Methods in Cell Biology The Neuronal Cytoskeleton, Motor Proteins, and Organelle Trafficking in the Axon Volume 131 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 The Neuronal Cytoskeleton, Motor Proteins, and Organelle Trafficking in the Axon Volume 131 Edited by K Kevin Pfister Department of Cell Biology, Charlottesville, 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 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, 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 2016 Copyright © 2016 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: 978-0-12-803344-9 ISSN: 0091-679X For information on all Academic Press publications visit our website at http://store.elsevier.com Contributors Stefanie Alber Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel C.J Alexander Cell Biology and Physiology Center, National Heart, Lung Blood Institute, National Institutes of Health, MD, USA Adam W Avery Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA Peter W Baas Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA Alexandre D Baffet Department of Pathology and Cell Biology, Columbia University, New York, NY, USA Lisa Baker Marine Biological Laboratory, Woods Hole, MA, USA Gary Banker Jungers Center for Neurosciences Research, Oregon Health and Science University, Portland, OR, USA Marvin Bentley Jungers Center for Neurosciences Research, Oregon Health and Science University, Portland, OR, USA Mark M Black Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA, USA Kiev R Blasier Department of Cell Biology, University of Virginia, Charlottesville, VA, USA Scott T Brady Marine Biological Laboratory, Woods Hole, MA, USA; Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Anthony Brown Department of Neuroscience, The Ohio State University, Columbus, OH, USA xiii xiv Contributors Kristy J Brown Research Center for Genetic Medicine, Children’s National Health System, Washington, DC, USA; Department of Integrative Systems Biology, Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA Alma L Burlingame Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA John C Cain Department of Cell Biology, University of Virginia, Charlottesville, VA, USA Aure´lie Carabalona Department of Pathology and Cell Biology, Columbia University, New York, NY, USA Anaeăl Chazeau Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Michael Chein Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Tiago J Dantas Department of Pathology and Cell Biology, Columbia University, New York, NY, USA David D Doobin Department of Pathology and Cell Biology, Columbia University, New York, NY, USA Ella Doron-Mandel Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel Catherine M Drerup Department of Cell, Developmental and Cancer Biology, School of Medicine, Oregon Health & Science University, Portland, OR, USA Noelle D Dwyer Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA, USA Mike Fainzilber Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel Contributors J Daniel Fenn Department of Neuroscience, The Ohio State University, Columbus, OH, USA Xiaoqin Fu Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Kathlyn J Gan Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada Archan Ganguly Department of Pathology, University of California, San Diego, La Jolla, CA, USA Shani Gluska Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel J.A Hammer, III Cell Biology and Physiology Center, National Heart, Lung Blood Institute, National Institutes of Health, MD, USA Thomas S Hays Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA Erika L.F Holzbaur Department of Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; Neuroscience Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Casper C Hoogenraad Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Daniel J Hu Department of Pathology and Cell Biology, Columbia University, New York, NY, USA Chung-Fang Huang Jungers Center for Neurosciences Research, Oregon Health and Science University, Portland, OR, USA; National Laboratory Animal Center, NARLabs, Taipei, Taiwan Ariel Ionescu Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel xv xvi Contributors Kerstin M Janisch Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA, USA Minsu Kang Marine Biological Laboratory, Woods Hole, MA, USA; Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Lukas C Kapitein Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Eugene A Katrukha Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Noopur V Khobrekar Department of Pathology and Cell Biology, Columbia University, New York, NY, USA Eva Klinman Department of Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; Neuroscience Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Kelsey