The Cell as a Unit of Health and Disease PDF

See TARGETED THERAPY available online at www.studentconsult.com C H A P T E R The Cell as a Unit of Health and Disease Richard N. Mitchell...

See TARGETED THERAPY available online at www.studentconsult.com C H A P T E R The Cell as a Unit of Health and Disease Richard N. Mitchell 1 CHAPTER CONTENTS The Genome 1 Cytoskeleton 11 Modular Signaling Proteins, Hubs, and Noncoding DNA 1 Cell-Cell Interactions 12 Nodes 19 Histone Organization 3 Biosynthetic Machinery: Endoplasmic Transcription Factors 19 Micro-RNA and Long Noncoding RNA 4 Reticulum and Golgi 13 Growth Factors and Receptors 20 Micro-RNA 4 Waste Disposal: Lysosomes and Extracellular Matrix 21 Long Noncoding RNA 5 Proteasomes 14 Components of the Extracellular Gene Editing 6 Cellular Metabolism and Matrix 23 Cellular Housekeeping 6 Mitochondrial Function 15 Maintaining Cell Populations 25 Plasma Membrane: Protection and Cellular Activation 16 Proliferation and the Cell Cycle 25 Nutrient Acquisition 8 Cell Signaling 17 Stem Cells 28 Membrane Transport 9 Signal Transduction Pathways 17 Regenerative Medicine 29 Pathology literally translates as the study of suffering (Greek breathtaking level of complexity far beyond the linear pathos = suffering, logos = study); more prosaically, and as sequence of the genome. The potential of these powerful applied to modern medicine, it is the study of disease. Virchow innovations to explain disease pathogenesis and drive was prescient in asserting that disease originates at the therapeutic discovery excites and inspires scientists and the cellular level, but we now appreciate that cellular pathologies lay public alike. arise from perturbations in molecules (genes, proteins, and metabolites) that influence cell survival and behaviors. Thus Noncoding DNA the foundation of modern pathology is understanding the cellular and molecular aberrations that give rise to diseases. The human genome contains some 3.2 billion DNA It is illuminating to consider these abnormalities in the base pairs. Yet, within the genome there are only about context of normal cellular structure and function, which is 20,000 protein-encoding genes, constituting just 1.5% the subject of this introductory chapter. of the genome. These are the blueprints that instruct the It is unrealistic (and even undesirable) to condense the assembly of the enzymes, structural elements, and signaling vast and fascinating field of cell biology into a single chapter. molecules within the 50 trillion cells that make up the human Consequently, rather than attempting a comprehensive body. Although 20,000 underestimates the actual number review, the goal here is to survey basic principles and of encoded proteins (many genes produce multiple RNA highlight recent advances that are relevant to the mechanisms transcripts that translate to different protein isoforms), it of disease that are emphasized throughout the rest of the is nevertheless startling to realize that worms, which are book. 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 THE GENOME molecules expressed in humans. What then separates humans from worms? The sequencing of the human genome at the beginning of The answer is not completely known, but evidence the 21st century represented a landmark achievement of suggests that much of the difference lies in the 98.5% of biomedical science. Since then the rapidly declining cost the human genome that does not encode proteins. The of sequencing, the burgeoning computational capacity to function of such long stretches of DNA (so-called genome mine the ensuing data, and the expanding toolkits to analyze “dark matter”) was mysterious for many years. However, functional outputs (genomics, proteomics, and metabolomics) over 85% of the human genome is ultimately transcribed; promise to revolutionize our understanding of health and nearly 80% is devoted to regulation of gene expression. It disease. The emerging information has also revealed a 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 organ- Special structural regions of DNA, in particular, telomeres isms, it is the noncoding regions of the genome that provide (chromosome ends) and centromeres (chromosome the critical “architectural planning.” Practically stated, the “tethers”). A major component of centromeres is so-called difference between worms and humans apparently lies satellite DNA, consisting of large arrays—up to megabases more in the genomic “blueprints” than in the construction in length—of repeating sequences (from 5 bp up to 5 kb). materials. Although classically associated with spindle apparatus There are five major classes of functional non–protein- attachment, satellite DNA is also important in maintaining coding sequences in the human genome (Fig. 1.1): the dense, tightly packed organization of heterochromatin Promoter and enhancer regions that provide binding sites (discussed later). for transcription factors. Binding sites for factors that organize and maintain higher Many genetic variations (polymorphisms) associated with order chromatin structures. diseases are located in non–protein-coding regions of the Noncoding regulatory RNAs. Over 60% of the genome genome. Thus variation in gene regulation may prove to is transcribed into RNAs that are never translated be more important in disease causation than structural but regulate gene expression through a variety of mecha- changes in specific proteins. Another surprise that emerged nisms. The two best-studied varieties—micro-RNAs from genome sequencing is that any two humans are typically (miRNAs) and long noncoding RNAs (lncRNAs)—are more than 99.5% DNA-identical (and are 99% sequence- described later. identical with chimpanzees)! Thus individual variation, Mobile genetic elements (e.g., transposons) make up more including differential susceptibility to diseases and envi- than a third of the human genome. These “jumping genes” ronmental