Methods in enzymology, volume 547

METHODS IN ENZYMOLOGY Editors-in-Chief JOHN N ABELSON and MELVIN I SIMON Division of Biology California Institute of Technology Pasadena, California ANNA MARIE PYLE Departments of Molecular, Cellular and Developmental Biology and Department of Chemistry Investigator Howard Hughes Medical Institute Yale University Founding Editors SIDNEY P COLOWICK and NATHAN O KAPLAN 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 32 Jamestown Road, London NW1 7BY, UK The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2014 Copyright © 2014 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 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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: 978-0-12-801415-8 ISSN: 0076-6879 For information on all Academic Press publications visit our website at store.elsevier.com CONTRIBUTORS Hossein Aleyasin Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, and Fishberg Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA Ishraq Alim Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, USA A Ambrus Department of Medical Biochemistry, Semmelweis University, and MTA-SE Laboratory for Neurobiochemistry, Budapest, Hungary Estela Area-Gomez Department of Neurology, Columbia University Medical Center, New York, USA Sandra R Bacman Department of Neurology, University of Miami School of Medicine, Miami, Florida, USA Stephen D Baird Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada Irene Bolea Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, USA David C Chan Division of Biology and Biological Engineering, and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA Guo Chen State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, China Linbo Chen State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, China Quan Chen State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, and State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Andy Cheuk-Him Ng Children’s Hospital of Eastern Ontario Research Institute, and Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada xiii xiv Contributors Megan M Cleland Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA Swathi Devireddy Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA Ajit S Divakaruni Department of Pharmacology, University of California, San Diego, California, USA Du Feng Guangdong Key laboratory of Age-related Cardiac-cerebral Vascular Disease, Institute of Neurology, Guangdong Medical College, Zhanjiang, Guangdong Province, China David A Ferrick Seahorse Bioscience, Billerica, Massachusetts, USA Wen-Biao Gan Department of Physiology and Neuroscience, Skirball Institute, New York University School of Medicine, New York, USA Elisabeth Garland-Kuntz Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA Shealinna X Ge Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research (STAR), University of Maryland School of Medicine, Baltimore, Maryland, USA Roberta A Gottlieb Department of Molecular Cardiobiology, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA Hengchang Guo Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, and Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA Renee E Haskew-Layton Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, and Department of Health and Natural Sciences, Mercy College, Dobbs Ferry, New York, USA Riikka H Haămaălaăinen Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland Peter J Hollenbeck Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA Gregory P Holmes-Hampton Molecular Medicine Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA Contributors xv Martin Jastroch Institute for Diabetes and Obesity, Helmholtz Zentrum Muănchen, German Research Center for Environmental Health, Neuherberg, Germany Mariusz Karbowski Center for Biomedical Engineering and Technology, and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA Adam L Knight Synaptic Function Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA Pin-Chao Liao Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA Mei-Yao Lin Synaptic Function Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA Lei Liu State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Jordi Magrane´ Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, USA Giovanni Manfredi Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, USA Thomas Misgeld German Center for Neurodegenerative Diseases (DZNE); Munich Center for Systems Neurology (SyNergy), and Institute of Neuronal Cell Biology, Technische Universitaăt Muănchen, Munich, Germany Carlos T Moraes Department of Neurology, University of Miami School of Medicine, Miami, Florida, USA Anne N Murphy Department of Pharmacology, University of California, San Diego, California, USA David G Nicholls Department of Clinical Sciences in Malm€ o, Unit of Molecular Metabolism, Lund University Diabetes Centre, CRC, Malm€ o, Sweden, and Buck Institute for Research on Aging, Novato, California, USA Dominik Paquet Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, and German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Alexander Paradyse Department of Pharmacology, University of California, San Diego, California, USA xvi Contributors Guy A Perkins National Center for Microscopy and Imaging Research, University of California, San Diego, California, USA Anh H Pham Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA Milena Pinto Department of Neurology, University of Miami School of Medicine, Miami, Florida, USA Gabriela Pluci nska Munich Center for Systems Neurology (SyNergy), Munich, Germany Brian M