Ladt Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA Zofia M Lasiecka Children’s National Medical Center, Washington, DC, USA Seung Joon Lee Department of Biological Sciences, University of South Carolina, Columbia, SC, USA Lanfranco Leo Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA Min-gang Li Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA Judy S Liu Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Contributors James B Machamer Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Katalin F Medzihradszky Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA David J Mitchell Department of Cell Biology, University of Virginia, Charlottesville, VA, USA Paula C Monsma Department of Neuroscience, The Ohio State University, Columbus, OH, USA Gerardo Morfini Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA; Marine Biological Laboratory, Woods Hole, MA, USA Kanneboyina Nagaraju Research Center for Genetic Medicine, Children’s National Health System, Washington, DC, USA; Department of Integrative Systems Biology, Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA Alex V Nechiporuk Department of Cell, Developmental and Cancer Biology, School of Medicine, Oregon Health & Science University, Portland, OR, USA Amanda L Neisch Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA Jeffrey J Nirschl Department of Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; Neuroscience Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Juan A Oses Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA Eran Perlson Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel K Kevin Pfister Department of Cell Biology, University of Virginia, Charlottesville, VA, USA xvii xviii Contributors Sree Rayavarapu Research Center for Genetic Medicine, Children’s National Health System, Washington, DC, USA; Department of Integrative Systems Biology, Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA Mitchell W Ross Department of Cell Biology, University of Virginia, Charlottesville, VA, USA Nimrod Rotem Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Subhojit Roy Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA; Department of Pathology, University of California, San Diego, La Jolla, CA, USA Philipp Schaătzle Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Michael A Silverman Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada; Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada; Brain Research Centre, University of British Columbia, Vancouver, BC, Canada Yuyu Song Marine Biological Laboratory, Woods Hole, MA, USA; Yale School of Medicine, Department of Genetics and Howard Hughes Medical Institute, Boyer Center, New Haven, CT, USA Jeffery L Twiss Department of Biological Sciences, University of South Carolina, Columbia, SC, USA Atsuko Uchida Department of Neuroscience, The Ohio State University, Columbus, OH, USA Richard B Vallee Department of Pathology and Cell Biology, Columbia University, New York, NY, USA Bettina Winckler Department of Neuroscience, University of Virginia Medical School, Charlottesville, VA, USA 496 Volumes in Series Volume 96 (2010) Electron Microscopy of Model Systems Edited by Thomas Muăeller-Reichert Volume 97 (2010) Microtubules: In Vivo Edited by Lynne Cassimeris and Phong Tran Volume 98 (2010) Nuclear Mechanics & Genome Regulation Edited by G.V Shivashankar Volume 99 (2010) Calcium in Living Cells Edited by Michael Whitaker Volume 100 (2010) The Zebrafish: Cellular and Developmental Biology, Part A Edited by: H William Detrich III, Monte Westerfield and Leonard I Zon Volume 101 (2011) The Zebrafish: Cellular and Developmental Biology, Part B Edited by: H William Detrich III, Monte Westerfield and Leonard I Zon Volume 102 (2011) Recent Advances in Cytometry, Part A: Instrumentation, Methods Edited by Zbigniew Darzynkiewicz, Elena Holden, Alberto Orfao, William Telford and Donald Wlodkowic Volume 103 (2011) Recent Advances in Cytometry, Part B: Advances in Applications Edited by Zbigniew Darzynkiewicz, Elena Holden, Alberto Orfao, Alberto Orfao and Donald Wlodkowic Volume 104 (2011) The Zebrafish: Genetics, Genomics and Informatics 3rd Edition 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Microscopy, 4th Edition Edited by Greenfield Sluder and David E Wolf Volume 115 (2013) Microtubules, in Vitro, 2nd Edition Edited by John J Correia and Leslie Wilson 497 498 Volumes in Series Volume 116 (2013) Lipid Droplets Edited by H Robert Yang and Peng Li Volume 117 (2013) Receptor-Receptor Interactions Edited by P Michael Conn Volume 118 (2013) Methods for Analysis of Golgi Complex Function Edited by Franck Perez and David J Stephens Volume 119 (2014) Micropatterning in Cell Biology Part A Edited by Matthieu Piel and Manuel The´ry Volume 120 (2014) Micropatterning in