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Robbins and Cotran. Where an effective understanding of disease begins. A current and accurate understanding of pathophysiology is integral to effective practice in every specialty. No source delivers that understanding better than the Robbins and Cotran family! Robbins Basic Pathology, Robbins Essential Pathology, and their many companion resources provide a clear, up-to-date explanation of disease mechanisms you need to succeed. Robbins Basic Pathology, 10th Edition ISBN: 9780323353175 Key Features: • Excellent art program boasts high-quality photomicrographs, gross photos, and radiologic images to supplement the world-class illustrations. • Bulleted summary boxes provide quick access to key information and easy review of key concepts. • Highlights pathogenesis, morphology, and pathophysiologic content throughout. Robbins Essential Pathology, 1st Edition ISBN: 9780323640251 Key Features: • The most concise Robbins text available, providing high-quality content and a case-based approach in a focused, multimedia learning package for coursework and exam preparation. • Focuses on the core knowledge of disease mechanisms and essential clinical aspects that medical students need to know. • Features more than 500 images and tables that illustrate key disorders and concepts. Explore the entire Robbins and Cotran Family at elsevierhealth.com/pathology ROBBINS&COTRAN PATHOLOGIC BASIS OF DISEASE RO B B I N S & C OT R A N PATHOLOGIC BASIS OF DISEASE TENTH EDITION Vinay Kumar, MBBS, MD, FRCPath Alice Hogge and Arthur A. Baer Distinguished Service Professor of Pathology Biologic Sciences Division and the Pritzker Medical School The University of Chicago Chicago, Illinois Abul K. Abbas, MBBS Emeritus Professor and Chair Department of Pathology University of California San Francisco San Francisco, California Jon C. Aster, MD, PhD Michael Gimbrone Professor of Pathology Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts Associate Editor Jerrold R. Turner, MD, PhD Profe; ssor of Pathology and Medicine Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts W I T H I L L U S T R AT I O N S B Y : James A. Perkins, MS, MFA Distinguished Professor of Medical Illustration Rochester Institute of Technology Rochester, New York Elsevier 1600 John F. Kennedy Blvd. Ste 1800 Philadelphia, PA 19103-2899 ROBBINS & COTRAN PATHOLOGIC BASIS OF DISEASE, TENTH EDITION INTERNATIONAL EDITION Copyright © 2021 by Elsevier, Inc. All rights reserved. ISBN: 978-0-323-53113-9 ISBN: 978-0-323-60992-0 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). Notice Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or contributors 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. Previous editions copyrighted 2015, 2010, 2004, 1999, 1994, 1989, 1984, 1979, 1974. International Standard Book Number: 978-0-323-53113-9 Publisher: Jeremy Bowes Content Development Director: Rebecca Gruliow Publishing Services Manager: Catherine Jackson Health Content Management Specialist: Kristine Feeherty Design Direction: Brian Salisbury Printed in Canada Last digit is the print number: 9 8 7 6 5 4 3 2 1 DEDICATION To Our teachers For inspiring us To Our students For constantly challenging us To our spouses Raminder Kumar Ann Abbas Erin Malone For their unconditional support Contributors Mahul B. Amin, MD Karen M. Frank, MD, PhD, D(ABMM) Douglas C. Anthony, MD, PhD Ryan M. Gill, MD, PhD Chairman Department of Pathology College of Medicine University of Tennessee Health Science Center Memphis, Tennessee The Lower Urinary Tract and Male Genital System Professor Pathology, Laboratory Medicine, and Neurology Warren Alpert Medical School of Brown University; Chief of Pathology Lifespan Academic Medical Center Providence, Rhode Island Peripheral Nerves and Skeletal Muscles Anthony Chang, MD Professor Department of Pathology The University of Chicago Chicago, Illinois The Kidney Nicole A. Cipriani, MD Professor of Pathology University of California San Francisco School of Medicine San Francisco, California Liver and Gallbladder Marc K. Halushka, MD, PhD Deputy Director of Education Department of Pathology Professor of Pathology The Johns Hopkins Hospital Baltimore, Maryland Blood Vessels Andrew Horvai, MD, PhD Associate Professor of Pathology The University of Chicago Chicago, Illinois Head and Neck Professor Department of Pathology University of California San Francisco San Francisco, California Bones, Joints, and Soft Tissue Tumors Andrew J. Connolly, MD, PhD Aliya N. Husain, MBBS Professor of Pathology University of California San Francisco San Francisco, California The Heart Lora Hedrick Ellenson, MD Professor Department of Pathology The University of Chicago Chicago, Illinois Diseases of Infancy and Childhood; The Lung Attending Physician and Director of Gynecologic Pathology Department of Pathology Memorial Sloan Kettering Cancer Center New York, New York The Female Genital Tract Sanjay Kakar, MD Robert Folberg, MD Selene C. Koo, MD, PhD Departments of Ophthalmology and Pathology Beaumont Health - Royal Oak Royal Oak, Michigan The Eye vi Chief Department of Laboratory Medicine Clinical Center National Institutes of Health Bethesda, Maryland Infectious Diseases Professor of Pathology University of California San Francisco School of Medicine San Francisco, California Liver and Gallbladder Assistant Professor Department of Pathology The Ohio State University; Pathologist Department of Pathology and Laboratory Medicine Nationwide Children’s Hospital Columbus, Ohio Diseases of Infancy and Childhood Contributors Zoltan G. Laszik, MD, PhD Professor of Pathology University of California San Francisco San Francisco, California The Kidney Alexander J. Lazar, MD, PhD Professor Departments of Pathology, Genomic Medicine, Dermatology, and Translational Molecular Pathology The University of Texas MD Anderson Cancer Center Houston, Texas The Skin Susan C. Lester, MD, PhD Assistant Professor Breast Pathology Services Department of Pathology Harvard Medical School Brigham and Women’s Hospital Boston, Massachusetts The Breast Mark W. Lingen, DDS, PhD, FRCPath Professor of Pathology The University of Chicago Chicago, Illinois Head and Neck Anirban Maitra, MBBS Professor Pathology and Translational Molecular Pathology The University of Texas MD Anderson Cancer Center Houston, Texas The Pancreas; The Endocrine System Marta Margeta, MD, PhD Associate Professor of Pathology University of California San Francisco School of Medicine San Francisco, California The Central Nervous System Alexander J. McAdam, MD, PhD Associate Professor of Pathology Department of Pathology Harvard Medical School; Medical Director Clinical Microbiology Laboratory Boston Children’s Hospital Boston, Massachusetts Infectious Diseases Richard N. Mitchell, MD, PhD Lawrence J. Henderson Professor of Pathology Member of the Harvard/MIT Health Sciences and Technology Faculty Department of Pathology Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts The Cell as a Unit of Health and Disease; Blood Vessels; The Heart George Jabboure Netto, MD Chair, Department of Pathology University of Alabama at Birmingham School of Medicine Birmingham, Alabama The Lower Urinary Tract and Male Genital System Scott A. Oakes, MD Professor and Vice Chair of Research and Academic Affairs Department of Pathology University of Chicago Pritzker School of Medicine Chicago, Illinois Cell Injury, Cell Death, and Adaptations Arie Perry, MD Professor of Pathology and Neurological Surgery Director of Neuropathology University of California San Francisco San Francisco, California The Central Nervous System Edyta C. Pirog, MD Associate Professor Department of Pathology and Laboratory Medicine Weill Cornell Medicine-New York Presbyterian Hospital New York, New York The Female Genital Tract Peter Pytel, MD Professor Department of Pathology University of Chicago Chicago, Illinois Peripheral Nerves and Skeletal Muscles vii Preface As we launch the tenth edition of Pathologic Basis of Disease we look to the future of pathology as a discipline and how this textbook can remain most useful to readers in the twenty-first century. It is obvious that an understanding of disease mechanisms is based more than ever on a strong foundation of basic science. We have always woven the relevant basic cell and molecular biology into the sections on pathophysiology in various chapters. In the previous edition we went one step further and introduced a new chapter at the very beginning of the book titled “The Cell as a Unit of Health and Disease.” We are delighted that the chapter was found useful by many students and faculty. Because progress in basic cell biology is moving at a very brisk pace, the chapter has been updated significantly. In the preface of the very first edition (1957), Stanley Robbins wrote: “The pathologist is interested not only in the recognition of structural alterations, but also in their significance, i.e., the effects of these changes on cellular and tissue function and ultimately the effect of these changes on the patient. It is not a discipline isolated from the living patient, but rather a basic approach to a better understanding of disease and therefore a foundation of sound clinical medicine.” We hope we continue to illustrate the principles of pathology that Dr. Robbins enunciated with such elegance and clarity over half a century ago. This edition, like all previous ones, has been extensively revised, and some areas have been completely rewritten. A few examples of significant changes are as follows: • Chapter 2 has been updated to include novel pathways of cell death beyond the long-established pathways of necrosis and apoptosis. Indeed, the distinction between these two is being blurred. Autophagy, which has begun to take center stage in diseases ranging from aging to cancer and neurodegeneration, has been revised, as have the possible molecular mechanisms of aging. • In Chapter 3, we have married morphology with molecular mechanisms. Thus, different patterns of inflammation can be related to distinct molecular pathways. • Chapter 5 includes a discussion of gene editing technology and a revised and updated section on molecular diagnosis. • Chapter 7 has been extensively revised to incorporate new insights into cancer pathogenesis, the interplay between cancer cells and host immunity, and cancer precision diagnostics. • Chapter 10, covering pediatric diseases, includes discussion of newly approved therapies targeting specific mutated forms of the CFTR transporter. • Chapter 11, covering vascular diseases, includes discussion of clonal hematopoiesis, a newly emerging risk factor for atherosclerosis and other inflammatory disorders. viii • Chapters 18 and 28, covering diseases of the liver and the central nervous system, have a fresh look brought in by new contributors. • In addition to the revision and reorganization of the text, many new photographs and schematics have been added and a large number of the older “gems” have been enhanced by digital technology. We made the changes highlighted above while remaining focused on the same longstanding overarching goals, which serve as our guiding principles: • To integrate into the discussion of pathologic processes and disorders the newest established information available—morphologic as well as molecular. • To organize information into logical and uniform presentations, facilitating readability, comprehension, and learning. • To maintain the book at a reasonable size and yet provide adequate discussion of significant lesions, processes, and disorders. Indeed, despite the addition of new information, we are happy to state that the overall length of the book is unchanged. One of our most challenging tasks is to decide what to eliminate to make room for key new findings. • To place great emphasis on clarity of writing and proper use of language in the recognition that struggling to comprehend is time-consuming and wearisome and gets in the way of the learning process. • To make this text useful to students throughout all of their years in medical school and into their residencies—but, at the same time, to provide sufficient detail and depth to meet the needs of more advanced readers. We have repeatedly been told by readers that up-todatedness is a special feature that makes this book very valuable. We have strived to remain current by providing new information from the recent literature, up to the current year, and by adding coverage of the COVID-19 epidemic. We are now into the