde trafficking analysis, see also Integrin recycling analysis CI-M6PR, 201e203, 202f experimental analysis, 206e207 experimental strategy reagents and materials, 203e204 integrin a5b1 cartoon depicting recycling, 202f methods antibody labeling to monitor retrograde trafficking of CI-M6PR, 204e205 cell plating, 204 fixing and blocking of cells, 205 prepare and stain with primary antibody, 205e206 prepare and stain with secondary fluorescentconjugated antibodies, 206 RNAiMAX transfection on coverslips, 204 Ribonucleic acid (RNA), 53 rme-1, 186e188, 187f RNA, see Ribonucleic acid (RNA) RNA interference (RNAi), 201 RNAi, see RNA interference (RNAi) RNAi-mediated depletion of RalA, 314e315 RNAiMAX transfection on coverslips, 204 ROI, see Region of interest (ROI) Room temperature (RT), 219, 240 RPE, see Retinal pigment epithelium (RPE) RT, see Room temperature (RT) S Saponin permeabilization workflow, 328e329 sb-Tf, see Sulfotag and biotin-Tf (sb-Tf) SDS, see Sodium dodecyl sulfate (SDS) SDS-PAGE, see Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SE, see Sorting endosome (SE) Sec7, 106 protein expression, 110e111 protein purification, 111e112 purification of Sec7(203e2009), 106 protein expression, 109 protein purification, 109e110 virus preparation, 107e109 Semiautomated endosome membrane tubule segmentation, 170f Fiji macro script for, 173e174 SEP, see Super-ecliptic pHluorin (SEP) Sholl analysis, 172e173 signal sequence (ss), 335 Signal to noise ratio (SNR), 220 Single-event resolution, quantifying recycling at, 339 considerations, 341 flow of experiments, 339e341 results, 341 siRNA, see Small interfering RNA (siRNA) Slow recycling endosomes, 36 Small GTPase crosstalk variation, 121 SMALL GTPases myristoylated Arf1 purification, 112e114 soluble constructs purification, 112 Small interfering RNA (siRNA), 203 SNARE protein family, SNR, see Signal to noise ratio (SNR) SNX, see Sorting nexin (SNX) Sodium dodecyl sulfate (SDS), 292 Index Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 85, 351e352 Solubilization, 116e117 Soluble constructs purification, 112 Sorting endosome (SE), 140e141, 272 Sorting nexin (SNX), 82 Sorting signals, 82 SpH, see Superecliptic pHluorin (SpH) ss, see signal sequence (ss) Stain cells, 135e136 Staining isolated centrosomes, methods for, 55 Step-by-step endosome membrane tubule “tracing”, 171e173 STX4, see Syntaxin-4 (STX4) Sulfotag and biotin-Tf (sb-Tf), 283 recycling, 283e285 Super-ecliptic pHluorin (SEP), 14e15 Superecliptic pHluorin (SpH), 335 Syntaxin-4 (STX4), 61 T TBS, see Tris buffer saline (TBS) TCA, see Trichloroacetic acid (TCA) TCL, see Total cell lysates (TCL) TEM, see Transmission electron microscope (TEM) Tf, see Transferrin (Tf) Tfn-568, loading endosomes with, 48f Tfn-HRP-loaded endosomes, 52f TfR, see Transferrin receptor (TfR) TfR-rich perinuclear common recycling endosomes (CRETfR), 272e273 TGN, see trans-Golgi network (TGN) Thin layer chromatography (TLC), 76 3T3-L1 cells, 85 3T3-L1 preadipocytes maintenance and differentiation, 312 RNAi-mediated depletion of RalA in mature, 314e315 Time-lapse analysis, 29 of endosome and actin dynamics during, 27 analyzing actin dynamics, 29e30 and apical lumen formation in 3D cultures, 30e32 RAB11/FIP3-endosome transport analysis, 27e29 Time-lapse imaging, 194e195 TIRFM, see Total internal reflection fluorescence microscopy (TIRFM) Tissue culture supplies, 310 TLC, see Thin layer chromatography (TLC) TM proteins, see Transmembrane proteins (TM proteins) Total cell lysates (TCL), 313 Total internal reflection fluorescence microscopy (TIRFM), 335 Trafficking of receptors, 200 trans-Golgi network (TGN), 16, 103, 128, 201e203, 274 Transcytotic assay, 283e285 Transfections, 84e85, 134 Transferrin (Tf), 11, 92, 142, 158e160, 218, 239 recycling, 92, 93f recycling through endocytic compartments, 168 slow Tf recycling route analysis, 242 material, 242 methods, 243e244 results, 244 ultrastructural localization, 241e242 uptake into endocytic compartments, 168 Transferrin receptor (TfR), 82, 128, 143e144, 239e240, 272 TfR-1, 248 Transmembrane proteins (TM proteins), recycling cargo assays experimental strategies and considerations, 192e193 hTAC, 192 MIG-14-GFP, 193e194 principles, 191e192 Transmission electron microscope (TEM), 53, 160 TRE, see Tubular recycling endosomes (TRE) Trichloroacetic acid (TCA), 260, 350 precipitation, 263 reagents to, 256e257 Tris buffer saline (TBS), 350 Tubular recycling endosomes (TRE), 141e142 Tubulation factors, 163 V Virus preparation, 107 bacmid preparation, 107 insect cell transfection, 107e109 viral amplification, 109 W WASH, see Wiskott-Aldrich syndrome protein and SCAR Homologue (WASH) WASH domain localization in fixed cells cell plating, Tf uptake and anti-WASH immunofluorescence, 227 considerations, 231 image analysis, 228e230, 229f 395 396 Index WASH domain localization in fixed cells (Continued) imaging, 227 object-based colocalization method, 226 quantitative 3D analysis, 225e226 reagents and instruments, 226e227 results, 230e231 WASH microdomains live imaging, 222e223, 224f, see also Endosome tubulation imaging cell imaging, 223 cell plating and Tf uptake, 223 considerations, 225 image analysis and results, 223e225 reagents and instrument, 223 Western blot analysis, 245 Wheat germ agglutinin (WGA), 275e276, 351 Wild-type receptor (WT receptor), 324 Wiskott-Aldrich syndrome protein and SCAR Homologue (WASH), 200e201, 216e217 complex, 201 function, 201 WASH-mediated actin polymerization, 217 WT receptor, see Wild-type receptor (WT receptor) Y Yolk protein (YP170), 182 uptake assay in C elegans oocyte, 184e186 YP170::GFP transport assay sensitivity, 186 ... experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety... bringing nutrients into the cell on recycling surface receptors (Maxfield & McGraw, 2004) Transferrin receptors in complex with the iron chelating protein transferrin traverse this network, bringing... the change in amount of transferrin in a cell as follows: the rate of change in the amount of the transferrin in the cell over time is proportional to the amount of transferrin in the cell, that