Cell Biology Part B Edited by Matthieu Piel and Manuel The´ry Volume 121 (2014) Micropatterning in Cell Biology Part C Edited by Matthieu Piel and Manuel The´ry Volume 122 (2014) Nuclear Pore Complexes and Nucleocytoplasmic Transport - Methods Edited by Vale´rie Doye Volume 123 (2014) Quantitative Imaging in Cell Biology Edited by Jennifer C Waters and Torsten Wittmann Volume 124 (2014) Correlative Light and Electron Microscopy II Edited by Thomas Muăller-Reichert and Paul Verkade Volume 125 (2015) Biophysical Methods in Cell Biology Edited by Ewa K Paluch Volume 126 (2015) Lysosomes and Lysosomal Diseases Edited by Frances Platt and Nick Platt 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 Volume 130 (2015) Sorting and Recycling Endosomes Edited by Wei Guo 499 Index Note: Page numbers followed by “f” and “t” indicate figures and tables respectively A Acquire timelapse, 258e259 Actin, 92 Affinity chromatography, 471e475, 472f axoplasm, 474 beads, conjugation to, 474 materials, 471e473 preparation, 473e474 washing and elution, 475 Alzheimer’s disease (AD), 278e279 Ab oligomers impair axonal transport, 427e428 AbO preparation and application, 436e437 unmodified and fluorescent Ab oligomers, preparation and application of, 437 unmodified and fluorescent Ab peptides, monomerization and storage of, 436e437 AbO sources and preparations, 432 cell viability, assessment of, 443e445, 444fe445f healthy and diseased neurons, fast axonal transport in, 426e427 imaging axonal transport, approaches to, 428e432 invertebrate techniques, 428e429 mammalian in vitro techniques, 430e432, 431t mammalian in vivo techniques, 429e430 kymograph generation and analysis, 441 live imaging, 437e441 chamber assembly, 439e441, 440f equipment setup, 438 imaging chamber, preparation of, 438e439 organelles implicated, live imaging of, 432e433 primary neuronal culture, 433e434 problems and troubleshooting, 442e443 transfections, 434e436 transfection protocol, 434e436 AmaxaÒ, 172 Amicon Ultrafree-MC 0.1 mm centrifugal concentrator (EMD Millipore), 56 Amino acids, Amyloid precursor protein (APP), 422f density, 421e422 motility, 422 velocity, 421 Anterior spiracles, 289e290 Apical endfoot, 234e235 Argus 20, 334 Astrocytes, 394 Autophagosomes-LC3, 419f density, 418 motility, 418 velocity, 418 Axiomat, 334 Axonal actin imaging actin probes, 98e99, 99f dynamics, 102e103, 104fe105f low expressers, selection, 99e102, 101f neuronal morphology and axon selection Lipofectamine, 95 neuron suitable/unsuitable imaging, 97f specific criteria, 97e98 synaptophysin vesicles, 97f Axonal jams, 292, 293f Axonal transport, 344e345 cargo, selection of, 411e414 cargo motion, properties of, 412 density, 413, 413f motility, 414 velocity, 413 defined, imaging, basics of, 414e415 live-cell imaging axons, neurofilaments transported in, 8e10 axoplasm, SCb and soluble proteins movement of, 12e14 cytoskeletal polymers, movement of, microtubules, 10e12 vesicular cargoes, FC and movement of, 4fe5f, 7e8 methods data analysis, see Data analysis live-cell imaging, see Live-cell imaging primary neuron culture, 271, 272f motility, distinct patterns of, 415e422 amyloid precursor protein, see Amyloid precursor protein (APP) autophagosomeseLC3, see AutophagosomesLC3 late endosomes/lysosomeseLAMP1, see Late endosomes/lysosomes-LAMP1 late endosomes/lysosomeseRab7, see Late endosomes/lysosomes-Rab7 mitochondriaeMITO, see Mitochondria-MITO 501 502 Index Axonal transport (Continued) signaling endosomeseTrkB, see Signaling endosomes-TrkB pulse-labeling studies of, 2e7, 4fe5f transport analysis, 415 zebrafish confocal imaging, mounting for, 316e318, 317f kymograph analysis, 320e323, 321f, 324f microinjection, deoxyribonucleic acid for, 314 monomeric red fluorescent protein-tagged dynein light intermediate chain transport, 318e320, 319f mRFP-Dync1li1 and lysosome associated membrane protein 1-GFP cotransport, 323e326, 325f zygotic microinjection, see Zygotic microinjection Axoplasm axonal transport, 344e345 buffers, 335e336 challenges, 335 dissection and extrusion, 332f, 341f Dumont forceps, 338 P100 Petri dish, 340 squid mantle, 338, 340f stellate ganglion and giant axons, 336, 339f Vannas-style iris scissors, 340e342 dynabeads, preclearing with, 474 materials, 338t perfusions, 335, 342e344 RNA-bound beads, 474 sciatic nerves, axonal proteins from materials, 469 procedure, 470e471 vesicle trafficking, 334 B Biotin-UTP, in vitro transcription with, 473 Biotinylated RNA oligonucleotides, 473e474 Brain-derived neurotrophic factor (BDNF), 432e433 Brain slices, live imaging of medium preparation and slicing, 356e357 screening and slice preparation, 357e358 small molecule inhibitors, treating brain slices with, 358 Buffer X, 335e336 C Cables/spikes, 101e102 