digital age, and so the text will be available online to those who own the print version. Such access gives the reader the ability to search across the entire text, bookmark passages, add personal notes, use PubMed to view references, and exploit many other exciting features. In addition, also available online are case studies developed by one of us (VK), and revised by Dr. Alex Gallan from the University of Chicago. The cases are designed to enhance and reinforce learning by challenging students to apply their knowledge to solve clinical cases. To assist in the classroom, we have also made the images available for instructors on the Evolve website. Instructors may register Preface at https://evolve.elsevier.com/ to gain access to the images for teaching purposes. All three of us have reviewed, critiqued, and edited each chapter to ensure the uniformity of style and flow that have been the hallmarks of the book. Together, we hope that we have succeeded in equipping the readers with the scientific basis for the practice of medicine and in whetting their appetite for learning beyond what can be offered in any textbook. Vinay Kumar Abul K. Abbas Jon C. Aster ix Acknowledgments First and foremost, all four of us offer thanks to our contributing authors for their commitment to this textbook. Many are veterans of previous editions; others are new to the tenth edition. All are acknowledged in the table of contents. Their names lend authority to this book, for which we are grateful. As in previous editions, the four of us have chosen not to add our own names to the chapters we have been responsible for writing, in part or whole. We welcome to this edition Dr. Jerry Turner in the capacity of Associate Editor. Jerry is a veteran of Robbins texts having written the chapter on diseases of the gastrointestinal tract in previous editions. His editing has strengthened several chapters. Many colleagues have enhanced the text by reading various chapters and providing helpful critiques in their area of expertise. They include Dr. Celeste Thomas, University of Chicago; Dr. Meenakshi Jolly, Rush University, Chicago; Dr. Richard Aster, Blood Research Institute, Milwaukee; and Dr. Suneil Koliwad, UCSF. Many colleagues provided photographic gems from their collections. They are individually acknowledged in the text. All of the graphic art in this book was created by Mr. James Perkins, Distinguished Professor of Medical Illustration at Rochester Institute of Technology. His ability to convert complex ideas into simple and aesthetically pleasing sketches has considerably enhanced this book. x Many individuals associated with our publisher, Elsevier, need our special thanks. Outstanding among them is Kristine Feeherty, Health Content Management Specialist, and our partner in the production of this book. Her understanding of the needs of the authors, promptness in responding to requests (both reasonable and unreasonable), and cheerful demeanor went a long way in reducing our stress and making our lives less complicated. Jim Merritt handed over the charge to Jeremy Bowes, Publisher. Our thanks also go to Director of Content Development, Rebecca Gruliow, and Designer Brian Salisbury. Undoubtedly there are many others who may have been left out unwittingly—to them we say “thank you” and tender apologies for not acknowledging you individually. We also want to acknowledge many readers—students, residents, and faculty members—scattered around the globe whose comments improve the book. We are impressed by their careful reading of the text. Efforts of this magnitude take a toll on the families of the authors. We thank our spouses, Raminder Kumar, Ann Abbas, Erin Malone, and Judy Turner, for their patience, love, and support of this venture, and for their tolerance of our absences. Vinay Kumar Abul K. Abbas Jon C. Aster Jerrold R. Turner Contents CHAPTER 1 The Cell as a Unit of Health and Disease 1 Richard N. Mitchell CHAPTER 2 Cell Injury, Cell Death, and Adaptations 33 Scott A. Oakes CHAPTER 3 Inflammation and Repair 71 CHAPTER 4 Hemodynamic Disorders, Thromboembolic Disease, and Shock 115 CHAPTER 5 Genetic Disorders 141 CHAPTER 6 Diseases of the Immune System 189 CHAPTER 7 Neoplasia 267 CHAPTER 8 Infectious Diseases 339 Karen M. Frank • Alexander J. McAdam CHAPTER 9 CHAPTER 10 Environmental and Nutritional Diseases 405 Diseases of Infancy and Childhood 453 Aliya N. Husain • Selene C. Koo CHAPTER 11 Blood Vessels 485 Richard N. Mitchell • Marc K. Halushka CHAPTER 12 The Heart 527 Richard N. Mitchell • Andrew J. Connolly CHAPTER 13 Diseases of White Blood Cells, Lymph Nodes, Spleen, and Thymus 583 CHAPTER 14 Red Blood Cell and Bleeding Disorders 635 CHAPTER 15 The Lung 673 Aliya N. Husain CHAPTER 16 Head and Neck 731 Mark W. Lingen • Nicole A. Cipriani CHAPTER 17 The Gastrointestinal Tract 753 xi xii Contents CHAPTER 18 Liver and Gallbladder 823 Ryan M. Gill • Sanjay Kakar CHAPTER 19 The Pancreas 881 Anirban Maitra CHAPTER 20 The Kidney 895 Anthony Chang • Zoltan G. Laszik CHAPTER 21 The Lower Urinary Tract and Male Genital System 953 George Jabboure Netto • Mahul B. Amin CHAPTER 22 The Female Genital Tract 985 Lora Hedrick Ellenson • Edyta C. Pirog CHAPTER 23 The Breast 1037 Susan C. Lester CHAPTER 24 The Endocrine System 1065 Anirban Maitra CHAPTER 25 The Skin 1133 Alexander J. Lazar CHAPTER 26 Bones, Joints, and Soft Tissue Tumors 1171 Andrew Horvai CHAPTER 27 Peripheral Nerves and Skeletal Muscles 1217 Peter Pytel • Douglas C. Anthony CHAPTER 28 The Central Nervous System 1241 Marta Margeta • Arie Perry CHAPTER 29 The Eye Robert Folberg 1305 See TARGETED THERAPY available online at www.studentconsult.com The Cell as a Unit of Health and Disease C H A P T E R 1 Richard N. Mitchell CHAPTER CONTENTS The Genome 1 Noncoding DNA 1 Histone Organization 3 Micro-RNA and Long Noncoding RNA 4 Micro-RNA 4 Long Noncoding RNA 5 Gene Editing 6 Cellular Housekeeping 6 Plasma Membrane: Protection and Nutrient Acquisition 8 Membrane Transport 9 Cytoskeleton 11 Cell-Cell Interactions 12 Biosynthetic Machinery: Endoplasmic Reticulum and Golgi 13 Waste Disposal: Lysosomes and Proteasomes 14 Cellular Metabolism and Mitochondrial Function 15 Cellular Activation 16 Modular Signaling Proteins, Hubs, and Nodes 19 Transcription Factors 19 Growth Factors and Receptors 20 Extracellular Matrix 21 Maintaining Cell Populations 25 Cell Signaling 17 Signal Transduction Pathways 17 Pathology literally translates as the study of suffering (Greek pathos = suffering, logos = study); more prosaically, and as applied to modern medicine, it is the study of disease. Virchow was prescient in asserting that disease originates at the cellular level, but we now appreciate that cellular pathologies arise from perturbations in molecules (genes, proteins, and metabolites) that influence cell survival and behaviors. Thus the foundation of modern pathology is understanding the cellular and molecular aberrations that give rise to diseases. It is illuminating to consider these abnormalities in the context of normal cellular structure and function, which is the subject of this introductory chapter. It is unrealistic (and even undesirable) to condense the vast and fascinating field of cell biology into a single chapter. Consequently, rather than attempting a comprehensive review, the goal here is to survey basic principles and highlight recent advances that are relevant to the mechanisms of disease that are emphasized throughout the rest of the book. THE GENOME The sequencing of the human genome at the beginning of the 21st century represented a landmark achievement of biomedical science. Since then the rapidly declining cost of sequencing, the burgeoning computational capacity to mine the ensuing data, and the expanding toolkits to analyze functional outputs (genomics, proteomics, and metabolomics) promise to revolutionize our understanding of health and disease. The emerging information has also revealed a Components of the Extracellular Matrix 23 Proliferation and the Cell Cycle 25 Stem Cells 28 Regenerative Medicine 29 breathtaking level of complexity far beyond the linear sequence of the genome. The potential of these powerful innovations to explain disease pathogenesis and drive therapeutic discovery excites and inspires scientists and the lay public alike. Noncoding DNA The human genome contains some 3.2 billion DNA base pairs. Yet, within the genome there are only about 20,000 protein-encoding genes, constituting just 1.5% of the genome. These are the blueprints that instruct the assembly of the enzymes, structural elements, and signaling molecules within the 50 trillion cells that make up the human body. Although 20,000 underestimates the actual number of encoded proteins (many genes produce multiple RNA transcripts that translate to different protein isoforms), it is nevertheless startling to realize that worms, which are composed of fewer than 1000 cells and have 30-fold smaller genomes also have about 20,000 protein-encoding genes. Many of these proteins are recognizable homologs of molecules expressed in humans. What then separates humans from worms? The answer is not completely known, but evidence suggests that much of the difference lies in the 98.5% of the human genome that does not encode proteins. The function of such long stretches of DNA (so-called genome “dark matter”) was mysterious for many years. However, over 85% of the human genome is ultimately transcribed; nearly 80% is devoted to regulation of gene expression. It follows that while proteins provide the building blocks and 1 2 CHAPTER 1 The Cell as a Unit of Health and Disease machinery required for assembling cells, tissues, and organisms, it is the noncoding regions of the genome that provide the critical “architectural planning.” Practically stated, the difference between worms and humans apparently lies more in the genomic “blueprints” than in the construction materials. There are five major classes of functional non–proteincoding sequences in the human genome (Fig. 1.1): • Promoter and enhancer regions that provide binding sites for transcription factors. • Binding sites for factors that organize and maintain higher order chromatin structures. • Noncoding regulatory RNAs. Over 60% of the genome is transcribed into RNAs that are never translated but regulate gene expression through a variety of mechanisms. The two best-studied varieties—micro-RNAs (miRNAs) and long noncoding RNAs (lncRNAs)—are described later. • Mobile genetic elements (e.g., transposons) make up more than a third of the human genome. These “jumping genes” can move around the genome during evolution, resulting in variable copy number and positioning even among closely related species (e.g., humans and other primates). Although implicated in gene regulation and chromatin organization, the function of mobile genetic elements is not well established. Heterochromatin Nucleolus Euchromatin Nucleus • Special structural regions of DNA, in particular, telomeres (chromosome ends) and centromeres (chromosome “tethers”). A major component of centromeres is so-called satellite DNA, consisting of large arrays—up to megabases in length—of repeating sequences (from 5 bp up to 5 kb). Although classically associated with spindle apparatus attachment, satellite DNA is also important in maintaining the dense, tightly packed organization of heterochromatin (discussed later). Many genetic variations (polymorphisms) associated with diseases are located in non–protein-coding regions of the genome. Thus variation in gene regulation may prove to be more important in disease causation than structural changes in specific proteins. Another surprise that emerged from genome sequencing is that any two humans are typically more than 99.5% DNA-identical (and are 99% sequenceidentical with chimpanzees)! Thus individual variation, including differential susceptibility to diseases and environmental stimuli, is encoded in less than 0.5% of our DNA (representing about 15 million bp). The two most common forms of DNA variation in the human genome are single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). • SNPs are variants at single nucleotide positions and are almost always biallelic (only two choices exist at a given Heterochromatin (dense, inactive) Euchromatin (disperse, active) Nucleosome DNA Transcription PremRNA Cell p arm Promoter Exon mRNA 5’ UTR Centromere Chromosome Enhancer Intron Splicing Intron q arm Telomeres Exon Open-reading frame Exon Intron 3’ UTR Translation Protein Figure 1.1 The organization