Polster Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research (STAR), and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA Rajiv R Ratan Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, USA Brian A Roelofs Center for Biomedical Engineering and Technology, and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA Tracey A Rouault Molecular Medicine Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA Robert A Screaton Children’s Hospital of Eastern Ontario Research Institute; Department of Biochemistry, Microbiology, and Immunology; Department of Cellular and Molecular Medicine, and Department of Pediatrics, University of Ottawa, Ottawa, Ontario, Canada Zu-Hang Sheng Synaptic Function Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA Orian S Shirihai Department of Medicine, Obesity and Nutrition Section, The Mitochondria Affinity Research Collaborative, Evans Biomedical Research Center, Boston University School of Medicine, Boston, Massachusetts, USA, and Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be’er-Sheva, Israel Tao Sun Synaptic Function Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA Contributors xvii Hyun Sung Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA Mathew Tantama Department of Chemistry, Purdue University, West Lafayette, Indiana, USA Wing-Hang Tong Molecular Medicine Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA L Tretter Department of Medical Biochemistry, Semmelweis University, and MTA-SE Laboratory for Neurobiochemistry, Budapest, Hungary Kyle M Trudeau Department of Medicine, Obesity and Nutrition Section, The Mitochondria Affinity Research Collaborative, Evans Biomedical Research Center, Boston University School of Medicine, Boston, Massachusetts, USA Hideo Tsukada Central Research Laboratory, Hamamatsu Photonics K.K., Hamakita, Shizuoka, Japan Sion L Williams Department of Neurology, University of Miami School of Medicine, Miami, Florida, USA Sheng Xie Department of Radiology, China-Japan Friendship Hospital, BeiJing, China Gary Yellen Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA Weiling Zhang State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Bing Zhou Synaptic Function Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA Yushan Zhu State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, China PREFACE Although mitochondrial dysfunction is evident in intractable diseases such as Alzheimer’s, Parkinson’s, and mitochondrial diseases, there is a lack of therapeutic approaches that target the underlying pathogenic mechanisms To identify rational therapeutic approaches, it is critical to clearly understand many aspects of mitochondrial biology in the brain, including normal and aberrant movement, turnover, metabolism, and the feasibility of genetic modification Current interest in mitochondrial dynamics, bioenergetics, and metabolism in the brain has been greatly facilitated by rapidly evolving techniques allowing assessment of mitochondria within the context of intact cells and tissues In no other cell type besides a neuron is the importance of mitochondrial trafficking and dynamic changes in morphology so readily apparent given the distances mitochondria must traverse between the cell body and a synapse From a cell biology perspective, neurons must overcome unique challenges so that mitochondria can reach the nerve terminals and meet the energy demands of these electrically excitable cells Thus, this volume begins with a collection of excellent chapters addressing current techniques for the measurement of mitochondrial turnover, fusion and fission, and trafficking Mitochondrial degradation through the autophagic pathway, a process termed mitophagy, is thought to be a quality control mechanism that is critical for maintaining a healthy population of mitochondria within cells Chapters 1–3 detail methods to identify molecules involved in mitophagy and to measure the turnover of mitochondria Mitochondrial fusion is a central aspect of mitochondrial dynamics, and Chapter describes a method to directly quantify mitochondrial fusion in living, cultured cells Mitochondrial transport is critical to the function of neurons, and multiple animal models have been developed to study this process Chapters 5–9 detail methods to study mitochondrial transport in the neurons of mouse, fly, and zebrafish The second half of this volume addresses methods to assess mitochondrial function from diverse perspectives As proteomic technologies have advanced, so has the need to identify the precise localization of newly discovered mitochondrial proteins, and a technique to so is addressed in Chapter 10 The juxtaposition of mitochondrial outer membranes with specialized areas of the endoplasmic reticulum is a topic of interest to those studying signaling events and lipid homeostasis, and thus methods for the xix xx Preface study of mitochondria-associated membranes are addressed in Chapter 11 The importance of mitochondrial reactive oxygen species production and iron metabolism to normal cell function and disease pathogenesis continues to be a topic of interest, yet remains challenging to assess, which is a topic of Chapters 12–15 The advent of plate-based technologies for the measurement of cellular bioenergetic function has revolutionized the rate of acquisition and scope of data detailing how cells meet their energy requirements Expert guidance for interpretation of plate-based respirometry and extracellular acidification data is provided in Chapter 16 Chapter 17 details an exciting fluorescence imaging approach to providing real-time