Caenorhabditis elegans, 312e313 Calmodulin-regulated spectrin-associated protein (CAMSAP), 128e129 Cargo axonal transport motility, distinct patterns of amyloid precursor protein, see Amyloid precursor protein (APP) autophagosomeseLC3, see Autophagosomes-LC3 late endosomes/lysosomeseLAMP1, see Late endosomes/lysosomes-LAMP1 late endosomes/lysosomeseRab7, see Late endosomes/lysosomes-Rab7 mitochondriaeMITO, see Mitochondria-MITO signaling endosomeseTrkB, see Signaling endosomes-TrkB selection of, 411e414 cargo motion, properties of, 412 density, 413, 413f motility, 414 velocity, 413 Casein kinase (CK2), 201 CellaxessÒ, 166 Cell counter, 247 Cell fractionation, 3, Cell viability, assessment of, 443e445, 444fe445f Cerebral cortex brain sample preparation brain slices, live imaging of, 356e358 developing brain sections, immunohistochemistry of, 355e356 microscopy fixed imaging, 358e359 live imaging, 359e361, 360f in utero electroporation surgical procedure, 351e353, 353f timing considerations, 353e355, 354f Chromatic aberration correction, 143 Cilia index, 246 Class IV dendritic arborization neurons materials and equipment, 299e300 methods dendritic arbor reconstruction, 302e303 larvae for imaging, 300, 301f live imaging, 300e301, 302f morphology, 303e305, 304fe305f UASetransgene expression, 299 Coimmunoprecipitation (co-IP), 480e481 axoplasm, preparation of, 480 beads, incubation of antibodyeprotein mixture with, 481 Index materials, 480 protein extract, incubation of antibody with, 480 protein G beads, preparation of, 480 washing and elution, 481 Compartmental microfluidic chambers axonal transport, 380f compartment partition and Quantum Dot-NGF, 378 live imaging, 379 data analysis, 383e384, 384f DRG explant cultures, 376e378 image analysis, 379e383 mold diagram, 367e368, 369f mold dimension measurement, 367e368, 371t mold fabrication alignment markers coating, 371e372 development, 373 fluoropolymer coating and plate attachment, 373 groove layer, 372 hard bake, 373 new wafer cleaning, 370 photolithography, 369e373 set up mask aligner, 372 soft bake, 372 neuronal growth factor, 367 neurons advantage, 366e367 characteristics, 366 PDMS casting and platform assembly, 367e368 chlorotrimethylsilane treatment, 374 microfluidic chamber preparation, 370f, 375 plate coating, 375e376 preparation and casting, 374 pressurized air, 374 protocol flow chart, 367e368, 368f well and cave pattern punching, 367e368, 370f Concentrated peptides, 155e156 Corpus callosum, 152, 157f Cortical neuroepithelial progenitors, 234e235, 235f Cortical neuron cultures, 27e28, 28t Banker method, 36 coverslip sandwich assembly, 36, 36f glial sandwich cultures, 37, 40e42 preparation, 40 suspension preparation, 37e40, 38f Cortical root ganglion neurons, 458 Cortical slab preparation, 236e240, 237fe238f Cultured neurons binocular dissecting microscope, 25 cortical neuron cultures, 27e28, 28t, 36e42 coverslips and culture dishes, 29e33 dorsal root ganglion neuron cultures, 28e29, 29t, 42e44 live-cell imaging, 392 acquisition, 404e405 compartment-specific fluorescent dyes, 402e403 data analysis, 406 imaging conditions, 404 imaging set-up, 403e404 mouse hippocampal culture, P0 pups, 394e395 rat hippocampal culture, E18 embryos, 392e394 visualizing proteins of interest, 395e403 SCG cultures, 34e36 superior cervical ganglion neuron cultures, 26e27, 26t Cytokinesis cortical slab preparation, 236e240, 237fe238f data analysis, 245e247, 248f imaging, 241e245, 242f, 244f immunohistochemistry, 240e241 Kif20bmagoo mutants, 245e250, 248f Kif20b mutations, 236 mammals, 235e236 neural epithelia, 234e235, 235f Cytoplasmic dynein, 7, 157e158, 254, 312, 461e462 calcium phosphate transfection, 255e258 fluorescent dynein ICs membrane-bounded organelle markers, 259e260 regulatory constructs, 260 with fluorescent microscopy, 258e259 image and data analysis with cargo markers, 262 density of dynein puncta, 262 dynein puncta, 261e262 kymographs, 264, 265f MetaMorph, 261 motility and velocity, 264 primary hippocampal neurons, 255 transfection methods and siRNA, 261 electroporation, 260e261 Lipofecatamine 2000, 260 nucleofection, 260e261 Cytoskeletal-associated protein glycine-rich (CAP-Gly), 270e271 D Data analysis materials, 273e274 503 504 Index Data analysis (Continued) retrograde flux, kymograph analysis of, 274 ddaC neurons, see Class IV dendritic arborization neurons Deltavision Elite microscope, 241e243 Dendrites, 128 Deoxyribonucleic acid, 314 Dextran conjugates, 402e403 Differential interference contrast (DIC), 293 Digital multimirror device (DMD), 75e76 Distal bouton accumulation analysis, 298e299 Dorsal root ganglion (DRG), 411, 412f, 458 explant cultures, 376e378, 377f neuron cultures, 28e29, 29t long-term myelinating DRG cocultures, 