stimuli, is encoded in less than 0.5% of our DNA can move around the genome during evolution, resulting (representing about 15 million bp). in variable copy number and positioning even among The two most common forms of DNA variation in the closely related species (e.g., humans and other primates). human genome are single nucleotide polymorphisms Although implicated in gene regulation and chromatin (SNPs) and copy number variations (CNVs). organization, the function of mobile genetic elements is SNPs are variants at single nucleotide positions and are not well established. almost always biallelic (only two choices exist at a given Heterochromatin Nucleolus Heterochromatin Euchromatin Euchromatin Nucleus (dense, inactive) (disperse, active) Nucleosome DNA Transcription Promoter Exon Exon Enhancer Exon Pre- Cell mRNA Intron Intron Splicing Intron p arm q arm mRNA Telomeres 5’ UTR 3’ UTR Open-reading frame Centromere Translation Chromosome 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 3 site within the population, such as A or T). Over 6 million thread, the entire genome can be packed into a nucleus human SNPs have been identified, with many showing as small as 7 to 8 μm in diameter. In most cases, this wide variation in frequency in different populations. structured DNA, termed chromatin, is not wound uni- SNPs occur across the genome—within exons, introns, formly. Thus at the light microscopic level, nuclear intergenic regions, and coding regions. chromatin is recognizable as cytochemically dense and Roughly 1% of SNPs occur in coding regions, which transcriptionally inactive heterochromatin and disperse, is about what would be expected by chance, since transcriptionally active euchromatin (see Fig. 1.1). In coding regions comprise about 1.5% of the genome. general, only the regions that are “unwound” are available SNPs located in noncoding regions can occur within for transcription. Chromatin structure can therefore regu- genomic regulatory elements, thereby altering gene late transcription independent of traditional promoters expression; in such instances, SNPs influence disease and DNA-binding elements and, due to variations between susceptibility directly. cell types, helps to define cellular identity and activity. Some SNPs, termed “neutral” variants, are thought Histones are not static, but rather are highly dynamic to have no effect on gene function or individual structures regulated by a host of nuclear proteins. phenotype. Thus chromatin remodeling complexes can reposition Even “neutral” SNPs may be useful markers if they nucleosomes on DNA, exposing (or obscuring) gene happen to be coinherited with a disease-associated regulatory elements such as promoters. “Chromatin polymorphism as a result of physical proximity. In writer” complexes, on the other hand, carry out over 70 other words, the SNP and the causative genetic factor different histone modifications generically denoted as are in linkage disequilibrium. “marks.” Such covalent alterations include methylation, The effect of most SNPs on disease susceptibility is acetylation, or phosphorylation of specific amino acids weak, and it remains to be seen if identification of within histones. such variants, alone or in combination, can be used Actively transcribed genes in euchromatin are associ- to develop effective strategies to identify those at risk ated with histone marks that make the DNA accessible and, ultimately, prevent disease. to RNA polymerases. In contrast, inactive genes have CNVs are a form of genetic variation consisting of different histone marks that enable DNA compaction into hetero- numbers of large contiguous stretches of DNA; these chromatin. Histone marks are reversible through the can range from 1000 base pairs to millions of base pairs. activity of “chromatin erasers.” Still other proteins CNVs can be biallelic and simply duplicated or, alterna- function as “chromatin readers,” binding histones that tively, deleted in some individuals. At other sites there bear particular marks and thereby regulating gene are complex rearrangements of genomic material, with expression. multiple variants in the human population. Histone methylation. Both lysines and arginines can be CNVs are responsible for between 5 million and 24 methylated by specific writer enzymes; methylation of million base pairs of sequence difference between any histone lysine residues can lead to transcriptional activa- two individuals. tion or repression, depending on which histone residue Approximately 50% of CNVs involve gene-coding is marked. sequences; thus CNVs may underlie a large portion Histone acetylation. Lysine residues are acetylated by of human phenotypic diversity. histone acetyltransferases (HATs), whose modifications It is important to note that alterations in DNA sequence tend to open the chromatin and increase transcription. cannot by themselves explain the diversity of phenotypes In turn, these changes can be reversed by histone deacety- in human populations; moreover, classic genetic inheritance lases (HDACs), leading to chromatin condensation. cannot explain differing phenotypes in monozygotic twins. Histone phosphorylation. Serine residues can be modified The answers to these conundrums probably lie in epigenetics— by phosphorylation; depending on the specific residue, heritable changes in gene expression that are not caused the DNA may be opened for transcription or condensed by variations in DNA sequence (see the following section). and inactive. DNA methylation. High levels of DNA methylation in Histone Organization gene regulatory elements typically result in transcriptional silencing. Like histone modifications, DNA methylation Even though virtually all cells in the body have the same is tightly regulated by methyltransferases, demethylating genetic composition, differentiated cells have distinct enzymes, and methylated-DNA-binding proteins. structures and functions that