measurement of the ATP/ATP ratio The discovery of treatments for mitochondrial diseases requires improved methods for modeling the defects in patients’ cells which is addressed in Chapter 19, and Chapter 18 describes an exciting approach for engineering a mitochondrial targeted endonuclease to rectify defects in mitochondrial DNA Both chapters hold promise for the design of future therapeutic intervention for these devastating diseases The final two chapters (Chapters 20 and 21) address assessment of mitochondrial function in vivo using PET and MRI approaches, representing technologies that may become not only important diagnostic tools but also the means to monitor the efficacy of potential therapeutic responses We hope these chapters will provide a resource and practical guidelines for those interested in further characterization of normal and dysregulated mitochondrial function and lead to new insights into the pathogenic mechanisms operating in neurodegenerative disorders ANNE N MURPHY DAVID C CHAN CHAPTER ONE High-Content Functional Genomic Screening to Identify Novel Regulators of the PINK1–Parkin Pathway Andy Cheuk-Him Ng*,†, Stephen D Baird*, Robert A Screaton*,†,{,},1 *Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada † Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada { Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada } Department of Pediatrics, University of Ottawa, Ottawa, Ontario, Canada Corresponding author: e-mail address: rob@arc.cheo.ca Contents Introduction and Theory General Screen Design Strategy 2.1 Biological systems 2.2 Screen libraries 2.3 Choice of assays 2.4 Follow-up validation RNAi Screen for Genes Involved Required for PARK2 Translocation 3.1 Assay design 3.2 Execution of genome-wide screen 3.3 Follow-up validation Equipment Materials 5.1 Solutions and buffers Genome Screen Protocol 6.1 Duration 6.2 Sample preparation 6.3 Microscope and algorithm settings 6.4 Data analysis Acknowledgments References 3 6 7 8 10 10 11 13 15 18 18 Abstract PINK1/PARK6 and Parkin/PARK2 are amongst the most commonly mutated genes associated with recessive forms of familial Parkinson's disease Recent evidence indicates Methods in Enzymology, Volume 547 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-801415-8.00001-1 # 2014 Elsevier Inc All rights reserved 472 Skimina, T A., 265 Skjoerringe, T., 286–287 Skladal, D., 402–403 Sklar, L A., 213–214 Skujat, D., Skulachev, V P., 201t, 210–211 Sliter, D A., Smirnova, E., 59–60 Smirnova, N A., 254, 255, 256–258, 257f, 258f, 259–261 Smith, A M., 339 Smith, C L., 61–62, 66–67, 70–71 Smith, G F., 278t Smith, P M., 379 Smith, S R., 284f, 287–288 Soejima, A., 389 Soernaes, R., 283–284 Sokoloski, E A., 227 Soldner, F., 401–402 Solimena, M., 30–31 Solomon, E I., 294t Solomon, G G., 252–253 Sommer, C A., 401 Sone, M., 403–405, 404t Song, P., 40–42, 45–47, 49–51 Song, Q., 48–49 Song, S., Song, Y., 78, 266 Sonnay, S., Sorber, C W., 287–288 Sosinsky, G E., 166, 178 Soubannier, V., 50–51 Sowa, M E., Spangenburg, E E., 323f Sparman, M., 404–405, 406 Spartalian, K., 298t Spiro, T G., 290 Sprecher, S G., 133 Squirrell, J M., 265 Srikun, D., 265 Srinivasan, V J., 265 Srivastava, S., 374–376, 379–381, 389 St Pierre, J., 201t, 213–214 Stadtman, E R., 253 Staerk, J., 401 Stafa, K., Stampfl, A P., 289 Stanika, R I., 244 Author Index Stanley, I A., 310–311 Starkov, A A., 201t, 208, 210–211, 213, 215, 226, 233 Staroverov, D B., 49, 99–100, 266 Stegman, L D., 254–255 Steipe, B., 49 Stemmler, T L., 282–283 Stevens, D A., 40 Stevens, R D., 310–311 Steward, O., 122 Stewart, B., 279–280 Stewart, D P., 70–71 Stewart, J B., 404–405 Stewart, M G., 82–83, 152 Stiles, K M., 2–3 Stiles, L., 320 Stoica, R., 193–194 Stokes, N., 2–3 Stolz, A., 40 Stolz, D B., 40–42, 50–51 Stone, D., 266–267 Stone, J M., 423–424 Stookey, L L., 278–279, 278t Stoppani, A O M., 429 Stoppini, L., 112, 113–114 Storm, T A., 382 Stormo, G D., 2–3 Stotland, A., 23, 24–27, 28–29, 30–31, 47 Stout, C D., 276 St-Pierre, F., 356–357 Stracka, D., 185–187 Streit, W J., 289 Strickler, J H., 265 Strobel, G., 332 Struyvenberg, P A., 402–403 Studer, D., 287 Stuurman, N., 159 Subramanian, M., 310–311 Suen, D F., 2, 22–23, 40, 50–51 Suga, A., 406 Sugita, S., 406 Sugiura, Y., 61 Sujkowski, A., 23, 27, 30–31, 47 Sukumar, M., 310–311 Sullivan, G J., 406 Sultan, F., 120–121 Sumalekshmy, S., 282–283, 289 Sumi, Y., 286 Author Index Sun, C W., 252–253, 266 Sun, J., 294t Sun, T., 78, 79, 90–94, 91f, 93f Sunada, Y., 402 Sundaresan, M., 252–253 Sundquist, W I., Sung, H., 132–148 Sung, Y H., 384–385 Suomalainen, A., 59–60, 66–67, 400, 402–406, 404t Supuran, C T., 340 Surerus, K K., 298t Suryo Rahmanto, Y., 283–284 Suski, J M., 187 Sutachan, J J., 87 Sutphin, P D., 339 Suzuki, N., 133, 434–435 Suzuki, T., 286 Svistunenko, D A., 293–295 Swaminathan, R., 65–66 Sweet, I R., 315 Sweitach, P., 340 Swistowski, A., 314, 345 Sydow, A., 153 Szabadkai, G., 90 Szabo, I., 175, 206–207 Szczerbowska-Boruchowska, M., 289 Szlyk, B., 310–311, 362 Szweda, L I., 217–218 Szymczak, A L., 385–387 T Tabata, T., 401 Tachibana, M., 404–405, 406 Taivassalo, T., 283–284 Takacs, K., 210–211, 210f Takahashi, K., 400–401, 408 Takahashi, S., 440, 441 Takai, D., 389 Takamatsu, H., 423–424 Takamatsu, T., 290 Takanaga, H., 369 Takemitsu, M., 379, 389 Takenaka, S., 43–45 Talbot, D A., 213–214 Tamarit, J., 276, 277–279, 278t Tambini, M D., 182, 188–189, 193–195 Tamura, Y., 473 Tan, P., 23, 28, 30–31 Tanabe, K., 400–401, 408 Tanaka, A., 2, 22–23, 40, 50–51 Tanaka, K., 40, 61, 420 Tanaka, M., 120–121, 379–381, 392 Tang, T., 315 Tang, Y., 389 