42e44 NbActiv4TMculture medium, 42 short-term nonmyelinating DRG cultures, 44 Doublecortin (Dcx) gene defined, 152, 153f mass spectrometry data analysis, 156 data validation of, 156 defined, 155e156 sample preparation for, 154e156 method animal handling, 153 generating 13C-LYSINE-C57BL/6-SILAC mice, 154 tissue collection, 154 roles, 152 Drift correction, 142 Drosophila, 234, 312e313, 428e429 Drosophila melanogaster, see also Neuronal intracellular transport, Drosophila UAS-Gal4 system, 279 DsRed2, 45e46 DualView (Photometrics), 259e260 Dynamic instability, 128e129 Dynein/Dynactin, 293e295 E Ectopic protein expression, 110 Electron multiplying cooled CCD (EMCCD), 70 Electroporation, 49e52, 165e166, 260e261 End-binding (EB) proteins, 128e129 Endoplasmic reticulum (ER), 179 Endosomes function, 390 imaging, see Live-cell imaging role, 390 Epifluorescence microscopy, 461 Eppendorf system, 54 F Fab fragments, 401 F-actin, 92 Fast axonal transport (FAT), 200e202, 208 Fast component (FC), 4fe5f, 7e8 FAT, see Fast axonal transport (FAT) FC, see Fast component (FC) Fetal bovine serum (FBS), 27 FIJI, data processing and analysis with, 119e123, 121f image processing, 120e122 kymographs, analyzing dynamics with, 121f, 122e123 Fixed-field tracking, 79e81 Fluorescence photoactivation, 61e65 experiments, 77e78 Fluorescence photobleaching, 61 experiments, 76e77 Fluorescence recovery after photobleaching (FRAP), 405 Fluorescent-tagged proteins, 430 Focus drift, 73e74 G G-actin, 93 GAGG, see Glycine rich-linker sequence (GAGG) Glass-bottomed dishes, 30f coverslips attachment, paraffin wax, 31 drilling the holes, 30e31 sterilizing and coating coverslips, 32e33 Glial sandwich cultures, 37, 40e42 Glycine rich-linker sequence (GAGG), 113 Golgi-derived vesicles, Green fluorescent protein (GFP), 164, 166e168 H Hank’s balanced salt solution (HBSS), 39, 180, 182, 190e191 Hard-coated filters, 68e69 Hemocytometer, 35e36 HibernateTM, 66 Hippocampal dissection, 116e117 Hippocampal neurons, 220, 455, 458 Hippocampal tissue, 455e456 Hippocampi, 395 Human cytomegalovirus (CMV), 47 I Image data quantification, 246e247 ImageJ, 334 Image processing, 120e122, 245e246 running average subtraction, 122 Index standard average subtraction, 122 Imaging axonal transport, approaches to, 428e432 invertebrate techniques, 428e429 mammalian in vitro techniques, 430e432, 431t mammalian in vivo techniques, 429e430 Imaging chamber, 118 Immunocytochemistry (ICC), 156, 166e168, 169f blocking, 139 buffers and equipment, 137 direct/indirect ICC, 139 GA/extraction fixation, 138 MeOH/PFA fixation, 138 post ICC fixation and sample preservation, 139 quenching autofluorescence and permeabilization, 138 solutions, 137 Immunohistochemistry, 240e241 developing brain sections fixation method, 355 histological sections, preparation of, 355e356 immunostaining conditions, 356 screening, 356 Immunoprecipitation analyses, 6, 475e479 axoplasm, preparation of, 476 beads, antibodyeprotein mixture incubation with, 478 cDNA synthesis, 479 coimmunoprecipitation (co-IP), 477f materials, 476 protein extract, antibody with, 476e478 protein G beads, preparation of, 478 qPCR reaction, 479 RNA extraction, 478e479 washing and elution, 478 Internal ribosome entry site (IRES), 112fe113f, 113 In utero electroporation surgical procedure, 351e353, 353f timing considerations, 353e355, 354f IP2 software, 156 IRES, see Internal ribosome entry site (IRES) Iris diaphragm method, 75 K KHCN262S, 280 Kinesin light chain (KLC), 200e201 Kinesin translocation, 219f axons and dendrites, 220 cJun N-terminal kinase 3, 221e224 hippocampal neurons, 221, 222f KIF13A, 221 KIF21B, 221 materials and equipment, 229 microtubules, 224e225 neurite tips, 221 pattern and efficiency, 226e227 population assays, 218e219 serine 176, 221e224, 223f time-lapse imaging, 227e229 KLC, see Kinesin light chain (KLC) Kymograph analysis, 297f, 320e323, 321f, 324f analyzing dynamics with, 121f, 122e123 flux, 298 MetaMorph software, 295e296 run length, 296 segmental velocity, 296e298 stationary/paused vesicles, 298 transport dynamics and directionality, 295e296 KYMOMAKER, 274 Kynurenic acid, 255 L Laminin (LMN), 455e456 Laser scanning method, 76 Late endosomes/lysosomes-LAMP1, 418f density, 417 motility, 417 velocity, 416e417 Late endosomes/lysosomes-Rab7, 417f density, 416 motility, 416 velocity, 416 Leibovitz’s L-15 culture medium, 26e27 Lifeact, 93 Lipofecatamine 2000, 260 Lipofection, 52 Live-cell imaging, 117e119, 392 acquisition, 404e405 buffers, 117 camera, 70e71 cells and axonal regions, 71e72 choice of objective, 69e70 compartment-specific fluorescent dyes, 402e403 data analysis, 406 dynein, see Cytoplasmic dynein fluorescence photoactivation experiments, 77e78 fluorescence photobleaching experiments, 