of nuclear DNA. At the light microscopic level, the nuclear genetic material is organized into dispersed, transcriptionally active euchromatin and densely packed, transcriptionally inactive heterochromatin; chromatin can also be mechanically connected with the nuclear membrane, and membrane perturbation can thus influence transcription. Chromosomes (as shown) can be visualized only during mitosis. During mitosis, they are organized into paired chromatids connected at centromeres; the centromeres act as the locus for the formation of a kinetochore protein complex that regulates chromosome segregation at metaphase. The telomeres are repetitive nucleotide sequences that cap the termini of chromatids and permit repeated chromosomal replication without deterioration of genes near the ends. The chromatids are organized into short “P” (“petite”) and long “Q” (next letter in the alphabet) arms. The characteristic banding pattern of chromatids has been attributed to relative GC content (less GC content in bands relative to interbands), with genes tending to localize to interband regions. Individual chromatin fibers are comprised of a string of nucleosomes— DNA wound around octameric histone cores—with the nucleosomes connected via DNA linkers. Promoters are noncoding regions of DNA that initiate gene transcription; they are on the same strand and upstream of their associated gene. Enhancers can modulate gene expression over distances of 100 kb or more by looping back onto promoters and recruiting additional factors that drive the expression of pre–messenger RNA (mRNA) species. Intronic sequences are spliced out of the pre-mRNA to produce the final message that is translated into protein—without the 3′–untranslated region (UTR) and 5′-UTR. In addition to the enhancer, promoter, and UTR sequences, noncoding elements, including short repeats, regulatory factor binding regions, noncoding regulatory RNAs, and transposons, are distributed throughout the genome. The genome site within the population, such as A or T). Over 6 million human SNPs have been identified, with many showing wide variation in frequency in different populations. • SNPs occur across the genome—within exons, introns, intergenic regions, and coding regions. • Roughly 1% of SNPs occur in coding regions, which is about what would be expected by chance, since coding regions comprise about 1.5% of the genome. • SNPs located in noncoding regions can occur within genomic regulatory elements, thereby altering gene expression; in such instances, SNPs influence disease susceptibility directly. • Some SNPs, termed “neutral” variants, are thought to have no effect on gene function or individual phenotype. • Even “neutral” SNPs may be useful markers if they happen to be coinherited with a disease-associated polymorphism as a result of physical proximity. In other words, the SNP and the causative genetic factor are in linkage disequilibrium. • The effect of most SNPs on disease susceptibility is weak, and it remains to be seen if identification of such variants, alone or in combination, can be used to develop effective strategies to identify those at risk and, ultimately, prevent disease. • CNVs are a form of genetic variation consisting of different numbers of large contiguous stretches of DNA; these can range from 1000 base pairs to millions of base pairs. CNVs can be biallelic and simply duplicated or, alternatively, deleted in some individuals. At other sites there are complex rearrangements of genomic material, with multiple variants in the human population. • CNVs are responsible for between 5 million and 24 million base pairs of sequence difference between any two individuals. • Approximately 50% of CNVs involve gene-coding sequences; thus CNVs may underlie a large portion of human phenotypic diversity. It is important to note that alterations in DNA sequence cannot by themselves explain the diversity of phenotypes in human populations; moreover, classic genetic inheritance cannot explain differing phenotypes in monozygotic twins. The answers to these conundrums probably lie in epigenetics— heritable changes in gene expression that are not caused by variations in DNA sequence (see the following section). • • • • Histone Organization Even though virtually all cells in the body have the same genetic composition, differentiated cells have distinct structures and functions that arise as a result of lineagespecific gene expression programs. Such cell type–specific differences in transcription and translation depend on epigenetic factors (literally, factors that are “above genetics”) that can be conceptualized as follows (Fig. 1.2): • Histones and histone-modifying factors. Nucleosomes consist of DNA segments 147 bp long that are wrapped around a central core structure of highly conserved low molecular weight proteins called histones. The resulting DNA-histone complex resembles a series of beads joined by short DNA linkers. The naked DNA of a single human cell is about 1.8 m long. By winding around histones, like spools of • thread, the entire genome can be packed into a nucleus as small as 7 to 8 µm in diameter. In most cases, this structured DNA, termed chromatin, is not wound uniformly. Thus at the light microscopic level, nuclear chromatin is recognizable as cytochemically dense and transcriptionally inactive heterochromatin and disperse, transcriptionally active euchromatin (see Fig. 1.1). In general, only the regions that are “unwound” are available for transcription. Chromatin structure can therefore regulate transcription independent of traditional promoters and DNA-binding elements and, due to variations between cell types, helps to define cellular identity and activity. Histones are not static, but rather are highly dynamic structures regulated by a host of nuclear proteins. Thus chromatin remodeling complexes can reposition nucleosomes on DNA, exposing (or obscuring) gene regulatory elements such as promoters. “Chromatin writer” complexes, on the other hand, carry out over 70 different histone modifications generically denoted as “marks.” Such covalent alterations include methylation, acetylation, or phosphorylation of specific amino acids within histones. Actively transcribed genes in euchromatin are associated with histone marks that make the DNA accessible to RNA polymerases. In contrast, inactive genes have histone marks that enable DNA compaction into heterochromatin. Histone marks are reversible through the activity of “chromatin erasers.” Still other proteins function as “chromatin readers,” binding histones that bear particular marks and thereby regulating gene expression. Histone methylation. Both lysines and arginines can be methylated by specific writer enzymes; methylation of histone lysine residues can lead to transcriptional activation or repression, depending on which histone residue is marked. Histone acetylation. Lysine residues are acetylated by histone acetyltransferases (HATs), whose modifications tend to open the chromatin and increase transcription. In turn, these changes can be reversed by histone deacetylases (HDACs), leading to chromatin condensation. Histone phosphorylation. Serine residues can be modified by phosphorylation; depending on the specific residue, the DNA may be opened for transcription or condensed and inactive. DNA methylation. High levels of DNA methylation in gene regulatory elements typically result in transcriptional silencing. Like histone modifications, DNA methylation is tightly regulated by methyltransferases, demethylating enzymes, and methylated-DNA-binding proteins. Chromatin organizing factors. Much less is known about these proteins, which are believed to bind to noncoding regions and control long-range looping of DNA, thus regulating the spatial relationships between enhancers and promoters that control gene expression. Deciphering the mechanisms that allow epigenetic factors to control genomic organization and gene expression in a cell-type-specific fashion is an extraordinarily complex proposition. Despite the intricacies, there is already ample evidence that dysregulation of the “epigenome” has a central role in malignancy (Chapter 7), and emerging data indicate 3 CHAPTER 1 The Cell as a Unit of Health and Disease A Core DNA (1.8 turns) DNA H2A Nucleosome 4 H4 Histone protein core H2B H3 Linker DNA H1 Linker DNA B Heterochromatin (inactive) Linker histone H1 Euchromatin (active) Methylation Acetylation H1 H1 H1 H1 Figure 1.2 Histone organization. (A) Nucleosomes are comprised of octamers of histone proteins (two each of histone subunits H2A, H2B, H3, and H4) encircled by 1.8 147 bp DNA loops. Histones sit on 20- to 80-nucleotide stretches of linker DNA between nucleosomes. Histone subunits are positively charged, thus allowing compaction of negatively charged DNA. (B) The relative state of DNA unwinding (and thus access for transcription factors) is regulated by histone modification, including acetylation, methylation, and/or phosphorylation; these “marks” are dynamically written and erased. Certain marks such as histone acetylation “open up” the chromatin structure, whereas others such as methylation of particular histone residues condense DNA to silence genes. DNA can also be methylated, leading to transcriptional inactivation. that many other diseases are associated with inherited or acquired epigenetic alterations. Unlike genetic changes, many epigenetic alterations (e.g., histone acetylation and DNA methylation) are reversible and amenable to therapeutic intervention; HDAC and DNA methylation inhibitors are already being tested in the treatment of various forms of cancer. Micro-RNA and Long Noncoding RNA Genes can also be regulated by noncoding RNAs. These genomic sequences are transcribed but not translated. Although many distinct families of noncoding RNAs exist, we will discuss only two examples here: small RNA molecules called microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) (>200 nucleotides in length). Micro-RNA miRNAs do not encode proteins; they modulate translation of target messenger RNAs (mRNAs). Posttranscriptional silencing of gene expression by miRNA is a fundamental and well-conserved mechanism of gene regulation present in all eukaryotes (plants, animals, and fungi). Even bacteria have a primitive version of the same machinery that they use to protect themselves against foreign DNA (e.g., phage and virus DNA). The profound influence of miRNAs on protein expression allows these relatively short RNAs (22 nucleotides on average) to be critical regulators of developmental pathways as well as pathologic conditions (e.g., cancer). The human genome encodes almost 6000 miRNA genes, about 30% of the total number of protein-coding genes. Individual miRNAs can regulate multiple protein-coding genes, allowing each miRNA to coregulate entire programs of gene expression. Transcription of miRNA genes produces a primary transcript (pri-miRNA) that is processed into progressively smaller segments, including trimming by the enzyme Dicer. This generates mature single-stranded miRNAs of 21 to 30 nucleotides that associate with a multiprotein aggregate called RNA-induced silencing complex (RISC) (Fig. 1.3). Subsequent base pairing between the miRNA strand and its target mRNA directs the RISC to either induce mRNA cleavage or repress its translation. In this way the target mRNA is posttranscriptionally silenced. Small interfering RNAs (siRNAs) are short RNA sequences that can be introduced experimentally into cells where they The genome serve as substrates for Dicer and interact with RISC, thereby reproducing endogenous miRNAs function. Synthetic siRNAs that target specific mRNA species are powerful laboratory tools to study gene function (so-called knockdown technology) and are also being studied as potential therapeutic agents to silence pathogenic genes (e.g., oncogenes that drive neoplastic transformation). miRNA gene pri-miRNA Long Noncoding RNA Target gene pre-miRNA Export protein pre-miRNA Dicer Recent studies have further identified an untapped universe of lncRNAs—by some calculations, the number of lncRNAs may exceed coding mRNAs by 10-fold to 20-fold. lncRNAs modulate gene expression by several mechanisms (Fig. 1.4). As one example, lncRNAs can bind to chromatin and restrict RNA polymerase from accessing coding genes within that region. The best-known example is XIST, which is transcribed from the X chromosome and plays an essential role in the physiologic X chromosome inactivation that occurs in A. Gene activation