Tannahill, G M., 310–311 Tanner, K G., 226 Tanner, S D., 281 Tannock, I., 338–339, 340 Tantama, M., 356, 369 Taroni, F., 118–119 Tasaki, I., 132 Tatsuta, T., 22–23 Tauer, U., 119 Tavernarakis, N., 22 Tavitian, B., 421–422 Taylor, C., 201t Taylor, G M., 2–3 Taylor, R W., 374 Tea, J S., 48–49 Teardo, E., 175 Teclemariam-Mesbah, R., 286 Telser, J., 293–295, 294t Templin, A T., 338 Tennant, D A., 310–311 Tennant, M E., 78, 98–99 Teperino, R., 310–311, 330–331, 339 Tepikin, A V., 181–182 Teplitz, R L., 389 Terauchi, Y., 252–253 Terman, A., 22–23 Terman, J R., 252–253 Terrenoire, C., 402, 406 Terry, J., 289 Terskikh, A V., 23, 28, 30–31, 266 Thatcher, J W., 59 Thevenaz, P., 125, 161 Thiagarajan, P., 50–51 Thiel, K., 191 Thomas, J P., 203t Thompson, C B., 418 Thompson, J W., 252 Thompson, S M., 112 Thorburn, D R., 282–283, 402–403 Thornell, L E., 283–284 Thornton, C., 23, 24–27, 28–29, 30–31, 47 474 Thorson, L., 43–45 Thul, P J., 191 Tian, J H., 77–78, 77f, 83–84, 90–92 Tian, L., 356–357 Tian, W., 45–47 Tieu, Q., 59–60 Tifft, C., 403 Tilley, M., 245 Tiranti, V., 389 Tiwari, P., 98–99 Togawa, H., 357 Tohgi, H., 440, 441 Tollefson, K E., 233 Tol€ o, J., 356–357 Toloe, J., 357 Tomita, A., 120–121 Tomoda, K., 400–401, 408 Tondera, D., 70–71 Tong, W H., 283–284 Toni, N., 70–71 Toompuu, M., 389 Torricelli, J R., 87 Toth, R., 48–49, 51 Townes, T M., 252–253 Toyooka, M., 434–435 Tran, D D., 98–99 Trautwein, A X., 298t Treberg, J R., 201t, 315 Tremblay, S., 310–311 Tretter, L., 202t, 208–209, 209f, 210–211, 210f, 213, 216, 217–218 Treutelaar, M K., 338 Treuting, P., 237 Trifunovic, A., 404–405 Trigo, D., 152, 157 Trounce, I A., 385, 389 Trubert, C L., 287–288 Trudeau, K., 23, 25f, 26f, 28, 29–31, 32f, 35–36, 47 Trudeau, K M., 22–27, 28–36 Trumpower, B L., 292 Trushina, E., 98–99 Tsai, G E., 260 Tsang, C P., 298t Tsch€ op, M H., 310–311, 339, 340, 345, 346–347 Author Index Tsujikawa, T., 434–435 Tsukada, H., 418–420, 421–422, 423–424, 426, 428–429 Tsukahara, S., 434–435 Tsukamoto, T., 327–330 Tsukita, K., 402 Tudor, E L., 78, 98–99 Tulyathan, O., 266 Tung, K W., 402, 406 Turcotte, S., 339 Turnbull, D M., 374 Turner, B A., 139, 141–142, 148 Turner, I M., 294t, 298t Turrens, J F., 201t, 203t Twelvetrees, A E., 35–36, 78, 90 Twig, G., 22, 23, 25f, 26f, 27, 28, 29–31, 32f, 35–36, 47 Twining, B S., 289 Tyagi, N K., 78 Tyurina, Y Y., 49, 50–51 U Uematsu, H., 434–435 Uematsu, T., 422 Ullrich, C., 113–114 Umemura, K., 422, 423–424 Unser, M., 125, 161 Urano, Y., 187 Ure, D., 98–99 Ursini, F., 203t Ushio-Fukai, M., 203t V Vaandrager, A B., 188 Va´ghy, P L., 336 Vaidya, K S., 339 Valdebenito, R., 369 Vale, R., 159 Valente, E M., Valera, V A., 339 Van de Bittner, G C., 266 van den Ouweland, J M., 402–403 van der Bliek, A M., 50–51, 59–60 van der Krogt, G N., 30–31 van der Laan, M., 22 van der Meulen, M C., 288 van der Windt, G J., 310–311 van Giesen, L., 133 475 Author Index van Gramberg, A., 282 Van Hattum, J., 287–288 Van Laar, V S., van Manen, H J., 290 Van Remmen, H., 203t van Slageren, J., 294t Vance, D E., 183–184, 187 Vance, J E., 183, 184, 185–187 Vander Heiden, M G., 418 Vanin, E F., 385–387 Vankatachalam, V., 356–357 Vargas, M E., 153 Varnai, P., 182, 193–194 Vasquez-Vivar, J., 201t, 227, 228 Vaughan-Jones, R D., 340 Vaz, W L., 183 Velasquez-Castano, J C., 266 Venditti, P., 201t, 217 Venediktova, N., 379–381 Venken, K J., 121–122 Verburg, J., 147 Vercesi, A E., 200, 227–228 Verkhusha, V V., 99–100 Verkman, A S., 65–66 Verschoor, M J., 281–282 Verstreken, P., 121–122 Vidensky, S., 402 Vignali, K M., 385–387 Villarino, A V., 310–311, 330–331 Vincent, A I., 384 Vinogradov, S., 278–279, 278t Vitelli, C., 241–242, 247, 314, 316, 345 Vives-Bauza, C., Vo, T D., 384, 385–387 Voelker, D R., 187, 188 Voeltz, G K., 181–182, 194–195 Vogel, R., 203t Vogelstein, B., 392 Vogt, S., 282, 288, 289 Volk, B., 119 Volkow, N D., 423–424 von Zglinicki, T., 22–23, 31 Vongtragool, S., 294t Voronina, S G., 181–182 Voth, M., 58 Votteler, J., Votyakova, T V., 209, 213 Vries-Smits, A M., 252–253 Vuong, N., 43–45 W Wabitsch, M., 310–311, 339, 340, 345, 346–347 Wabnig, S., 356–357 Wada, H G., 337, 340 Wada, K., 422 Wagner, D., 289 Wagner, J A., 59 Wai, T., 59, 70–71 Wakabayashi, J., 118–119 Wakabayashi, N., 118–119 Walberer, M., 421–422 Walke, W F., 440 Walker, J E., 331 Walkey, C J., 183–184 Wall, E A., 322, 323–324 Wallace, D C., 315–316, 385, 389 Walsh, A., 279–280 Walter, L., 182, 193–194 Walther, T C., 191 Wang, A., 229f, 231f, 233, 234, 235–237, 242, 243, 247 Wang, C., 50–51 Wang, D., 22–23, 31 Wang, G., 244 Wang, H., 384–385 Wang, J., 384, 385–387 Wang, K., 402, 406 Wang, L., 385 Wang, R., 310–311 Wang, T., 344–345 Wang, V Y., 119 Wang, W Q., 3–4 Wang, X., 2, 35–36, 90, 98–99, 132–133, 139, 148, 376–377 Wang, Y., 49, 70–71, 152, 385–387 Wang, Z X., 438, 441, 442f Warburg, O., 421–422 Ward, C W., 323f Ward, M W., 316, 318f Ward, T H., 60–61 Warren, L., 401 Waseem, T., 356 Wasiak, S., 98 Wasti, A T., 310–311 476 Watanabe, A., 408 Watanabe, M., 423–424 Waterhouse, R N., 420–421 Watt, I N., 331 Watts, C A., 244 Waymire, K G., 400 Weaver, D., 90, 182, 193–194 Weaver, J L., 317 Webb, W W., 265 Wegener, E., Wehler, M., 244 Wehrli, F W., 435 Weijzen, S., 252–253 Weinberg, F., 326 Weinberg, S., 326 Weisiger, R A., 226 Weiss, P., 132 Weisskoff, R M., 435 Wellington, A., 132–134, 138, 141–142 Wenz, T., 379 Werth, M T., 294t West, M., 194–195 Westenskow, P D., 406 Wexler, E., 418–420 Weyer, A., 119–120 Whatley, S A., 201t Wheaton, W W., 326 White, C W., 217–218, 243 White, E., 310–311 White, J G., 265 White, K., 139 White, K L M., 276 White, R