76e77 focus drift, 73e74 GFP-NFM, 65e66 imaging conditions, 404 imaging media, 66 imaging set-up, 403e404 immunostaining, 65e66 505 506 Index Live-cell imaging (Continued) materials, 271 microscope, 68e69, 69f mouse hippocampal culture, P0 pups, 394e395 near-simultaneous dual-channel imaging, 94 neuronal culture, 94 neuron transfection, 94e95 optical setup, 93, 94f organotypic slice cultures, lentivirus transduction efficiency in, 119, 120f perfusion system and imaging chamber, 118 photobleaching and image acquisition, 272e273 photobleaching and photodamage, 74 photobleaching assays, general considerations for, 271e272 rat hippocampal culture, E18 embryos, 392e394 spatially selective illumination, 74e76 spinning disk confocal microscopy, 118 temperature, CO2, and humidity, 66e68 air stream incubators, 68 imaging chambers, 67e68 objective heaters, 68 stage-top incubators, 66e67 time-lapse/streaming image acquisition, 72e73 visualizing proteins of interest electroporation, 398e399 exogenously expressing proteins, 395e396 fluorophore-coupled antibodies/ligands, 401e402 infection with lentivirus, 399e401 labeling endogenous proteins/compartments, 395e396 labeling transmembrane proteins, 401e402 lipofection, 396e398 nucleofection, 398e399 Loligo pealeii, 332e333, 332f Long-term myelinating DRG cocultures, 42e44 Lonza (Amaxa), 260e261 Lysis buffer (LB), 206 LysoSensors, 402e403 Lysosomes/late endosomes (LLEs), 459e460 LysoTrackers, 402e403 M Magnetofection, 52e53 Manganese (Mn2+)-enhanced magnetic resonance imaging (MEMRI), 429e430 MARCKS, see Myristoylated alanine-rich C-kinase substrate (MARCKS) Mass spectral data, 156 Mass spectrometry data analysis, 156 data validation of, 156 defined, 155e156 sample preparation for, 154e156 MatrigelTM, 28e29 Membrane-bound organelles (MBOs), 200e201, 428 MetaMorph, 258, 287, 295e296 Metamorph, 334 Methanol (MeOH) fixation, 131 MethocelTM, 27 Microinjection, deoxyribonucleic acid for, 314 Micromanipulator, 55 Microscopy fixed imaging, 358e359 live imaging, 359e361, 360f Microtubule (MT) cytoskeleton FIJI, data processing and analysis with, 119e123, 121f image processing, 120e122 kymographs, analyzing dynamics with, 121f, 122e123 live cell imaging, 117e119 buffers, 117 organotypic slice cultures, lentivirus transduction efficiency in, 119, 120f perfusion system and imaging chamber, 118 spinning disk confocal microscopy, 118 organotypic slice cultures, 114e117 buffers/solutions and equipment, 115e116 hippocampal dissection and slice preparation, 116e117 slice infection and organotypic cultures maintenance, 117 +TIP markers using lentivirus, 110e114 buffers and solutions, 111 pSIN-TRE-S vector, cloning GFP and MARCKS-TagRFP into, 111e113 virus production, 114 tissue culturing, 109 Microtubule-related proteins, 171e172 Midbody axis ratio, 246 Midbody index, 246 MilliQ water, 392 Minimal Essential Medium (MEM), 27e28 Mitochondria, 7e8 Mitochondria-MITO, 420f density, 419 motility, 420 velocity, 419 Mitotic index, 246 Index Monomeric red fluorescent protein-tagged dynein light intermediate chain transport, 318e320, 319f Motoneurons, axonal transport defects axonal jams, 292 data analyses, 295e298 dissection pin preparation NaOH solutin, 289 positive battery terminal, 288, 288f tungsten wire, 287e289 V battery, 287e288 distal bouton accumulation analysis, 298e299 larval dissections, 291f anatomical features, 290f anterior spiracles, 289e290 stereomicroscope, 289e290 live imaging, 292 materials and equipment, 284e287, 286f methods, 286f, 287 organelle/vesicle motility, in axons, 292 synaptic terminals, 283e284, 293e295, 294f UAS-Gal4 system, 284 MT, see Neuronal microtubule (MT) MT-associated proteins (MAPs), 128e129, see also Neuronal microtubule (MT) Multifield tracking, 81 Myristoylated alanine-rich C-kinase substrate (MARCKS), 111e113 N NbActiv4TM, 27e28 Neural epithelia, 234e235 Neurobasal/B27TM, 27e28 Neurofilament fusion proteins, 44 assembly competence, 48e49, 48f DsRed2, 45e46 fusion and expression construct, 46e47, 47f green fluorescence, 45e46 mCherry, 45e46 neurofilament subunit, 44e45 PAGFP, 46 Neurofilament protein L (NFL), 24 Neurofilament transport, 8e10, see also Live-cell imaging culturing neurons binocular dissecting microscope, 25 cortical neuron cultures, 27e28, 28t, 36e42 coverslips and culture dishes, 29e33 dorsal root ganglion neuron cultures, 28e29, 29t, 42e44 SCG cultures, 34e36 superior cervical ganglion neuron cultures, 26e27, 26t fixed-field tracking, 79e81 function, 24 kymograph analysis, 82e83 live-cell imaging camera, 70e71 cells and axonal regions, 71e72 choice of objective, 69e70 fluorescence photoactivation experiments, 77e78 fluorescence photobleaching experiments, 76e77 focus drift, 73e74 GFP-NFM, 65e66 imaging media, 