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Methods in Molecular Biology 1474 Steven D. Schwartzbach Omar Skalli Thomas Schikorski Editors High-Resolution Imaging of Cellular Proteins Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire , AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 High-Resolution Imaging of Cellular Proteins Methods and Protocols Edited by Steven D. Schwartzbach Department of Biological Sciences, The University of Memphis, Memphis, TN, USA Omar Skalli Department of Biological Sciences, The University of Memphis, Memphis, TN, USA Thomas Schikorski Department of Anatomy, Universidad Central del Caribe, Bayamon, PR, USA Editors Steven D . S chwartzbach Omar S kalli Department of Biological Sciences Department of Biological Sciences The University of Memphis The University of Memphis Memphis, TN , USA Memphis, T N , USA Thomas Schikorski Department of Anatomy Universidad Central del Caribe Bayamon, PR, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6350-8 ISBN 978-1-4939-6352-2 (eBook) DOI 10.1007/978-1-4939-6352-2 Library of Congress Control Number: 2016946471 © Springer Science+Business Media New York 2 016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC New York Prefa ce Cell biology is the study of the structure and function of cells, cellular organelles, and subcellular structures. Originally, cellular functions were studied without a detailed knowledge of the structures involved and structures were described without an understanding of their function. Imaging techniques that simultaneously studied structure and function or at least correlated function with structure eventually closed this disparity. Several technologies have been greatly responsible for this progress by providing high-resolution images rich in functional information. Over the past decades, these technologies and their combinations have provided possibilities previously unthinkable. This book contains a collection of timely techniques and methods that have been instrumental in the evolution of microscopy from a purely descriptive technique to one of four-dimensional imaging in living organisms. The biochemist and molecular biologist determines the functions of the molecules and macromolecular complexes found within cellular structures. They isolate individual cellular constituents and reconstruct vital cellular processes. These in vitro experiments provide a valuable understanding of cellular function. But, biochemistry lacks the potential to place this knowledge into the cellular context of cell types and cellular compartments. Electron microscopy (EM) was the fi rst technique to bridge the gap between biochemistry, molecular biology, and cellular context by localizing macromolecules to cellular structures. EM collected valuable information about proteins contained within a structure and their spatial relationships with other proteins and structures. The combination of immunolabeling with EM made it even possible to localize specifi c proteins of known function to subcellular structures. Immunoelectron microscopy can be used to study virtually every unicellular and multicellular organism. The only requirements are suitable protocols and the availability of an antibody to the molecule whose structural location is to be deter- mined. Although introduced decades ago, this technology is far from obsolete because of its nanometer precision in localizing proteins in cells and tissues. More recently, labeling methods for scanning EM and for serial sections and electron tomography were developed and used to visualize specifi c biomolecules within the three-dimensional structure of organelles and subcellular compartments. Computer-assisted image acquisition and analysis greatly contributed to this development. Advances in light microscopy soon made it a competitive alternative to EM for studies correlating structure and function. The introduction of video cameras marked a break- through adding a new dimension, time, to microscopic observations of structure in a living cell. The development of fl uorescent dyes that could be conjugated to antibodies and dyes localized to specifi c subcellular compartments further advanced live cell imaging. To fully utilize the potential of these probes confocal and two-photon microscopes were designed. These microscopes overcame the limitations of standard fl uorescent microscopes by increas- ing the localization accuracy in tissue and the resolution in the z -axis. The next innovation that boosted functional live cell imaging was the discovery of the green fl uorescent protein (GFP). The ability to use DNA cloning methods to create constructs encoding a protein of interest fused to GFP opened the door to using live cells for studying the function of specifi c proteins. The development of a diverse color pallet of fl uorescent proteins v vi Preface and of methods to make fl uorescence dependent upon the interaction of two proteins as well as photocontrollable fl uorescent tags and the constant advances in designing ever faster and more sensitive cameras have greatly expanded the structure function information that can be obtained from live cell imaging. It did not take long before correlative methods were developed in which the distribu- tion of specifi c proteins was examined fi rst by confocal microscopy and then by EM. An example from our own work demonstrates how confocal and immunoelectron microscopy provide unexpected insights into structure-function relationships. Thus, immunoelectron microscopy fi rst demonstrated that the E uglena light harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) is present in the Golgi apparatus prior to its presence in the chloroplast. This fi nding was the impetus for detailed biochemical studies that eluci- dated a new mechanism for chloroplast protein import, namely transport from the ER to the Golgi apparatus to the chloroplast. This volume takes into account the increasingly multidisciplinary nature of microscopy by presenting three toolboxes. The molecular toolbox focuses on the development of molecular