F., 260–261 Whitnall, M., 283–284 Wibom, R., 283–284 Wieckowski, M R., 181–182, 183, 201t Wiederkehr, A., 29–30 Wiedermann, D., 434–435 Wiegand, U K., 30–31 Wiesinger, H., 203t Wikstrom, J D., 22, 27, 320 Wiley, S E., 318f, 322, 323–324, 326 Wilks, A., 294t Will, G., 276 Williams, M D., 203t Williams, R M., 265 Williams, S L., 379, 381–382, 383f, 385–387, 389, 390, 391–393, 391f Author Index Williamson, C D., 183 Williamson, R., 282–283 Willis, J B., 279–280 Wilson, G L., 379–381 Wilson, M T., 293–295 Windmiller, J., 405–406 Wingen, M., 168np Winger, J A., 276 Winklhofer, K F., 40, 70–71 Winter, D., 2, 98–99 Winterbourn, C C., 226 Winters, C A., 244 Wipf, P., 134–135, 147–148 Wisden, W., 119–120 Wiseman, H., 276 Wiwczar, J., 310–311, 362 Wojtczak, L., 201t Wojtovich, A P., 177 Wokosin, D L., 265 Wolf, A P., 423–424 Wong, E D., 59 Wong, Y L., 2, 98–99 Wood, A., 112 Woodward, J., 404–405, 406 Workman, C J., 385–387 Wortel, J., 286 Wredenberg, A., 404–405 Wu, B., 282–283 Wu, C F., 133 Wu, H., 40–42, 45–47, 49–50, 51 Wu, J., 44 Wu, J C., 400 Wu, L., 266 Wu, S., 237–238 Wu, W., 45–47 Wu, X S., 92–94 Wyrick, S., 260 X Xia, D F., 384, 385–387 Xia, J Q., 298t Xiao, J X., 435–437, 438, 441, 442f Xiao, L., 384–385 Xie, H., 339 Xie, S., 435–437, 438, 441, 442f Xie, Z., 98–99 Xiong, C., 356 Xu, H., 375 Author Index Xu, J., 92–94 Xu, L., 344 Xu, P., 23, 27, 30–31, 47 Y Yablonskiy, D A., 435–437 Yadava, N., 201t, 310, 320–321 Yaffe, M P., 59 Yakovlev, A G., 244 Yaku, H., 290 Yalamanchili, P., 418–420 Yamagishi, Y., 420 Yamakawa, D., 288 Yamakawa, T., 408 Yamamoto, A., 185–187, 366–367 Yamamura, Y., Yamanaka, S., 400–401 Yamano, K., 2, 3, 50–51 Yamaoka, Y., 290 Yamasoba, T., 389 Yan, Z., 27 Yang, C Y., 44 Yang, J., 177, 310–311, 401 Yang, K S., 253 Yang, L C., 289 Yang, P., 260 Yang, W., 92–94 Yang, Y., 356–357 Yano, M., 120–121 Yanushevich, Y G., 49 Yarbrough, D K., 359 Yaseen, M A., 265 Yavin, E., 276 Yazaki, Y., 252–253 Ye, B., 139, 148 Ye, C P., 315 Yellen, G., 356, 357–358, 366–367, 369 Yi, C X., 310–311, 339, 340, 345, 346–347 Yi, J., 369 Yi, M., 90 Yilmaz, O H., 344–345 Yin, X M., 40–42 Yin, Y., 252–253 Ylikallio, E., 402–403 Yokoyama, H., 288 Yoneda, M., 379–381, 392, 434–435 Yonetani, T., 298t Yonezawa, H., 440, 441 477 Yoo, S Y., 434–435 Yoshida, S., 120–121 Yoshihara, K., 244 Yoshihiro, K., 227–228 Yoshii, S R., 42–43, 48–49 Yoshikawa, E., 423–424 Yoshimori, T., 40–42, 366–367 Yoshimoto, T., 435 Yoshioka, N., 401 Youle, R J., 2, 22–23, 40, 50–51, 61–62, 66–67, 70–71 Young, A B., 112–113 Yu, D., 289 Yu, H., 356, 357 Yu, H B., 43–45 Yu, H H., 252–253 Yu, L., 70–71, 438 Yu, M., 418–420 Yu, W., Yu, Z., 310–311, 369 Yu, Z X., 252–253 Yue, Y., 382 Yumoto, H., 288, 289–290 Z Zach, J., 30–31 Zak, B., 278–279, 278t Zakaria, H M., 76–77, 83–84 Zakikhani, M., 339 Zala, D., 356, 357 Zald, P., 77–78, 77f, 83–84, 90–92 Zaman, K., 252–253 Zaraisky, A., 23, 28, 30–31 Zelickson, B R., 311, 319 Zeviani, M., 389 Zhang, C Y., 315 Zhang, H., 227 Zhang, J., 44, 379–381, 384–385, 392, 438, 441, 442f Zhang, J H., 5–6 Zhang, P., 284f, 287–288 Zhang, P W., 402 Zhang, X., 266, 384–385 Zhang, X D., 435–437, 438 Zhang, Y., 294t, 385 Zhang, Z., 118–119 Zhao, B S., 266 Zhao, C., 260–261, 338 Zhao, D., 438, 441, 442f 478 Zhao, F., 44 Zhao, H., 227 Zhao, Y., 339, 369 Zheng, C., 266 Zheng, H., 254–255 Zheng, J Q., 98–99 Zheng, L., 310–311, 330–331, 339 Zheng, Q., 40–42, 45–47, 49–51 Zhou, C., Zhou, H -M., 369 Zhou, K., 177 Zhou, T., 401 Zhou, Y., 121–122 Zhu, J., 40 Zhu, Y., 40 Author Index Zhu, Y B., 78 Zick, Y., 226 Zielonka, J., 226, 227, 228, 230–231, 243 Zik, E., 23, 25f, 26f, 28, 29–31, 32f, 35–36, 47 Zilva, J F., 337 Zimmer, J., 113–114 Zimmer, M., 213–214 Zinsmaier, K E., 132–134, 138, 141–142 Zipfel, W R., 265 Zitomer, R S., 276 Zoccarato, F., 203t, 215–216 Zoghbi, H Y., 119 Zoratti, M., 206–207 Zvyagin, S A., 294t SUBJECT INDEX Note: Page numbers followed by “f ” indicate figures and “t ” indicate tables A Adenosine triphosphate (ATP) ATP-to-ADP ratio brain energy metabolism, 356–357 PercevalHR biosensor (see PercevalHR biosensor) ratiometric image analysis, 364–365 extracellular acidification rate (ECAR), 345–347 Amplex Red/UltraRed method advantages, 206 disadvantages, 206 endogenous substrates, 206–207 mitochondrial membrane potential, 208–209 mitochondrial protein concentration, 206 principles, 205 respiratory substrates, 207 transmembrane pH difference, 209 Atomic absorption spectroscopy, 279–280 Autophagy, 24–25, 30–33 Axonal mitochondrial transport adult DRG neuron cultures materials, 84–85 procedures, 86–87 solutions, 85–86 adult MN cultures materials, 87 procedures, 88–89 solutions, 87–88 anterograde, 98 Charcot-Marie-Tooth, 98–99 embryonic neuron cultures preparation materials, 79 procedures, 80–81 solutions, 80 fusion and fission balance, 98 image acquisition, 82–83 image analysis, 83–84 KIF5 and dynein motors, 77–78 kinesins, 98 labeling and image, 89–90 microtubules (MTs), 77–78 MitoDendra anesthesia, 101 femoral nerve exposure, 102 image acquisition, 103–104, 106–108 image analysis, 104–105, 108 neuromuscular junctions (NMJ), 102–103 peripheral nerves exposure, 101–102, 102f PhAM mouse lines, 101 photoconversion, 105–106 suturing, 103 Thy1.2 promoter, 99–100 TM57 mitoDendra mouse line, 99–100, 100f MitoFish general considerations, 157–159 imaging protocol, 159–160 motility patterns, 78 primary hippocampal neurons transfection materials, 81 procedures, 81–82 regulation of, 76–77 synapses motile mitochondria, presynaptic activity, 92–94 motility patterns, presynaptic boutons, 90–92 B Biochemical methods