66 immunostaining, 65e66 microscope, 68e69, 69f photobleaching and photodamage, 74 spatially selective illumination, 74e76 temperature, CO2, and humidity, 66e68 time-lapse/streaming image acquisition, 72e73 multifield tracking, 81 neurofilament fusion proteins, 44 assembly competence, 48e49, 48f DsRed2, 45e46 fusion and expression construct, 46e47, 47f green fluorescence, 45e46 mCherry, 45e46 neurofilament subunit, 44e45 PAGFP, 46 NFL, 24 observing movement, 57e58, 57f fluorescence photoactivation, 61e65 fluorescence photobleaching, 61 naturally occurring gaps, 58e61 pulse-escape fluorescence photoactivation analysis, 83e86 software, 78e79 stop and-go motility, 24 structural role, 24 transfecting neurons, 49, 50t electroporation, 49e52 lipofection, 52 magnetofection, 52e53 nuclear injection, 53e57, 54f plasmid purification, 49 Neuronal growth factor (NGF), 367 Neuronal intracellular transport, Drosophila Alzheimer’s disease, 278e279 class IV dendritic arborization neurons 507 508 Index Neuronal intracellular transport, Drosophila (Continued) materials and equipment, 299e300 methods, 300e305 UASetransgene expression, 299 Dynein/Dynactin, 280 KHCN262S, 280 motoneurons, axonal transport defects axonal jams, 292 data analyses, 295e298 dissection pin preparation, 287e289, 288f distal bouton accumulation analysis, 298e299 larval dissections, 289e290, 290fe291f live imaging, 292 materials and equipment, 284e287, 286f methods, 286f, 287 organelle/vesicle motility, in axons, 292 synaptic terminals, 283e284, 293e295, 294f UAS-Gal4 system, 284 RNAi, intracellular transport, 279 materials and equipment, 281e282 methods, 282e283 UAS-dicer2, 281 Spastin, 278e279 Unc-104, 280 Neuronal microtubule (MT) actin ring-like structures, 130 axons, 129 CAMSAP, 128e129 cytoskeletal components, 131 dendrites, 129 dSTORM, 131e133 dynamic instability, 128e129 EB3-GFP and CAMSAP2-GFP, 133 EM experiments, 130e131 end-binding (EB) proteins, 128e129 image acquisition, 139e140 buffer and equipment, 139e140 imaging buffer preparation, 140 mounting, 140 sample incubation with fiducial markers, 140 scanning fiducial markers for chromatic aberration correction, 141 solutions, 139e140 image analysis and reconstruction chromatic aberration correction, 143 drift correction, 142 fitting/reconstruction, 141 kinesin translocation, 224e225 methanol fixation, 131 paraformaldehyde, 131 plus-end preservation and antibody specificity, 133, 134fe135f sample preparation culturing and transfecting primary hippocampal neurons, 135 ICC, 137 SIM, 129e130 SMLM, 129e130, 132f STED, 129e130 Neuronal/nonneuronal cells data analysis, 461e463 microscopy/imaging, 459e461, 459t lysosomes/late endosomes, 460 virus transport, 461 microscopy, preparing cells for hippocampal and cortical neuron cultures, 455e457, 456f nonneuronal cell cultures, 454e455 motor protein activity, inhibition of drugs, 458e459 microinjection, 458 protein expression, dominant negative and knockdown, 457e458 Neurons advantage, 366e367 characteristics, 366 imaging neuronal culture, 94 transfection, 94e95 Neurotrophin-3 (NT-3), 28e29 Nuclear injection, 53e57, 54f Nucleofection, 166, 260e261 NucleofectorÒ, 172 O Organelle traffic, 334 Organelle/vesicle motility, in axons, 292 kymograph analyses, 295e298, 297f Organotypic slice cultures, 114e117 buffers/solutions and equipment, 115e116 hippocampal dissection and slice preparation, 116e117 slice infection and organotypic cultures maintenance, 117 P Papain solution, 180 Papain stop solution, 180 Index Paraformaldehyde (PFA), 131, 238 Particle tracking data, 462e463, 462f Pasteur pipette, 35 Perfusion system, 118 Phosphorylation axonal kinase activity, specific proteins effects on, 203e208 materials, 206e207 procedure, 207e208 axonal proteins, metabolic labeling of, 208e214 materials, 208e211 metabolic labeling and immunoprecipitation, 211e214 isolated squid axoplasm preparation, 202, 203fe205f Photoactivatable green fluorescent protein (PAGFP), 46 Plasmid-based approach, 186e188 Plasmid purification, 49 pLL system, see Posterior lateral line (pLL) system Plus-end tracking proteins (+TIP) markers, 110e114 buffers and solutions, 111 pSIN-TRE-S vector, cloning GFP and MARCKSTagRFP into, 111e113 virus production, 114 Polydimethylsiloxane (PDMS) casting, 367e368 and platform assembly chlorotrimethylsilane treatment, 374 microfluidic chamber preparation, 370f, 375 plate coating, 375e376 preparation and casting, 374 pressurized air, 374 Poly-L-lysine (PLL), 455e456 Polymerase chain reaction (PCR), 166e168, 167f Posterior lateral line (pLL) system, 313e314, 313f, 319f Primary cultured neurons, 165e166 Primary hippocampal neurons (E17-19), 255 Primary mixed cerebellar cultures cells for nucleofection, preparation of, 183e184 E18 