lncRNA Target mRNA Ribonucleoprotein transcription complex Gene activation miRNA Unwinding of miRNA duplex RISC complex B. Gene suppression Imperfect match Decoy lncRNA Gene suppression Perfect match Target mRNA Translational repression mRNA cleavage C. Promote chromatin modification Methylation, acetylation Ribosome GENE SILENCING Figure 1.3 Generation of microRNAs (miRNAs) and their mode of action in regulating gene function. Transcription of a miRNA produces a primary miRNA (pri-miRNA), which is processed within the nucleus to form pre-miRNA composed of a single RNA strand with secondary hairpin loop structures and stretches of double-stranded RNA. After export out of the nucleus via specific transporter proteins, pre-miRNA is trimmed by the cytoplasmic Dicer enzyme to generate mature double-stranded miRNAs of 21 to 30 nucleotides. The miRNA subsequently unwinds and the single strands are incorporated into multiprotein RNA-induced silencing complexes (RISC). Base pairing between single-stranded miRNA and the targeted messenger RNA (mRNA) directs RISC to either cleave or repress translation of the mRNA, resulting in posttranscriptiontional silencing. D. Assembly of protein complexes Act on chromatin structure Multi-subunit complex Figure 1.4 Roles of long noncoding RNAs (lncRNAs). (A) lncRNAs can facilitate transcription factor binding and thus promote gene activation. (B) Conversely, lncRNAs can preemptively bind transcription factors to inhibit transcription. (C) Histone and DNA modification by acetylases or methylases (or deacetylases and demethylases) may be directed by lncRNA binding. (D) In other instances, lncRNAs can act as scaffolds to stabilize secondary or tertiary structures and multisubunit complexes that influence chromatin architecture or gene activity. (Modified from Wang KC, Chang HY: Molecular mechanisms of long noncoding RNAs, Mol Cell 43:904, 2011.) 5 6 CHAPTER 1 The Cell as a Unit of Health and Disease females. XIST itself escapes X inactivation but forms a repressive “cloak” on the X chromosome from which it is transcribed, resulting in gene silencing. Conversely, many enhancers are actually sites of lncRNA synthesis. In this case the lncRNAs expand transcription from gene promoters via a variety of mechanisms (see Fig. 1.4). Homologous gRNA sequence Cas9 protein gRNA Gene Editing An exciting new development that allows high-fidelity genome editing may usher in the next era of the molecular revolution. This advance comes from a wholly unexpected source: the discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (Cas), such as the Cas9 nuclease. These are linked genetic elements that endow prokaryotes with a form of acquired immunity to phages and plasmids. Bacteria use the system to sample the DNA of infecting agents and integrate portions into their genomes as CRISPRs. These CRISPR segments are subsequently transcribed and processed into guide RNA sequences that bind and direct the Cas9 nuclease to specific sites (e.g., a phage sequence) so that it can be cleaved to disable the infecting agent. Gene editing repurposes this process by using artificial 20-base guide RNAs (gRNAs) that bind Cas9 and are complementary to a targeted DNA sequence (Fig. 1.5). Cas9 then induces double-stranded DNA breaks at the site of gRNA binding. Repair of the highly specific cleavages can lead to random disruptive mutations (through nonhomologous end joining) or can introduce new genetic material with precision (by homologous recombination). Both the guide sequences and the Cas enzyme, either as a coding DNA (cDNA) or a protein, can be easily introduced into cells. The potential application to genetic engineering, due to the impressive specificity of the Cas9 system (up to 10,000-fold better than other previous editing systems), has led to great excitement. Applications include inserting specific mutations in cells and tissues to model cancers and other diseases and rapidly generating transgenic animal models from edited embryonic stem cells. CRISPR also makes it possible to selectively edit mutations that cause hereditable disease, or—perhaps more worrisome—to just eliminate less “desirable” traits. Predictably the technology has inspired a vigorous debate regarding the ethics of its use. CELLULAR HOUSEKEEPING Normal functioning and intracellular homeostasis depend on a variety of fundamental cell housekeeping functions that all differentiated cells must perform to maintain viability and normal activity. These include protection from the environment, nutrient acquisition, metabolism, communication, movement, renewal of senescent molecules, molecular catabolism, and energy generation. Many of the normal housekeeping functions of the cell are compartmentalized within membrane bound intracellular organelles (Fig. 1.6). By isolating certain cellular functions within distinct compartments, potentially injurious degradative enzymes or toxic metabolites can be kept at usefully high concentrations without risking damage to more delicate intracellular constituents. Moreover, Cleavage Double-stranded DNA Target genomic sequence Double-stranded DNA break NHEJ HDR Insertion/ deletion Donor DNA DNA with random mutation DNA with specific mutation Figure 1.5 Gene editing with clustered regularly interspersed short palindromic repeats (CRISPRs) and the nuclease Cas9. In bacteria, DNA sequences consisting of CRISPRs are transcribed into guide RNAs (gRNAs) with a constant region and a variable sequence of approximately 20 bases. The gRNA constant regions bind to Cas9, while the variable regions form heteroduplexes with homologous DNA sequences of interest; the Cas9 nuclease then cleaves the bound DNA to produce a double-stranded DNA break. In nature, bacteria use the CRISPR/Cas9 system to protect against phages and plasmids; CRISPR sequences from previous assaults are transcribed into gRNA from the bacterial genome. These bind to pathogen nucleotide sequences and form a complex with the Cas9 nuclease that leads to cleavage and, ultimately, destruction of the invader’s DNA. To perform gene editing, gRNAs are designed with variable regions that are homologous to a specific DNA sequence of interest; coexpression of the gRNA and Cas9 then leads to efficient and highly specific cleavage of the target sequence. In the absence of homologous DNA, the doublestranded break is repaired by nonhomologous end-joining (NHEJ), an error-prone mechanism that typically introduces disruptive insertions or deletions (indels). Conversely, in the presence of homologous “donor” DNA that spans the region target by the CRISPR/Cas9 complex, cells instead can use homologous DNA recombination (HDR) to repair the break. HDR is less efficient than NHEJ but has the capacity to introduce precise changes in DNA sequence. Potential applications of CRISPR/Cas9 coupled with HDR include repair of inherited genetic diseases and the creation of pathogenic mutations in inducible pluripotent stem cells. compartmentalization also allows the creation of unique intracellular environments (e.g., low pH or high calcium) that permit more efficient functioning of certain enzymes or metabolic pathways. New proteins destined for the plasma membrane or secretion are physically assembled in the rough endoplasmic Cellular housekeeping Relative volumes of intracellular organelles (hepatocyte) Compartment Cytosol Mitochondria Rough ER Smooth ER, Golgi Nucleus Endosomes Lysosomes Peroxisomes % total volume 54% 22% 9% 6% 6% 1% 1% 1% Golgi apparatus number/cell 1 1700 1 1 1 200 300 400 role in the cell metabolism, transport, protein translation energy generation, apoptosis synthesis of membrane and export protein protein modification, sorting, catabolism cell regulation, proliferation, DNA transcription intracellular transport and export cellular catabolism very long-chain fatty acid metabolism Rough Free endoplasmic ribosomes reticulum Nucleolus Nucleus Lysosome Mitochondrion Endosome Cytoskeleton Plasma membrane Peroxisome Microtubules Smooth endoplasmic reticulum Centrioles Figure 1.6 Basic subcellular constituents of cells. The table presents the number of the various organelles within a typical hepatocyte, as well as their volume within the cell. The figure shows geographic relationships but is not intended to be accurate to scale. ER, Endoplasmic reticulum. (Modified from Weibel ER, Stäubli W, Gnägi HR, et al: Correlated morphometric and biochemical studies on the liver cell. I. Morphometric model, stereologic methods, and normal morphometric data for rat liver. J Cell Biol 42:68, 1969.) reticulum (RER) and Golgi apparatus; proteins intended for the cytosol are synthesized on free ribosomes. Smooth endoplasmic reticulum (SER) is used for steroid hormone and lipoprotein synthesis and modification of hydrophobic compounds into water-soluble molecules for export. Cells catabolize the wide variety of molecules that they endocytose, as well as the entire repertoire of their own proteins and organelles—all of which are constantly being degraded and renewed. Breakdown of these constituents takes place at three different sites, ultimately serving different functions. • Proteasomes are “disposal” complexes that degrade denatured or otherwise “tagged” cytosolic proteins. In antigen-presenting cells, the resulting short peptides are presented in the context of class I or class II major histocompatibility molecules to help drive the adaptive immune response (Chapter 6). In other cases, proteasomal degradation of regulatory proteins or transcription factors can trigger initiation or suppression of signaling pathways. • Lysosomes are intracellular organelles containing degradative enzymes that permit digestion of a wide range of macromolecules, including proteins, polysaccharides, lipids, and nucleic acids. They are the site of senescent intracellular organelle breakdown (a process called autophagy) and where phagocytosed microbes are killed and catabolized. • Peroxisomes contain catalase, peroxidase, and other oxidative enzymes; they play a specialized role in the breakdown of very long-chain fatty acids, generating hydrogen peroxide in the process. The contents and location of cellular organelles are also highly regulated. Endosomal vesicles shuttle internalized material to the appropriate intracellular site(s), while other 7 8 CHAPTER 1 The Cell as a Unit of Health and Disease membrane-bound vesicles direct newly synthesized materials to the cell surface or specific organelles. Movement—of both organelles and proteins within the cell, as well as the entire cell in its environment—is accomplished by the cytoskeleton, which is composed of filamentous actin (microfilaments), keratins (intermediate filaments), and microtubules. These structural proteins also maintain cellular shape and intracellular organization, which are essential to generation and maintenance of cell polarity. This is particularly important in epithelium where the top of the cell (apical) and the bottom and sides of the cell (basolateral) are exposed to different environments and have distinct functions. Loss of polarity could, for example, disrupt vectorial transcellular transport in the intestine or renal tubule. Cell growth and maintenance require a constant supply of both energy and the building blocks that are needed for synthesis of macromolecules. Most of the adenosine triphosphate (ATP) that powers cells is generated via mitochondrial oxidative phosphorylation. Mitochondria also serve as an important source of metabolic intermediates needed for anabolic metabolism, are sites of synthesis of certain macromolecules (e.g., heme), and contain important sensors of cell damage that can initiate and regulate programmed cell death (e.g., apoptosis). In growing and dividing cells, all of these organelles have to be replicated (organellar biogenesis) and correctly apportioned in daughter cells following mitosis. Moreover, because the macromolecules and organelles have finite lifespans (mitochondria, for example, last only Plasma Membrane: Protection and Nutrient Acquisition Plasma membranes (and all other organellar membranes for that matter) are more than just static lipid sheaths. Rather, they are fluid bilayers of amphipathic phospholipids— hydrophilic head groups that face the aqueous environment and hydrophobic lipid tails that interact with each other to form a barrier to passive diffusion of large or charged molecules (Fig. 1.7). The bilayer has a remarkably heterogeneous composition of different phospholipids that vary by location and are also