flame AAS, 279–280 graphite furnace AAS, 279–280 ICP-MS, 281–282 ICP-OES, 280–281 in situ analysis advantage, 282 electron microscopy (see Electron microscopy) 479 480 Biochemical methods (Continued ) energy dispersive X-ray spectroscopy, 282 LA-ICP-MS, 282 multidimensional imaging techniques, 282 particle-induced X-ray emission, 282 secondary ion mass spectrometry, 282 SXRF, 282 total ion measurement acid digestion, 277–278 advantages and disadvantages, 279 colorimetric compounds, 277–278 compound structures, 278–279, 279f ligands, 277 membrane permeable chelators, 277 molar absorptivity values and wavelengths, 277–278, 278t Biophysical methods EPR application, 293 expensive technique, 293–295 features, 293–295 G values, 293, 294t oxidation and spin states, 293, 294t sensitive technique, 293–295 M€ ossbauer spectroscopy biological iron species, 297, 298t cost, 297 disadvantages, 297 experimental design, 296 generic H.S ferrous species, 297, 299f time-consuming technique, 297 UV-vis spectroscopy, 291–293 X-ray absorption spectroscopy, 295–296 B-score method (Bscore), 17 C CellHTS2 data files, 16 dataPath, 16–17 normalized percent inhibition (NPI), 17 parameter files, 15–16 Cellomics VTI software, 6–7 Charcot-Marie-Tooth, 98–99 Confocal microscope, 62–63 Correlated light and electron microscopy (CLEM) labeling, 166 See also Mini Singlet Oxygen Generator Subject Index D Dihydroethidium, 227 Doxycycline (DOX)-inducible promoter pulse-chase expression, 24–25, 25f reagents, 23, 24t Drosophila larval preparation dissection methods D42-GAL4UAS-mitoGFP, 134–135 dying-back neuropathy, 137–138 HL6 buffer composition, 134 larval preparation materials, 134 materials, 134 MitoGFP expression, 138f mounting, 135–137 rationale, 133–134 imaging anatomical features, 139 LSCM, 139–140 observation lifetime, 141 photobleaching, 140–141, 140f quantifying axonal transport duty cycle, 143–144 flux, 142 kymographs and single particle tracking, 141–142 mitochondrial density, 144 mitochondrial length, 144–145 mitochondrial membrane potential, 146 mitochondria percentage, 143 oscillatory mitochondria, 144 photobleaching, 145 reactive oxygen species, 147–148 run length, 144 TMRM imaging and analysis, 147 TMRM stock and solutions, 146 velocity, 143 E Electron energy loss spectroscopy (EELS), 287–288 Electron microscopic nonheme iron histochemistry cell accumulation iron, 284f, 285 historical, theoretical, and technical aspects, 285–286 “masked” and “not masked” Fe, 285 perfusion-Perls’ and perfusion-Turnbull staining, 286–287 481 Subject Index Perls’ acid ferrocyanide reaction, 285–286 Perls’ and Turnbull’s methods, 285–286 Perls’ and Turnbull’s stains, 285 Perls’ DAB-enhanced method, 286 Perls’ Prussian blue method, 285–286 Tirmann-Schmeltzer method, 286 Turnbull blue method, 286 Electron microscopy EELS, 287–288 electron microscopic nonheme iron histochemistry cell accumulation iron, 284f, 285 historical, theoretical, and technical aspects, 285–286 “masked” and “not masked” Fe, 285 perfusion-Perls’ and perfusionTurnbull staining, 286–287 Perls’ acid ferrocyanide reaction, 285–286 Perls’ and Turnbull’s methods, 285–286 Perls’ DAB-enhanced method, 286 Perls’ Prussian blue method, 285–286 Tirmann-Schmeltzer method, 286 Turnbull blue method, 286 Friedreich’s ataxia, 283–285 iron deposits, 283–284, 284f ISCU myopathy, 283–285 Perls’ diaminobenzidine (DAB) enhancement method, 283–284, 284f Perls’ method, 283–284, 284f Perls’ Prussian blue reaction, 283–284 sideroblastic anemia, 283–285 Electron paramagnetic resonance (EPR) application, 293 expensive technique, 293–295 features, 293–295 G values, 293, 294t oxidation and spin states, 293, 294t sensitive technique, 293–295 Electron spin resonance See electron paramagnetic resonance (EPR) Embryonic stem (ES) cells, 400–401 Extracellular acidification rate (ECAR) ATP demand, 345–347 glucose oxidation, 331 glycolytic turnover ATP hydrolysis, 335–338 canonical Embden–Meyerhof–Parnas pathway, 332 contributing factors, 344–345 CO2 production, 341–342 extracellular pH, 338–340 glycolysis-derived protons, 332–335 LDH, 332 qualitative assessment, CO2, 342–344 lactate anions, 331 pyruvate anions, 331 F 18 F-BCPP-EF See Mitochondrial complex I (MC-I) imaging Flavin mononucleotide (FMN) cofactor, 166–167 Fluorescent-activated cell sorting (FACs), 390 Friedreich’s ataxia (FRDA), 283–284 G Glucose oxidase (GOX), 217 H Hank’s Balanced Salt Solution (HBSS), 362 Hydrogen peroxide (H2O2) cell membrane production, 253 chemoselective fluorescent probes, 266–267 endogenous oxidative stress, 253–254 imaging studies microscope setup, 267–268 TPF imaging, 268–269 physiological vs pathological role, 253–254 R gracilis DAAO astrocyte cell cultures, 255 culture plates, 256 cytoplasm and mitochondria, 264 DAAO transduction, 256–257 D-Alanine and FAD concentrations, 258 expression location, 263–264 glutamate/HCA model, 261–262 heterologous cell-type culture, 259–260 H2O2 production and measurement, 259 HT22 cultures, 256 482 Subject Index total ion measurement acid digestion, 277–278 advantages and disadvantages, 279 colorimetric compounds, 277–278 compound structures, 278–279, 279f ligands, 277 membrane permeable chelators, 277 molar absorptivity values and wavelengths, 277–278, 278t Hydrogen peroxide (H2O2) (Continued ) targeting sequences, 262–264 viral construct generation, 256 two-photon microscopy, 265–266 tyrosine kinases, 253 I ImageJ software, 142 Induced pluripotent stem cells (iPSCs) embryonic stem cells, 400–401 mitochondrial disease disease models, 406 iPSCs generation, 408–411 mtDNA mutation, 