tools for microscopy. It will present methods for the expression of epitope-tagged proteins in animal cells. Methods for the production of antipeptide and polyclonal antibodies and how to conjugate colloidal gold to these proteins will also be presented. A molecular toolbox would be incomplete without the discussion of genetic tools that exploit viral vectors to optimize the transfer of genes into living cells. This technology is also addressed in the following toolboxes together with light and electron microscopic imaging. From the fl uorescent microscopy toolbox, this section presents methods that span diverse applications based on the use of conventional fl uorophores and expressed fl uorescent proteins such as GFP in plants, parasites, and animal cells. Fluorescence microscopy also enables monitoring protein-protein interactions in real time and bimolecular complementation methods enabling this feat will be presented. A pH-sensitive GFP variant is used to monitor exocytosis and endocytosis of synaptic vesicles in real time. How the traffi cking of proteins or organelles can be monitored by Fluorescence Recovery after Photobleaching and Fluorescence Redistribution after Photoactivation is presented. Finally one chapter presents the labeling of brain cells and the imaging of these cells in the living brain. From the EM toolbox, this section details methods for cryo-ultramicrotomy and rapid freeze-replacement fi xation which have the advantage of retaining protein antigenicity but at the expense of ultrastructural integrity as well as chemical fi xation methods that maintain structural integrity while sacrifi cing protein antigenicity. The toolbox also includes a proto- col for immunogold labeling of freeze-fracture replicas. This technique is known for its high sensitivity and its capability of localizing proteins to nanoscale protein assemblies like ion channels. Plants and algae contain cell walls, vacuoles, and other structures which present barriers to antibody penetration and complicate fi xation. Due to these problems, sepa- rate chapters will discuss fi xation and immunolabeling protocols for animals, plants, and yeast. Pre- and post-embedding immunogold labeling protocols will be presented. Pre- embedding methods perform immunogold labeling before ultrathin sections are prepared from resin-embedded samples resulting in greater sensitivity and better microstructure preservation. Post-embedding methods perform immunolabeling after ultrathin sections are prepared from resin-embedded samples resulting in decreased antigenicity. The detailed methods and notes will facilitate choosing the best method for the antibody and biological material to be studied. Extending these approaches, methods will be presented for immunogold labeling of two antigens for protein colocalization studies, for glycan localization, for nanogold enhancement allowing immunogold labeling using smaller gold particles Preface vii which more easily enter cells, and for immunogold scanning EM. For many years, the advances in genetics and functional imaging were not used to advance EM. Recently how- ever, these advances have been used to develop powerful EM techniques. Reporter genes suitable for EM and fi xation techniques that capture structure at a defi ned time point have been developed. Furthermore, these techniques are suitable for correlative light-electron microscopy. The volume presents two examples of these advances; the use of genetically engineered horseradish peroxidase as a genetically encoded label for electron microscopy and superfast fi xation for monitoring cellular processes second by second. It is our hope that the toolboxes created by this volume will be used by cell biologists interested in understanding structure-function relationships at the fundamental level as well as by cancer biologists, toxicologists, and microbiologists interested in understanding dis- ease mechanisms as a foundation to developing new therapies. Memphis, TN, USA Steven D. Schwartzbach Memphis, TN, USA Omar Skalli Bayamon, PR, USA Thomas Schikorski Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i PART I MOLECULAR TOOLBOX 1 Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture. . . . . . 3 Jay M. B hatt , M elanie L . S tyers , and Elizabeth Sztul 2 Antibody Production with Synthetic Peptides . . . . . . . . . . . . . . . . . . . . . . . . . 2 5 Bao-Shiang Lee , J in-Sheng Huang , Lasanthi P. Jayathilaka , Jenny L ee , and S halini G upta 3 P roduction and Purification of Polyclonal Antibodies . . . . . . . . . . . . . . . . . . . 4 9 Masami N akazawa , Mari M ukumoto , and K azutaka Miyatake 4 P reparation of Colloidal Gold Particles and Conjugation to Protein A/G/L, IgG, F(ab′)2, and Streptavidin . . . . . . . . . . . . . . . . . . . . . 6 1 Sadaki Y okota 5 H elper-Dependent Adenoviral Vectors and Their Use for Neuroscience Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 Mónica S. Montesinos , R achel Satterfield , and Samuel M. Young Jr. PART II FLUORESCENT MICROSCOPY TOOLBOX 6 Localizing Proteins in Fixed G iardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy . . . . . . . . . . . . . . . . . 93 Lilian Nyindodo-Ogari , Steven D . S chwartzbach , O mar Skalli , and Carlos E. E straño 7 U sing Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 13 Yong Cui , C aiji G ao , Qiong Z hao , and Liwen Jiang 8 U sing FRAP or FRAPA to Visualize the Movement of Fluorescently Labeled Proteins or Cellular Organelles in Live Cultured Neurons Transformed with Adeno-A ssociated Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . 1 25 Yaara T evet and Daniel G itler 9 B imolecular Fluorescence Complementation (BiFc) Analysis of Protein–Protein Interactions and Assessment of Subcellular Localization in Live Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 53 Evan P . S . P ratt , J ake L. Owens , Gregory H . Hockerman , and Chang-Deng Hu 10 Viral Injection and Cranial Window Implantation for In Vivo Two-Photon Imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Gordon B . S mith and David F itzpatrick ix