asymmetric—that is, membrane lipids preferentially associate with extracellular or cytosolic faces. Proper localization of these molecules is important for cell health. For example, specific phospholipids interact with particular membrane proteins and modify their distributions and functions. • Phosphatidylinositol on the inner membrane leaflet can be phosphorylated, serving as an electrostatic scaffold for intracellular proteins; alternatively, polyphosphoinositides can be hydrolyzed by phospholipase C to generate intracellular second signals like diacylglycerol and inositol trisphosphate. Extracellular protein Outside Phosphatidylcholine (outer mostly) about 10 days), mechanisms must also exist that allow for the recognition and degradation of “worn-out” cellular components. With this as a primer, we will now move on to discuss cellular components and their function in greater detail. Glycosylphosphatidylinositol (GPI) linked protein Glycolipids Sphingomyelin (outer mostly) Lipid raft P P Phosphatidylethanolamine (inner mostly) Phosphatidylserine (inner mostly) Phosphatidyl- Cholesterol inositol (both faces) (both faces) Transmembrane proteins Cytoplasm A B Lipid-linked protein Cytosolic protein Figure 1.7 Plasma membrane organization and asymmetry. (A) The plasma membrane is a bilayer of phospholipids, cholesterol, and associated proteins. The phospholipid distribution within the membrane is asymmetric due to the activity of flippases; phosphatidylcholine and sphingomyelin are overrepresented in the outer leaflet, and phosphatidylserine (negative charge) and phosphatidylethanolamine are predominantly found on the inner leaflet; glycolipids occur only on the outer face where they contribute to the extracellular glycocalyx. Although the membrane is laterally fluid and the various constituents can diffuse randomly, specific domains, for example cholesterol and glycosphingolipid-rich lipid rafts, can also form. (B) Membrane-associated proteins may traverse the membrane (singly or multiply) via α-helical hydrophobic amino acid sequences; depending on the membrane lipid content and relative hydrophobicity of protein domains, such proteins may have nonrandom distributions within the membrane. Proteins on the cytosolic face can be associated with the plasma membrane through posttranslational modifications (e.g., farnesylation) or addition of palmitic acid. Proteins on the extracytoplasmic face can associate with the membrane via glycosylphosphatidylinositol (GPI) linkages. Besides protein-protein interactions within the membrane, membrane proteins can also associate with extracellular and/or intracytoplasmic proteins to generate distinct domains (e.g., the focal adhesion complex). Transmembrane proteins can translate mechanical forces (e.g., from the cytoskeleton or extracellular matrix), as well as chemical signals across the membrane. Cellular housekeeping • Phosphatidylserine is normally restricted to the inner face where it confers a negative charge involved in electrostatic protein interactions; however, when flipped to the extracellular leaflet, it becomes a potent “eat me” signal during programmed cell death (e.g., apoptosis). In platelets, phosphatidylserine is also a cofactor in blood clotting. • Glycolipids and sphingomyelin are preferentially located on the extracellular face; glycolipids, including gangliosides with complex sugar linkages and terminal sialic acids that confer negative charges, support charge-based interactions that contribute to including inflammatory cell recruitment and sperm-egg fusion. composition. The latter strategy is used to maintain cell polarity (e.g., top/apical/free vs. bottom/basolateral/ bound to extracellular matrix [ECM]) in epithelial cells. Interactions of other membrane and cytosolic proteins with one another and the cytoskeleton also contributes to cell polarity. The extracellular face of the plasma membrane is diffusely decorated by carbohydrates, not only as complex oligosaccharides on glycoproteins and glycolipids, but also as polysaccharide chains attached to integral membrane proteoglycans. This glycocalyx can form a chemical and mechanical barrier. Despite substantial lateral fluidity, some membrane constituents concentrate into specialized domains (e.g., lipid rafts) that are enriched in glycosphingolipids and cholesterol. Since inserted membrane proteins have different intrinsic solubilities in domains with distinct lipid compositions, this membrane organization also impacts protein distribution. This geographic organization of plasma membrane components impacts cell-cell and cell-matrix interactions, intracellular signaling, and the specialized sites of vesicle budding or fusion. The plasma membrane is liberally studded with a variety of proteins and glycoproteins involved in (1) ion and metabolite transport; (2) fluid-phase and receptormediated uptake of macromolecules; and (3) cell-ligand, cell-matrix, and cell-cell interactions. The means by which these proteins associate with membranes frequently reflects function. For example, multiple transmembrane-spanning proteins are often pores or molecular transporters, while proteins that are superficially attached to the membrane via labile linkages are more likely to participate in signaling. In general, proteins associate with the lipid bilayer by one of four mechanisms. • Most proteins are integral or transmembrane proteins, having one or more relatively hydrophobic α-helical segments that traverse the lipid bilayer. • Proteins synthesized on free ribosomes in the cytosol may be modified posttranslationally by addition of prenyl groups (e.g., farnesyl, related to cholesterol) or fatty acids (e.g., palmitic or myristic acid) that insert into the cytosolic side of the plasma membrane. • Proteins on the extracellular face of the membrane may be anchored by glycosylphosphatidylinositol (GPI) tails that are added posttranslationally. • Peripheral membrane proteins may noncovalently associate with true transmembrane proteins. Although the barrier provided by plasma membranes is critical, transport of selected molecules across the lipid bilayer or to intracellular sites via vesicular transport is essential. Several mechanisms contribute to this transport. Many plasma membrane proteins function as large complexes; these may be aggregated either under the control of chaperone molecules in the RER or by lateral diffusion in the plasma membrane, followed by complex formation in situ. For example, many protein receptors (e.g., cytokine receptors) dimerize or trimerize in the presence of ligand to form functional signaling units. Although lipid bilayers are fluid within the plane of the membrane, components can be confined to discrete domains. This can occur by localization to lipid rafts (discussed earlier) or through intercellular protein-protein interactions (e.g., tight junctions) that establish discrete boundaries and also have unique lipid Membrane Transport Passive Diffusion. Small, nonpolar molecules like O2 and CO2 readily dissolve in lipid bilayers and therefore rapidly diffuse across them. Larger hydrophobic molecules, (e.g., steroid-based molecules like estradiol or vitamin D) can also cross lipid bilayers with relative impunity. While small polar molecules such as water (18 Da) can also diffuse across membranes at low rates, in tissues responsible for significant water movement (e.g., renal tubular epithelium), special integral membrane proteins called aquaporins form transmembrane channels for water, H2O2, and other small molecules. In contrast, the lipid bilayer is an effective barrier to the passage of larger polar molecules (>75 Da); at 180 Da, for example, glucose is effectively excluded. Lipid bilayers are also impermeant to ions due to their charge and hydration. Carriers and Channels (Fig. 1.8). Plasma membrane transport proteins are required for uptake and secretion of ions and larger molecules that are required for cellular function (e.g., nutrient uptake and waste disposal). Ions and small molecules can be transported by channel proteins and carrier proteins. Similar pores and channels also mediate transport across organellar membranes. These transporters that move ions, sugars, nucleotides, etc., frequently have exquisite specificities, and can be either active or passive (see below). For example, some transporters accommodate glucose but reject galactose. • Channel proteins create hydrophilic pores, which, when open, permit rapid movement of solutes (usually restricted by size and charge). • Carrier proteins bind their specific solute and undergo a series of conformational changes to transfer the ligand across the membrane; their transport is relatively slow. Solute transport across the plasma membrane is frequently driven by a concentration and/or electrical gradient between the inside and outside of the cell via passive transport (virtually all plasma membranes have an electrical potential difference across them, with the inside negative relative to the outside). In other cases, active transport of certain solutes (against a concentration gradient) is accomplished by carrier molecules (never channels) at the expense of ATP hydrolysis or a coupled ion gradient. For example, most apical nutrient 9 10 CHAPTER 1 Extracellular Carrier Energy Membrane The Cell as a Unit of Health and Disease Channel Endocytosis Exocytosis Coated pit Caveolin Coated vesicle Early endosome (low pH) Transcytosis Microbe Caveolae- Receptors Receptormediated mediated Cytosol Phagocytosis Receptor recycling Lysosome Phagosome Reconstitution Phagolysosome Late endosome Lysosome-late endosome fusion vesicle Undigested residual material Figure 1.8 Movement of small molecules and larger structures across membranes. The lipid bilayer is relatively impermeable to all but the smallest and/or most hydrophobic molecules. Thus the import or export of charged species requires specific transmembrane transporter proteins, vesicular traffic, or membrane deformations. From left to right in the figure: Small charged solutes can move across the membrane using either channels or carriers; in general, each molecule requires a unique transporter. Channels are used when concentration gradients can drive the solute movement; activation of the channel opens a hydrophilic pore that allows size-restricted and charge-restricted flow. Carriers are required when solute is moved against a concentration gradient; this typically requires energy expenditure to drive a conformational change in the carrier that facilitates the transmembrane delivery of specific molecules. Receptor-mediated and fluid-phase uptake of material involves membrane bound vesicles. Caveolae endocytose extracellular fluid, membrane proteins, and some receptor bound molecules (e.g., folate) in a process driven by caveolin proteins concentrated within lipid rafts. They can subsequently fuse with endosomes or recycle to the membrane. Endocytosis of receptor-ligand pairs often involves clathrin-coated pits and vesicles. After internalization the clathrin disassembles and individual components can be re-used. The resulting vesicle becomes part of the endocytic pathway, in which compartments are progressively more acidic. After ligand is released, the receptor can be recycled to the plasma membrane to repeat the process (e.g., iron dissociates from transferin at pH ~5.5; apotransferrin and the transferrin receptor then return to the surface). Alternatively, receptor and ligand complexes can eventually be degraded within lysosomes (e.g., epidermal growth factor and its receptor are both degraded, which prevents excessive signaling). Exocytosis is the process by which membrane-bound vesicles fuse with the plasma membrane and discharge their contents to the extracellular space. This includes endosome recycling (shown), release of undigested residual material from lysosomes, transcytotic delivery of vesicles, and export of secretory vacuole contents (not shown). Phagocytosis involves membrane invagination to engulf large particles and is most common in specialized phagocytes (e.g., macrophages and neutrophils). The resulting phagosomes eventually fuse with lysosomes to facilitate the degradation of the internalized material. Transcytosis can mediate transcellular transport in either apical-to-basal or basal-to-apical directions, depending on the receptor and ligand. transporters in the intestines and renal tubules exploit the extracellular to intracellular Na+ gradient to allow absorption even when intracellular nutrient concentrations