403–406 neurodegenerative disorders, 402 Yamanaka-cocktail, 401 Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) See Inductively coupled plasmaoptical emission spectroscopy (ICP-OES) Inductively coupled plasma-mass spectrometry (ICP-MS), 281–282 Inductively coupled plasma-optical emission spectroscopy (ICP-OES), 280–281 Iron metabolism biophysical methods EPR, 293–295 M€ ossbauer spectroscopy, 296–298 UV-vis spectroscopy, 291–293 X-ray absorption spectroscopy, 295–296 flame AAS, 279–280 graphite furnace AAS, 279–280 ICP-MS, 281–282 ICP-OES, 280–281 in situ analysis advantage, 282 confocal raman microscopy, 290 electron microscopy (see Electron microscopy) energy dispersive X-ray spectroscopy, 282 LA-ICP-MS, 282 multidimensional imaging techniques, 282 particle-induced X-ray emission, 282 secondary ion mass spectrometry, 282 SXRF, 282, 288–290 K KiNEDx robot arm, L Laser scanning confocal microscopy (LSCM), 139–140 M Mini Singlet Oxygen Generator (miniSOG) diaminobenzidine (DAB) polymerization, 166–167, 167f FMN cofactor, 166–167 mitochondrial calcium uniporter (MCU), 175 photooxidation protocol chimera construction and transfection notes, 169–174 for tissues, 174–175 properties, 167–168, 168t resolution, 168–169 Mitochondria-associated ER membranes (MAM) assays ACAT activity, 191–192 electron micrographs, 193–194 light microscopy, 194–195 protein concentration determination, 191 PtdSer synthesis, 187 characterization, 181–182 isolation crude mitochondria (CM), 184 differential centrifugation, 184 homogenization, 184 lipidomics analysis, 185 PEMT, 183–184 Percoll dilution, 185 liquid-ordered domain, 182 representation, 183f Subject Index Mitochondrial complex I (MC-I) imaging aging effects in living brain, 426, 427–429 ROS, 425–426 design and assessment, 419–421 stroke/ischemic damage, 422, 423–424 Mitochondrial DNA evaluation, 392–393 fluorescent-activated cell sorting, 390 “lastcycle hot” PCR, 391 mito-TALENs, cultured cells, 390–392 mtDNA heteroplasmy, 377–379 cybrid cells, 388–389 with mitochondrial-targeted nucleases, 380f mutant alleles, 379 TAL effector nucleases, 384–387 zinc-finger nucleases, 384 OXPHOS biogenesis, 374 quantitative southern blot analysis, 392 real-time quantitative PCR, 392 restriction endonucleases (REs) Drosophila melanogaster, 375 heteroplasmy shift, 379–382 PstI restriction, 375 TRE-mito-PstI mouse model, 377 TRE promoter, 376 Mitochondrial matrix-targeted photoactivatable green fluorescent protein (mito-PAGFP) Aequorea victoria, 60 Dnm1p, 59–60 Fzo1p, 59 Mgm1p, 59 mitochondria morphology, 58 OMM and IMM, 58–59 size estimation and network organization, 66–67 threonine 203 mutation, 60–61 time-lapse microscopy DNA concentration, 68–69 FuGENE6, 68–69 image resolution and cytotoxicity balance, 70 mito-YFP, 68–69 OMM-targeting C-terminal 21 amino acid, 69–70 483 transfected cells vs untransfected cells, 68–69 visualization and quantification, mitochondrial fusion chamber slides, 62 confocal microscope, 62–63 DsRED2 replacement, 63 laser power, 65–66 number of iterations, 65–66 polyethylene glycol (PEG) assay, 61–62 RNAi screen, 61 “yellow” mitochondria formation, 61–62 Zeiss LSM710 imaging system, 66 Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) MRI finding BOLD effects, 435 conventional MRI, 434 diffusion-weighted imaging, 434 magnetic inhomogeneities, 435 metabolic and oxidative defects, 434–435 theoretic signal-intensity model, 435–437 OEF imaging cerebral OEF, 439–442 positron emission tomography, 438 quantitative measurement, 437–438 ROIs, 438 Mitochondrial turnover See MitoTimer MitoDendra anesthesia, 101 femoral nerve exposure, 102 image acquisition, 103–104, 106–108 image analysis, 104–105, 108 neuromuscular junctions (NMJ), 102–103 peripheral nerves exposure, 101–102, 102f PhAM mouse lines, 101 photoconversion, 105–106 suturing, 103 Thy1.2 promoter, 99–100 TM57 mitoDendra mouse line, 99–100, 100f MitoFish axonal transport general considerations, 157–159 484 MitoFish (Continued ) imaging protocol, 159–160 mounting protocol materials, 155–157 method, 155–157 processing and quantification materials required, 161 method, 161 properties of, 153–155 Mitophagy, 22–23, 27, 30–31 fluorescence microscopy GFP-LC3 plasmid, 45–47, 45f ULK1 interaction, 45–47 inducer and inhibitors CCCP/FCCP, 50–51 ceramide, 51 CoQ supplementation, 50–51 FUNDC1/BNIP3L/NIX pathway, 49–50 ROS, 49 mitochondrial-targeting probes MitoTimer, 47 quantitative analysis, 48–49 mitochondria mass, 47 receptor and nonreceptor-mediated, 40 transmission electron microscopy, 40–42 western blot analysis BA1 treatment, 44 cell lysis, 44 FCCP induction, 42–43, 43f HRP-conjugated secondary antibodies, 44 immunoreactive bands visualization, 44 LC3 protein, 42–43 p62 degradation, 43–45 SDS–PAGE, 44 MitoSOX cortical neurons, 239f dye-free assay, 237–238 imaging studies antimycin A plus oligomycin, 241–242 exogenous factor, 242 Fluo4-FF, 247 mitochondrial fission/fusion, 242 rat cortical neurons, 244 micromolar concentrations, 239f mitochondrial localization, 245–247 MnTE-2-PyP, 235–237 Subject Index ROS detection antimycin, 233 cerebellar granule neurons, 228–230, 229f colocalization, 230 hydroethidine, 230–231 mitochondrial depolarization, 233–234 mitochondrial DNA-binding, 228–230 nonlinear fluorescence, 230–231 nucleic acid association, 228–230 SOD mimetic compounds, 235–237 MitoTimer autophagy inhibition, 24–25 expression length, 25–27 flow cytometry, 28–29 goal of, 22–23 high-resolution image analysis, 27 lifetime, 25–27, 26f mitochondrial dynamics, 22 mitochondrial-targeting probes, 