embryos, harvesting cerebella from, 181e183, 182f IF staining, 184e185 PN-specific promoter, nucleofection with, 184 replating cultures, 185e186 Protein Regulator of Cytokinesis (PRC1), 241e243 Protocol, 172e174 replating, 174 transfection, 172e174, 173f Pulse-escape fluorescence photoactivation analysis, 83e86 Purkinje neurons (PN) cerebellum, 178 dilute-lethal mouse, 178e179 material and methods, 180e190 IF staining, 188e190 media components, 180e181 PN-specific miRNA knockdown constructs, 186e188, 187t primary mixed cerebellar cultures, see Primary mixed cerebellar cultures primary mixed cerebellar cultures, 190e192 antibiotics, 190e191 papain treatment, 191, 191t specifically knock down proteins, custom plasmids to, 192e194, 193f Q QdotÒ605, 379 R RBP, see RNA-binding protein (RBP) Region of interest (ROI), 271 Replating, 169e170, 174 Ribonucleic acid (RNA) affinity chromatography, 471e475, 472f axoplasm, 474 beads, conjugation to, 474 materials, 471e473 preparation, 473e474 washing and elution, 475 coimmunoprecipitation (co-IP), 480e481 axoplasm, preparation of, 480 beads, incubation of antibodyeprotein mixture with, 481 materials, 480 protein extract, incubation of antibody with, 480 protein G beads, preparation of, 480 washing and elution, 481 immunoprecipitation, 475e479 axoplasm, preparation of, 476 beads, antibodyeprotein mixture incubation with, 478 cDNA synthesis, 479 coimmunoprecipitation (co-IP), 477f materials, 476 509 510 Index Ribonucleic acid (RNA) (Continued) protein extract, antibody with, 476e478 protein G beads, preparation of, 478 qPCR reaction, 479 RNA extraction, 478e479 washing and elution, 478 RNA-binding protein (RBP), 468e469, 470f, 471, 473e476, 480e481 RNAi, intracellular transport, 279 materials and equipment, 281e282 methods setup crosses, 282, 283f tail-flip phenotype, screen larvae, 283, 284f UAS-dicer2, 281 Running average subtraction, 122 S Self-inactivating (SIN) vector, 110 SEQUEST algorithm, 156 Serine 176, 221e224, 223f Serum-free DMEM/F12-based medium, 26e27 Short-term nonmyelinating DRG cultures, 44 Signaling endosomeseTrkB, 421f density, 420 motility, 420e421 velocity, 420 SILAC mice, 154 Simulated Emission Depletion Microscopy (STED), 129e130 Single molecule localization microscopy (SMLM), 129e130, 132f Slow component b (SCb), 12e14 Small interfering RNA (siRNA) choosing sequences, 165 microtubule-related proteins, 171e172 off-target effects, 170e172 primary cultured neurons, 165e166 protocol, 172e174 replating, 174 transfection, 172e174, 173f replating, 169e170 time frame in cell, 166e169, 167fe169f SOD1, see Superoxide dismutase (SOD1) Sodium dodecyl sulfate (SDS), 154e155 Soft-coated filters, 68e69 Spastin, 278e279 Spinning disk confocal microscopy, 118 Splitview MetaMorph, 262 Squid axoplasm system, Squid giant axon, VEC-DIC, 332e333 Standard average subtraction, 122 Stream acquisition, 258e259 Structured illumination microscopy (SIM), 129e130 Superior cervical ganglion (SCG ) cultures, 34e36 Superoxide dismutase (SOD1), 201e205 Superresolution microscopy, 92 SW13vim-cells, 49 Sylgard dish, 286f, 287 Synaptic terminals, 293e295, 294f T Tail-flip phenotype, 283, 284f Timed-pregnant females, 180 Time-lapse optical imaging, Tissue culturing, 109 Transfecting neurons, 49, 50t electroporation, 49e52 lipofection, 52 magnetofection, 52e53 nuclear injection, 53e57, 54f plasmid purification, 49 Transported pulse-labeled proteins, 2e3 Trituration, 393 Tubulin, 5e6, 10e12, 158e160 U UAS-Gal4 system, 279 Unc-104, 280 Utr-CH, 93, 101f V Vertebrate neuronal cultures, 164 Video-enhanced contrastedifferential interference contrast (VEC-DIC), 333, 428 microscope and imaging hardware, 333e334 procedures, 335 W Well punching, 375 Western blot analysis, 156 X Xenopus, 234 Y Yokogawa spinning disk confocal scanning unit, 118 Z Zebrafish confocal imaging, mounting for, 316e318, 317f kymograph analysis, 320e323, 321f, 324f Index microinjection, deoxyribonucleic acid for, 314 monomeric red fluorescent protein-tagged dynein light intermediate chain transport, 318e320, 319f mRFP-Dync1li1 and lysosome associated membrane protein 1-GFP cotransport, 323e326, 325f zygotic microinjection, see Zygotic microinjection Z phosphate transfection, 255e258 Zygotic microinjection, 314e316 materials, 315e316 511 ... diverse proteins move in SCb which include proteins of the actin and membrane cytoskeletons, enzymes of intermediary metabolism, proteins involved in membrane trafficking, and proteins that interact... 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... Typically, radioactive amino acids are injected into the environment surrounding the neuron cell bodies under study The amino acids are taken into the neurons and incorporated into proteins, some of which