exceed extracellular concentrations. This form of active transport does not use ATP directly, but depends on the Na+ gradient generated by Na+-Ka+ ATPase. Other transporters are ATPases. One example is the multidrug resistance (MDR) protein, which pumps polar compounds (e.g., chemotherapeutic drugs) out of cells and may render cancer cells resistant to treatment. Water movement into or out of cells is passive and directed by solute concentrations. Thus extracellular salt in excess of that in the cytoplasm (hypertonicity) causes net movement of water out of cells, while hypotonicity causes net movement of water into cells. Conversely, the charged metabolites and proteins within the cytoplasm attract charged counterions that increase intracellular osmolarity. Thus to prevent overhydration, cells must constantly pump out small inorganic ions (e.g., Na+)—typically through the activity of the membrane ion-exchanging ATPase. Loss of the ability to generate energy (e.g., in a cell injured by toxins or ischemia) therefore results in osmotic swelling and eventual cell rupture. Similar transport mechanisms also regulate concentrations of other ions (e.g., Ca2+ and H+). This is critical to many processes. For example, cytosolic enzymes are most active at pH 7.4 and are often regulated by Ca2+, whereas lysosomal enzymes function best at pH 5 or less. Uptake of fluids or macromolecules by the cell is called endocytosis. Depending on the size of the vesicle, endocytosis may be denoted pinocytosis (“cellular drinking”) or phagocytosis (“cellular eating”). Generally, phagocytosis is restricted to certain cell types (e.g., macrophages and Cellular housekeeping neutrophils) whose role is to specifically ingest invading organisms or dead cell fragments. Receptor-Mediated and Fluid-Phase Uptake (see Fig. 1.8) Certain small molecules—including some vitamins—bind to cell-surface receptors and are taken up through invaginations of the plasma membrane called caveolae. Uptake can also occur through membrane invaginations coated by an intracellular matrix of clathrin proteins that spontaneously assemble into a basket-like lattice which helps drive endocytosis (discussed more later). In both cases, activity of the “pinchase” dynamin is required for vesicle release. Macromolecules can also be exported from cells by exocytosis. In this process, proteins synthesized and packaged within the RER and Golgi apparatus are concentrated in secretory vesicles, which then fuse with the plasma membrane to expel their contents. Common examples include peptide hormones (e.g., insulin) and cytokines. Transcytosis is the movement of endocytosed vesicles between the apical and basolateral compartments of cells. This is a mechanism for transferring large amounts of intact proteins across epithelial barriers (e.g., ingested antibodies in maternal milk) or for rapid movement of large solute volumes. We now return to the specifics of endocytosis (see Fig. 1.8). • Caveolae-mediated endocytosis. Caveolae (“little caves”) are noncoated plasma membrane invaginations associated with GPI-linked molecules, cyclic adenosine monophosphate (cAMP) binding proteins, src-family kinases, and the folate receptor; caveolin is the major structural protein of caveolae, which, like membrane rafts (see above), are enriched in glycosphingolipids and cholesterol. Internalization of caveolae along with bound molecules and associated extracellular fluid is called potocytosis—literally “cellular sipping.” In addition to supporting transmembrane delivery of some molecules (e.g., folate), caveolae regulate transmembrane signaling and cellular adhesion via internalization of receptors and integrins. • Receptor-mediated endocytosis. Macromolecules bound to membrane receptors (such as transferrin or low-density lipoprotein [LDL] receptors) are taken up at specialized regions of the plasma membrane called clathrin-coated pits. The receptors are efficiently internalized by membrane invaginations driven by the associated clathrin matrix, eventually pinching off to form clathrin-coated vesicles. Trapped within these vesicles is also a gulp of the extracellular milieu (fluid-phase pinocytosis). The vesicles then rapidly lose their clathrin coating and fuse with an acidic intracellular structure called the early endosome; the endosomal vesicles undergo progressive maturation to late endosomes, ultimately fusing with lysosomes. In the acidic environment of the endosomes, LDL and transferrin receptors release their cargo (cholesterol and iron, respectively), which is then transported into the cytosol. After release of bound ligand, some receptors recycle to the plasma membrane and are reused (e.g., transferrin and LDL receptors), while others are degraded within lysosomes (e.g., epidermal growth factor receptor). In the latter case, degradation after internalization results in receptor downregulation that limits receptor-mediated signaling. Defects in receptor-mediated transport of LDL underlie familial hypercholesterolemia, as described in Chapter 5. Endocytosis requires recycling of internalized vesicles back to the plasma membrane (exocytosis) for another round of ingestion. This is critical, as a cell will typically ingest from the extracellular space the equivalent of 10% to 20% of its own cell volume each hour—amounting to 1% to 2% of its plasma membrane each minute! Without recycling, the plasma membrane would be rapidly depleted. Endocytosis and exocytosis must therefore be tightly coupled to avoid large changes in plasma membrane area. Cytoskeleton The ability of cells to adopt a particular shape, maintain polarity, organize intracellular organelles, and migrate depends on an intracellular scaffold of structural proteins that form the cytoskeleton (Fig. 1.9). Although the cytoskeleton can have an appearance similar to the beams and supports of a building, cytoskeletal structures are constantly elongating and shrinking; microfilaments and microtubules are most active, and their assembly and disassembly can drive cell migration. In eukaryotic cells, there are three major classes of cytoskeletal proteins. • Actin microfilaments are 5- to 9-nm diameter fibrils formed from the globular protein actin (G-actin), the most Microvilli Microtubules Tight junction Actin microfilaments Adherins junction Desmosome Gap junctions Intermediate filaments Hemidesmosome Basement membrane Integrins Figure 1.9 Cytoskeletal elements and cell-cell interactions. Interepithelial adhesion involves several different surface protein interactions at tight junctions, adherens junctions, and desmosomes; adhesion to the extracellular matrix involves cellular integrins (and associated proteins) within hemidesmosomes. The various adhesion proteins within the plasma membrane associate with actin microfilaments and intermediate filaments to provide a mechanical matrix for cell structure and signaling. Gap junctions do not impart structural integrity but allow cell-cell communication by the movement of small molecular weight and/or charged species. See text for details. 11 12 CHAPTER 1 The Cell as a Unit of Health and Disease abundant cytosolic protein in cells. G-actin monomers noncovalently polymerize into long filaments (F-actin) that intertwine to form double-stranded helices with a defined polarity. Although the details are (as always) more nuanced, new subunits are typically added at the “positive” end of the strand and removed from the “negative” end—a process referred to as actin treadmilling. Actin nucleating, binding, and regulatory proteins organize polymerization, bundling, and branching to form networks that control cell shape and movement. This complex and its association with motor proteins (e.g., myosin) is so precisely arrayed in skeletal and cardiac muscle that a banding pattern is apparent by light microscopy. ATP hydrolysis by myosin slides the actin filaments relative to one another to cause muscle contraction. Although less coordinated, myosins, of which there are many, are responsible for other functions that depend on actin contraction including vesicular transport, epithelial barrier regulation, and cell migration. • Intermediate filaments are 10-nm diameter fibrils that comprise a large and heterogeneous family that includes keratin proteins and nuclear lamins. Intermediate filaments predominantly form ropelike polymers and do not usually actively reorganize like actin and microtubules. This allows intermediate filaments to provide tensile strength so that cells can bear mechanical stress, e.g., in epithelia where intermediate filaments link desmosomes and hemidesmosomes (see Fig. 1.9). Individual intermediate filament proteins have characteristic tissue-specific patterns of expression that can be useful for assigning a cell of origin for poorly differentiated tumors. Examples include: • Vimentin, in mesenchymal cells (fibroblasts, endothelium). • Desmin in muscle cells forms the scaffold on which actin and myosin contract. • Neurofilaments are critical for neuronal axon structure and confer both strength and rigidity. • Glial fibrillary acidic protein is expressed in glial cells. • Cytokeratins are expressed in epithelial cells. There are at least 30 distinct different cytokeratins that are expressed in different cell lineages (e.g., lung vs. gastrointestinal epithelium). • Lamins are intermediate filament proteins that form the nuclear lamina, define nuclear shape, and can regulate transcription. • Microtubules are 25-nm-thick fibrils composed of noncovalently polymerized α- and β-tubulin dimers organized into hollow tubes. These fibrils are extremely dynamic and polarized, with “+” and “−” ends. The “−” end is typically embedded in a microtubule organizing center (MTOC or centrosome) near the nucleus, where it is associated with paired centrioles; the “+” end elongates or recedes in response to various stimuli by the addition or subtraction of tubulin dimers. Microtubules: • Serve as mooring lines for molecular motor proteins that use ATP to translocate vesicles, organelles, or other molecules around cells. There are two varieties of these motor proteins, kinesins and dyneins, that typically (but not exclusively) transport cargo in anterograde (− to +) or retrograde (+ to −) directions, respectively. • Mediate sister chromatid segregation during mitosis. • Form the core of primary cilia, single nonmotile projections on many nucleated cells that contribute to the regulation of cellular proliferation and differentiation (mutations in the proteins of the primary cilia complex lead to forms of polycystic kidney disease; see Chapter 20). • Can be adapted to form the core of motile cilia (e.g., in bronchial epithelium) or flagella (in sperm). Cell-Cell Interactions Cells connect and communicate with each other via junctional complexes that form mechanical links and facilitate receptor-ligand interactions. Similar complexes also mediate interaction with the ECM. Cell-cell junctions are organized into three basic types (see Fig. 1.9): • Occluding junctions (tight junctions) seal adjacent epithelial cells together to create a continuous barrier that restricts the paracellular (between cells) movement of ions and other molecules. Occluding junctions form a tight meshlike network (when viewed en face by freeze-fracture electron microscopy) of macromolecular contacts between neighboring cells; the complexes that mediate the cell-cell interactions are composed of transmembrane proteins including the tetraspanning claudin and tight junction– associated MARVEL protein (TAMP) families. These connect to a host of intracellular adaptor and scaffolding proteins, including the three members of the zonula occludens protein family (ZO-1, ZO-2, ZO-3) and cingulin. Besides forming a selectively permeable barrier that seals the space between cells (i.e., the paracellular space), this zone also represents the boundary that separates apical and basolateral membrane domains and helps to maintain cellular polarity. Nevertheless, these junctions are dynamic structures that can be modified to facilitate epithelial healing and inflammatory cell migration across epitheliallined mucosal surfaces. • Anchoring junctions (adherens junctions and desmosomes) mechanically attach cells—and their cytoskeletons—to other cells or the ECM. Adherens junctions are often closely associated with and beneath tight junctions. Desmosomes are more basal and form several types of junctions. When desmosomes attach the cell to the extracellular matrix (ECM) they are referred to