47 mitophagy, physiological rates, 22–23 488 nm laser excitation, 28 overexpression, 27 readout interpretion autophagy/mitophagy, 30–31 cellular level, 31–34, 34t green-to-red fluorescence transition kinetics, 30–31 green vs red appearance, 31, 32f increased reactive oxygen species (ROS), 29–30, 30f red/green ratio, 29–30 subcellular level, 34–36 steady-state expression, 25–27, 26f M€ ossbauer spectroscopy biological iron species, 297, 298t cost, 297 disadvantages, 297 experimental design, 296 generic H.S ferrous species, 297, 299f time-consuming technique, 297 O Organotypic slice cultures basal ganglia air-liquid interface, 112 culture media, 112 dissection and slicing, 116–118 485 Subject Index dissection buffer, 112 equipment, 112 excess dopamine, 112–113 instruments and reagents preparation, 114–115 preparation for dissection, 115–116 roller tube method, 112 sagittal slices, 113–114 surgical instruments, 112 cerebellum cytoarchitecture, 119 molecular markers, 119–120 preparation of, 120–121 Purkinje cells, 118–119 mitochondrial dynamics monitoring equipment, 122–123 kymographs generation, 124–127 live imaging, 123–124 media for imaging, 122–123 Oxygen consumption rate “ baseline ” feature, in Seahorse software absolute rates, 324 CLU201 hypothalamic astrocytes, 325–326 complex III inhibitor myxothiazol, 325–326 cellular respiration ATP-linked respiration, 314–315 basal respiration, 312–314 maximal respiration, 316–318 nonmitochondrial respiration, 320 proton leak-linked respiration, 315–316 reserve/spare respiratory capacity, 319–320 data analysis, 320–321 data presentation, 321 normalization methods cell number, 322–323 cellular protein, 322 mitochondrial content, 323–324 permeabilized cells cholesterol-dependent cytolysin, 326–327 complex I dysfunction, 327 genetic modification, 327 plasma membrane permeabilization, 326–327 substrate ranges, 330 TCA cycle flux, 327–330 P Pancreatic β-cells, 338 PARK2 translocation cellomics VTI software, 6–7 follow-up validation, 7–8 genome-wide screen execution, GFP Au4 -tagged Parkin, HeLa, 6–7 PercevalHR biosensor biosensor expression, 366–367 cultured cells, 361–363 fluorescent biosensor constructs, 361 live-cell imaging, 363–364 and MgATP-bound biosensor, 359 pH artifacts, 365–366 pH calibration, 367–369 pHRed, 360–361 reporting domain, 358 sensing domain, 357–358 spectral ratio, 359 Perls’ and Turnbull’s methods, 285–286 Perls’ diaminobenzidine (DAB) enhancement method, 283–284, 284f, 286 Perls’ method, 283–284, 284f Perls’ Prussian blue method, 283–284, 285–286 Phosphatidylethanolamine N-methyltransferase (PEMT), 183–184 Phosphatidylinositol 3,4,5-trisphosphate (PIP3), 253 Photobleaching, 140–141, 140f Photooxidation protocol chimera construction and transfection notes electron microscope sample preparation steps, 172 electron microscopy, 173 mersalyl acid, 170–171 photoconversion block, 170 photooxidation steps, 171 for tissues, 174–175 PINK1–Parkin pathway genome screen protocol cellHTS2, 15–18 duration, 10, 11t 486 PINK1–Parkin pathway (Continued ) microscope and algorithm settings, 13–14, 14f sample preparation, 11–12 high-throughput RNAi screening assay designing, 5, 5t equipment, 8, 9f follow-up validation, immortalized cell line vs primary cell line, 3, 4t PARK2 translocation, 6–8 screen libraries, 3–4, 4t solutions and buffers, 9–10, 10f Z’ factor, 5–6 Ser65 and ubiquitin, siRNA and shRNA, 2–3 Positron emission tomography (PET) See Mitochondrial complex I (MC-I) imaging PtdSer synthesis cell labeling, 188–189 lipid analysis, 190 PtdEtn conversion, 187 R Reactive oxygen species (ROS) homeostasis Amplex Red/UltraRed method (see Amplex Red/UltraRed method) enzymes and systems, role of, 200, 201t oxidative stress aconitase activity, 217–219 definition, 200 NADPH formation, 218–219 PHPA and homovanilic acid method, 213–214 residual H2O2 concentration Amplex Red, 216 electrode, 216 scopoletin, 215–216 spectrophotometric methods Amplex photometry, 214 cytochrome (cyt) c reduction, 214 spin-trapping methods, 215 Restriction endonucleases (REs) Drosophila melanogaster, 375 Subject Index heteroplasmy shift BALB/NZB mouse model, 381 jugular injection, 381–382 mouse-rat cybrid cell line, 379–381 recombinant rAAV9-mito-ApaLI-HA, 382, 383f TALENs, 382–383 zinc-finger nucleases, 382–383 PstI restriction, 375 TRE-mito-PstI mouse model, 377 S Seahorse XF technology, 338–340 Spectrophotometric methods Amplex photometry, 214 cytochrome (cyt) c reduction, 214 Spin-trapping methods, 215 Stroke, 422, 423–424 T Tirmann-Schmeltzer method, 286 Transcription activator-like effector nucleases (TALENs), 382–383 β-galactosidase enzyme activity, 387 Xanthomonas bacteria, 386f Transmission electron microscopy autophagosomal maturation, 40–42 lysosomes fusion, 40–42 mitochondrial structures identification, 40–42 tissue samples fixation, 40–42 in vitro cultured cells, 40–42 Turnbull blue method, 286 W Western blot analysis BA1 treatment, 44 cell lysis, 44 FCCP induction, 42–43, 43f HRP-conjugated secondary antibodies, 44 immunoreactive bands visualization, 44 LC3 protein, 42–43 p62 degradation, 43–45 SDS–PAGE, 44 ... 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... transferring into 37  C, 5% of CO2 incubator Incubate for 72 h Add 10 μL of 25 μM CCCP (final: μM) directly into the well to trigger GFP-Parkin translocation Return plates into TC incubator and incubate... the PINK1 and Parkin pathway We describe a method below using high-throughput RNA interference technology to interrogate the genome for novel components of the PINK1 and Parkin pathway INTRODUCTION