as hemidesmosomes (half a desmosome), since the other half of the desmosome is not present within the ECM. Both adherens junctions and desmosomes are formed by homotypic extracellular interactions between transmembrane glycoproteins called cadherins, on adjacent cells. • In adherens junctions the transmembrane adhesion molecules are associated with intracellular actin microfilaments through which they can also influence cell shape and/or motility. Loss of the epithelial adherens junction protein E-cadherin explains the discohesive invasion pattern of some gastric cancers and lobular carcinomas of the breast (Chapters 17 and 23). • In desmosomes the cadherins are linked to intracellular intermediate filaments, allowing extracellular forces to be mechanically communicated (and dissipated) over multiple cells. Cellular housekeeping • In hemidesmosomes the transmembrane connector proteins are called integrins; like desmosomal cadherins, these attach to intermediate filaments and link the cytoskeleton to the ECM. Focal adhesion complexes are composed of >100 proteins and localize at hemidesmosomes. Their component proteins can generate intracellular signals when cells are subjected to shear stress (e.g., endothelium in the bloodstream or cardiac myocytes in a failing heart). • Communicating junctions (gap junctions) permit the diffusion of chemical or electrical signals from one cell to another. The junction consists of a dense planar array of 1.5- to 2-nm pores (called connexons) formed by a pair of hexamers (one on each cell) of transmembrane connexin proteins. These form pores that permit passage of ions, nucleotides, sugars, amino acids, vitamins, and other small molecules; permeability of the junction is rapidly reduced by lowered intracellular pH or increased intracellular calcium. Gap junctions play a critical role in cell-cell communication. For example, gap junctions in cardiac myocytes allow cell-to-cell calcium fluxes that allow the many cells of the myocardium to behave as a functional syncytium with coordinated waves of contraction. Biosynthetic Machinery: Endoplasmic Reticulum and Golgi Apparatus All cellular constituents—including structural proteins, enzymes, transcription factors, and even the phospholipid membranes—are constantly renewed in an ongoing process balancing synthesis and degradation. The endoplasmic reticulum (ER) is the site for synthesis of all transmembrane proteins and lipids for plasma membrane and cellular organelles, including the ER itself. It is also the initial site of synthesis for secreted proteins. The ER is organized into a meshlike interconnected maze of branching tubes and flattened lamellae, forming a continuous sheet around a single lumen that is topologically equivalent to the extracellular environment. ER is composed of contiguous but distinct domains that are distinguished by the presence (RER) or absence (SER) of ribosomes (see Fig. 1.6). • Rough endoplasmic reticulum (RER): Membrane-bound ribosomes on the cytosolic face of RER translate mRNA into proteins that are extruded into the ER lumen or become integrated into the ER membrane. This process is directed by specific signal sequences on the N-termini of nascent proteins; synthesis of proteins with signal peptides is initiated on free ribosomes, but the complex then becomes attached to the ER membrane, and the protein is inserted into or passed across the ER membrane as it is translated. For proteins lacking a signal sequence, translation remains on free ribosomes in the cytosol, forming polyribosomes as multiple ribosomes attach to the mRNA; such transcribed proteins remain within the cytoplasm. Proteins inserted into the ER fold into their active conformation and can form polypeptide complexes (oligomerize); in addition, disulfide bonds are formed, and N-linked oligosaccharides (sugar moieties attached to asparagine residues) are added. Chaperone molecules assist in folding and retaining proteins in the ER until the modifications are complete and the proper conformation is achieved. If a protein fails to appropriately fold or oligomerize, it is retained and degraded within the ER. A good example of this is the most common mutation of the CFTR protein in cystic fibrosis. In mutant CFTR, a codon deletion leads to the absence of a single amino acid (Phe508) which results in its misfolding, ER retention and catabolism and therefore reduced surface expression. Moreover, excess accumulation of misfolded proteins— exceeding the capacity of the ER to edit and degrade them—leads to the ER stress response (also called the unfolded protein response [UPR]) (Fig. 1.10B). Detection of excess abnormally folded proteins leads to a reduction in protein synthesis overall with a concurrent increase in chaperone proteins; failure to correct the overload can trigger cell death through apoptosis (Chapter 2). • Golgi apparatus: From the RER, proteins and lipids destined for other organelles or extracellular export are shuttled into the Golgi apparatus. This consists of stacked cisternae that progressively modify proteins in an orderly fashion from cis (near the ER) to trans (near the plasma membrane). Cisternal progression, i.e., movement of cis-face cisternae to the trans aspect of the Golgi, akin to steps on an escalator, allows sequential processing of newly synthesized proteins and can be facilitated by small membrane-bound vesicles. Similar vesicles shuttle Golgiresident enzymes from trans to cis in order to maintain the different cisternal contents along this assembly line. As cisternae mature, the N-linked oligosaccharides originally added in the ER are pruned and extended in a stepwise fashion; O-linked oligosaccharides (sugar moieties linked to serine or threonine) are also appended. Some of this glycosylation is important in sorting molecules to lysosomes (via the mannose-6-phosphate receptor); other glycosylation adducts are important for cell-cell or cell-matrix interactions or for clearing senescent cells (e.g., platelets and erythrocytes). In the trans Golgi network, proteins and lipids are sorted and dispatched to other organelles, plasma membrane, or secretory vesicles. The Golgi complex is especially prominent in cells specialized for secretion, including goblet cells of the intestinal or bronchial epithelium, which secrete large amounts of polysaccharide-rich mucus. In plasma cells that secrete antibodies, the Golgi can be recognized as a perinuclear hoff on simple hematoxylin and eosin stains (Chapter 6). • Smooth endoplasmic reticulum (SER): In most cells, the SER is relatively sparse and primarily exists as the transition zone extending from RER to generate transport vesicles that carry newly synthesized proteins to the Golgi apparatus. The SER may, however, be particularly conspicuous in cells that synthesize steroid hormones (e.g., within the gonads or adrenals) or that catabolize lipid-soluble molecules (e.g., hepatocytes). Indeed, repeated exposure to compounds that are metabolized by the SER (e.g., phenobarbital, which is catabolized by the cytochrome P-450 system) can lead to SER hyperplasia. The SER is also responsible for sequestering intracellular calcium, which, when released into the cytosol, can mediate a number of responses to extracellular signals (including apoptotic cell death). In muscle cells, specialized SER called sarcoplasmic reticulum is responsible for the cyclic release and sequestration of calcium ions that regulate muscle contraction and relaxation, respectively. 13 14 CHAPTER 1 A The Cell as a Unit of Health and Disease LYSOSOMAL DEGRADATION Endoplasmic reticulum Nucleus Endocytosis Endosome Senescent organs Phagocytosis Denatured proteins Lysosomes LC3 Phagosome AUTOPHAGY HETEROPHAGY Autophagosome Phagolysosome Exocytosis B PROTEASOMAL DEGRADATION CYTOSOL Nascent peptide chains Chaperones Age, UV, heat, reactive oxygen species Folded Senescent or protein denatured protein Multiple ubiquitins E1, E2, E3 ligases Proteasome Peptide fragments Free ubiquitin Metabolic alterations (e.g., pH) Genetic mutations Viral infections ENDOPLASMIC RETICULUM “ER stress” (unfolded protein response) Excess misfolded protein APOPTOSIS Protein synthesis Chaperone production Figure 1.10 Intracellular catabolism. (A) Lysosomal degradation. In heterophagy (right side of panel A), lysosomes fuse with endosomes or phagosomes to facilitate the degradation of their internalized contents (see Fig. 1.8). The end products may be released into the cytosol for nutrition or discharged into the extracellular space (exocytosis). In autophagy (left side of panel A), senescent organelles or denatured proteins are targeted for lysosome-driven degradation as they are encircled within a double membrane vacuole derived from the endoplasmic reticulum and marked by LC3 protein (microtubuleassociated protein 1A/1B-light chain 3). Cell stress, such as nutrient depletion or some intracellular infections, can also activate the autophagocytic pathway. (B) Proteosomal degradation. Cytosolic proteins destined for turnover (e.g., transcription factors or regulatory proteins), senescent proteins, or proteins that have become denatured due to extrinsic mechanical or chemical stresses can be tagged by multiple ubiquitin molecules (through the activity of E1, E2, and E3 ubiquitin ligases). This marks the proteins for degradation by proteasomes, cytosolic multi-subunit complexes that degrade proteins to small peptide fragments. High levels of misfolded proteins within the endoplasmic reticulum (ER) trigger a protective unfolded protein response—engendering a broad reduction in protein synthesis, but specific increases in chaperone proteins that can facilitate protein refolding. If this is inadequate to cope with the levels of misfolded proteins it can lead to apoptosis. Waste Disposal: Lysosomes and Proteasomes Although cells rely primarily on lysosomes to digest internalized material and accumulated internal waste, there are multiple other routes to degrade intracellular macromolecules (see Fig. 1.10). These include proteosomes and peroxisomes. The latter are responsible for catabolism of long-chained fatty acids. • Lysosomes are membrane-bound organelles containing roughly 40 different acid hydrolases (i.e., that function best at pH ≤5); these include proteases, nucleases, lipases, glycosidases, phosphatases, and sulfatases. Lysosomal Cellular metabolism and mitochondrial function enzymes are initially synthesized in the ER lumen and then tagged with mannose-6-phosphate (M6P) within the Golgi apparatus. These M6P-modified proteins are subsequently delivered to lysosomes through trans Golgi vesicles that express M6P receptors. The other macromolecules destined for catabolism in the lysosomes arrive by one of three pathways (see Fig. 1.10). • Material internalized by fluid-phase or receptor-mediated endocytosis passes from plasma membrane to early and then late endosomes and ultimately arrives at the lysosome. These compartments are progressively acidified such that proteolytic enzymes become active in late endosome and lysosomes. • Senescent organelles and/or large, denatured protein complexes can be ferried into lysosomes by a process called autophagy (Chapter 2). Through a mechanism involving the products of a number of autophagyrelated (Atg) genes, obsolete organelles are corralled by a double membrane derived from the ER. The membrane progressively expands to encircle a collection of organelles and cytosolic constituents, forming the definitive autophagosome; these structures are targeted for eventual destruction by fusion with lysosomes. In addition to facilitating turnover of aged and/or defunct structures, autophagy can be used to preserve viability during nutrient depletion; is involved in protective responses to intracellular infections; participates in intracellular repair; and, under some circumstances, triggers programmed cell death (apoptosis). Autophagy is discussed in more detail in Chapter 2. • Phagocytosis of microorganisms or large fragments of matrix or debris occurs primarily in professional phagocytes (macrophages or neutrophils). The material is engulfed to form a phagosome that subsequently fuses with lysosomes. • Proteasomes play an important role in degrading cytosolic proteins (see Fig. 1.10); these include denatured or misfolded proteins as well as any other macromolecule whose lifespan needs to be regulated (e.g., signaling molecules). Many proteins destined for destruction are identified by covalently binding a small protein called ubiquitin. Polyubiquitinated molecules are unfolded and funneled into the polymeric proteasome complex—a cylinder containing multiple protease activities, each with its active site pointed at the hollow core. Proteasomes digest proteins into small (6 to 12 amino acids) fragments that can subsequently be degraded to their constituent amino acids and recycled. CELLULAR METABOLISM AND MITOCHONDRIAL FUNCTION Mitochondria evolved from ancestral prokaryotes that were engulfed by primitive eukaryotes about 1.5 billion years ago. Their origin explains why mitochondria contain their own DNA that encodes about 1% of total cellular protein and approximately 20% of the proteins involved in oxidative phosphorylation. Although their genomes are small, mitochondria can carry out all the steps of DNA replication, transcription, and translation using machinery similar to present-day bacteria; for example, mitochondria initiate protein synthesis with N-formylmethionine and are sensitive to some antibacterial antibiotics. Since mitochondria biogenesis requires a genetic contribution from preexisting mitochondria and the ovum contributes the vast majority of cytoplasmic organelles in the fertilized zygote, mitochondrial DNA is almost entirely maternally inherited. Nevertheless, because the protein constituents of mitochondria are encoded by both nuclear and mitochondrial DNA, mitochondrial disorders may be X-linked, autosomal, or maternally inherited. Mitochondria are impressively dynamic, constantly undergoing fission and fusion with other newly synthesized mitochondria; this supports their renewal and defends against degenerative changes that occur through ongoing oxygen free radical damage. Even so, mitochondria are short-lived—being degraded through autophagy (a process called mitophagy)—with estimated half-lives of 1 to 10 days, depending on the tissue, nutritional status, metabolic demands, and intercurrent injury. Besides providing the enzymatic machinery for oxidative phosphorylation (and thus the relatively efficient generation of energy from glucose and fatty acid substrates), mitochondria play a fundamental role in regulating apoptosis (Fig. 1.11). Details of mitochondrial functions follow: • Energy generation. Each mitochondrion has two separate membranes with distinct functions; the inner membrane contains the enzymes of the respiratory chain folded into cristae. This encloses a core matrix space that harbors the bulk of the enzymes of the glycolytic and tricarboxylic acid cycles, and these constitute the major working parts of the organelle. Outside the inner membrane is the intermembrane space, which is the site of nucleotide phosphorylation and is, in turn, enclosed by the outer membrane; the latter is studded with porin proteins that form voltage-dependent anion channels that are permeable to small (<5000 Da) molecules. As with other cellular membranes, larger molecules (and even some smaller polar species) require specific transporters. The major source of the energy that facilitates all cellular functions derives from oxidative metabolism. Mitochondria oxidize substrates to CO2, transferring the high-energy electrons from the original molecule (e.g., sugar) to molecular oxygen. Oxidation of various metabolites drives proton pumps that transfer H+ from the core matrix into the intermembrane space. As the H+ ions flow down their electrochemical gradient and out of the intermembrane space, the energy released is used to generate ATP. Notably, the electron transport chain need not necessarily be coupled to ATP generation. An inner membrane protein enriched in brown fat called thermogenin (also called uncoupling protein-1 [UCP-1]) is a hydrogen ion transporter that can dissipate the proton gradient, uncoupling it from oxidative phosphorylation. By this means, there is rapid substrate oxidation without ATP synthesis that allows tissues with high levels of UCP-1 to generate heat (nonshivering thermogenesis). As a natural (albeit usually low-level) by-product of substrate oxidation and electron transport, mitochondria are also an important source of reactive oxygen species (oxygen free radicals, hydrogen peroxide, etc.); importantly, hypoxia, toxic injury, or even mitochondrial aging can 15 16 CHAPTER 1 The Cell as a Unit of Health and Disease METABOLITES (glucose, glutamine, fatty acids) Intermembrane space (ATP generation) TCA cycle intermediates Lipid, amino acid and protein, and nucleic acid building blocks ATP GENERATION Inner membrane (respiratory chain proteins) APOPTOSIS SIGNALS Outer membrane Bax/Bak complexes Mitochondrion Core matrix (citric acid cycle enzymes) H+ H+ Mitochondrial outer membrane permeabilization (MOMP) Cytochrome C Injury Membrane permeability transition pores Stabilized apoptosome H+ ISCHEMIA, TOXIN ATP CELL DEATH (necrosis) Caspase cascade CELL DEATH (apoptosis) Figure 1.11 Roles of the mitochondria. Besides the efficient generation of adenosine triphosphate (ATP) from carbohydrate and fatty acid substrates, mitochondria also play an important role in intermediary metabolism—serving as the source of substrates used to synthesize lipids, proteins, and nucleotides. Significantly, they are centrally involved in cell life-and-death decisions: (1) toxic or ischemic injury will induce a membrane permeability transition that dissipates the intermembrane proton gradient, leading to cell death through loss of ATP generation; (2) intracellular signaling from both intrinsic and extrinsic sources can also result in the formation of oligomerized Bax and Bak protein pores on mitochondrial outer membrane permeabilization (MOMP) that facilitate release of cytochrome C from the electron transport chain proteins. Cytosolic cytochrome C stabilizes the multisubunit apoptosome to promote caspase activation and ultimately apoptotic cell death. TCA, Tricarboxylic acid. lead to oxidative stress, characterized by increases in intracellular reactive oxygen species. • Intermediate metabolism. Oxidative phosphorylation efficiently generates 36 to 38 ATP molecules per glucose molecule, but also “burns” substrates to their core CO2 and H2O, leaving no carbon moieties to use for building lipids and proteins. Consequently, to ensure the necessary building blocks for growth, rapidly dividing cells (both benign and malignant) increase their uptake of glucose and glutamine and switch to aerobic glycolysis, a phenomenon called the Warburg effect. In that setting, each glucose molecule is catabolized to lactic acid (even in the presence of adequate oxygen), generating only two net ATP molecules but “spinning off” intermediates that can be converted into new lipids, amino acids and proteins, and nucleic acids. Thus mitochondrial metabolism can be modulated to support either cellular energy maintenance or cellular proliferation. • Cell death. In addition to providing the ATP that enables the bulk of cellular activity, mitochondria are fundamental to cell survival. The role of mitochondria in cell death is detailed in Chapter 2 and briefly mentioned here. • Necrosis: External cellular injury (toxin, ischemia, trauma) can damage mitochondria, inducing the formation of mitochondrial permeability transition pores in the outer membrane. These channels allow the dissipation of the proton gradient so that subsequent mitochondrial ATP generation becomes impossible and the cell dies. • Apoptosis: Programmed cell death is a central feature of normal tissue development and turnover and can be triggered by extrinsic signals (including cytotoxic T cells or inflammatory cytokines) or intrinsic pathways (including DNA damage or intracellular stress). Mitochondria integrate intracellular proapoptotic and antiapoptotic effector signals to generate a final “go” or “no go” signal for apoptosis. A net proapoptotic signal results in mitochondrial outer membrane permeabilization (MOMP)—distinct from the mitochondrial permeability transition—that releases cytochrome C (and other proteins) into the cytoplasm. In turn, these proteins activate intracellular programmed cell death pathways. Notably, a failure of normal proapoptotic signaling (or too many antiapoptotic effectors) can underlie malignancy—even in the face of mutations that would otherwise trigger cellular suicide. Conversely, too strong an apoptotic signal (or lack of antiapoptotic effectors) may lead to premature cell death, as in neurodegenerative disorders (Chapter 28). CELLULAR ACTIVATION Intercellular communication is essential to multicellular organisms. At the most basic level, extracellular signals may determine whether a cell lives or dies, remains quiescent, Cellular activation or becomes active to perform its specific functions. Intercellular signaling is critical in the developing embryo and in maintaining tissue organization; it is also important in adult organisms, where intercellular signaling assures that all tissues act in appropriate concert (e.g., in response to local tissue trauma or a systemic infection). Loss of intercellular communication and “social controls” can also be reflected in unregulated growth (e.g., cancer) or in detrimental responses to extrinsic stress (e.g., shock). Cell Signaling Individual cells are chronically exposed to a remarkable cacophony of signals that must be integrated into rational output; some signals may induce a given cell type to differentiate, while others signal proliferation or direct the cell to perform specialized functions. Multiple signals at once, in a certain ratio, may trigger yet another totally unique response. Many cells require certain input just to continue living; in the absence of appropriate exogenous signals, they die by apoptosis. The signals that most cells respond to can be classified into several groups. • Danger and pathogens. Many cells have an innate capacity to sense and respond to damaged cells (danger signals), as well as foreign invaders such as microbes. The involved receptors are discussed in Chapters 3 and 6. • Cell-cell contacts, mediated through adhesion molecules and/or gap junctions. As mentioned previously, gap junction signaling is accomplished between adjacent cells via hydrophilic connexon channels that permit the movement of small ions (e.g., calcium), metabolites, and second messenger molecules (e.g., cAMP). • Cell-ECM contacts, mediated through integrins. We will return to a consideration of integrins in the context of leukocyte attachment to other cells during inflammation in Chapter 3. • Secreted molecules. The most important secreted molecules include growth factors (discussed later); cytokines, a term reserved for mediators of inflammation and immune responses (discussed also in Chapters 3 and 6); and hormones, which are secreted by endocrine organs (Chapter 24). Signaling pathways can also be classified based on the spatial relationships between the sending and receiving cells. • Paracrine signaling. Only cells in the immediate vicinity are affected. To accomplish this, there can be only minimal diffusion, after which the secreted signal is rapidly degraded, taken up by other cells, or trapped in the ECM. • Autocrine signaling occurs when molecules secreted by a cell affect that same cell. This can be a means to entrain groups of cells undergoing synchronous differentiation during development, or it can be used to amplify (positive feedback) or dampen (negative feedback) a particular response. • Synaptic signaling. Activated neurons secrete neurotransmitters at specialized cell junctions (i.e., synapses) onto target cells. • Endocrine signaling. A mediator is released into the bloodstream and acts on target cells at a distance. Regardless of the nature of an extracellular stimulus (paracrine, autocrine, synaptic, or endocrine), the signal that is conveyed is transmitted to the cell via specific receptor proteins. Signaling molecules (ligands) bind their respective receptors and initiate a cascade of intracellular events culminating in the desired cellular response. Ligands usually have high affinities for their receptors (≤10−8 M) and, at physiologic concentrations, bind receptors with exquisite specificity. Receptors may be present on the cell surface or within the cell (Fig. 1.12). • Intracellular receptors include transcription factors that are activated by lipid-soluble ligands that can easily transit plasma membranes; vitamin D and steroid hormones that activate nuclear hormone receptors are good examples. In other cases a small and/or nonpolar signaling ligand produced by one cell type can influence the activity of adjacent cells. Thus