arise as a result of lineage- Chromatin organizing factors. Much less is known about specific gene expression programs. Such cell type–specific these proteins, which are believed to bind to noncoding differences in transcription and translation depend on regions and control long-range looping of DNA, thus epigenetic factors (literally, factors that are “above genetics”) regulating the spatial relationships between enhancers that can be conceptualized as follows (Fig. 1.2): and promoters that control gene expression. Histones and histone-modifying factors. Nucleosomes consist of DNA segments 147 bp long that are wrapped around Deciphering the mechanisms that allow epigenetic factors a central core structure of highly conserved low molecular to control genomic organization and gene expression in a weight proteins called histones. The resulting DNA-histone cell-type-specific fashion is an extraordinarily complex complex resembles a series of beads joined by short DNA proposition. Despite the intricacies, there is already ample linkers. The naked DNA of a single human cell is about evidence that dysregulation of the “epigenome” has a central 1.8 m long. By winding around histones, like spools of role in malignancy (Chapter 7), and emerging data indicate 4 CHAPTER 1 The Cell as a Unit of Health and Disease A Core DNA DNA (1.8 turns) H2A Nucleosome Histone H4 protein H2B core H3 Linker DNA H1 Linker histone H1 Linker DNA B Heterochromatin Euchromatin (inactive) (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 have a primitive version of the same machinery that they acquired epigenetic alterations. Unlike genetic changes, many use to protect themselves against foreign DNA (e.g., phage epigenetic alterations (e.g., histone acetylation and DNA and virus DNA). The profound influence of miRNAs on methylation) are reversible and amenable to therapeutic protein expression allows these relatively short RNAs (22 intervention; HDAC and DNA methylation inhibitors are nucleotides on average) to be critical regulators of devel- already being tested in the treatment of various forms of opmental pathways as well as pathologic conditions (e.g., cancer. cancer). The human genome encodes almost 6000 miRNA genes, Micro-RNA and Long Noncoding RNA about 30% of the total number of protein-coding genes. Individual miRNAs can regulate multiple protein-coding Genes can also be regulated by noncoding RNAs. These genes, allowing each miRNA to coregulate entire programs genomic sequences are transcribed but not translated. of gene expression. Transcription of miRNA genes produces Although many distinct families of noncoding RNAs exist, a primary transcript (pri-miRNA) that is processed into we will discuss only two examples here: small RNA molecules progressively smaller segments, including trimming by the called microRNAs (miRNAs) and long noncoding RNAs enzyme Dicer. This generates mature single-stranded (lncRNAs) (>200 nucleotides in length). miRNAs of 21 to 30 nucleotides that associate with a multiprotein aggregate called RNA-induced silencing complex Micro-RNA (RISC) (Fig. 1.3). Subsequent base pairing between the miRNAs do not encode proteins; they modulate translation miRNA strand and its target mRNA directs the RISC to of target messenger RNAs (mRNAs). Posttranscriptional either induce mRNA cleavage or repress its translation. In silencing of gene expression by miRNA is a fundamental this way the target mRNA is posttranscriptionally silenced. and well-conserved mechanism of gene regulation present Small interfering RNAs (siRNAs) are short RNA sequences in all eukaryotes (plants, animals, and fungi). Even bacteria that can be introduced experimentally into cells where they  The genome 5 serve as substrates for Dicer and interact with RISC, thereby miRNA gene reproducing endogenous miRNAs function. Synthetic siRNAs that target specific mRNA species are powerful laboratory tools to study gene function (so-called knockdown technol- ogy) and are also being studied as potential therapeutic agents to silence pathogenic genes (e.g., oncogenes that drive pri-miRNA neoplastic transformation). Long Noncoding RNA Target gene 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 pre-miRNA modulate gene expression by several mechanisms (Fig. 1.4). Export As one example, lncRNAs can bind to chromatin and restrict protein 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 pre-miRNA physiologic X chromosome inactivation that occurs in Dicer A. Gene activation Ribonucleoprotein transcription complex lncRNA Target mRNA miRNA Gene activation Unwinding of miRNA duplex Decoy lncRNA RISC B. Gene suppression complex Gene suppression Imperfect Perfect match match Target mRNA Translational mRNA repression cleavage C. Promote chromatin modification Methylation, acetylation Ribosome GENE SILENCING D. Assembly of protein complexes Act on chromatin structure 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 Multi-subunit complex of the nucleus via specific transporter proteins, pre-miRNA is trimmed by the cytoplasmic Dicer enzyme to generate mature double-stranded Figure 1.4 Roles of long noncoding RNAs (lncRNAs). (A) lncRNAs can miRNAs of 21 to 30 nucleotides. The miRNA subsequently unwinds and facilitate transcription factor binding and thus promote gene activation. the single strands are incorporated into multiprotein RNA-induced (B) Conversely, lncRNAs can preemptively bind transcription factors to silencing complexes (RISC). Base pairing between single-stranded miRNA inhibit transcription. (C) Histone and DNA modification by acetylases or and the targeted messenger RNA (mRNA) directs RISC to either cleave methylases (or deacetylases and demethylases) may be directed by or repress translation of the mRNA, resulting in posttranscriptiontional lncRNA binding. (D) In other instances, lncRNAs can act as scaffolds to silencing. 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.) 6 CHAPTER 1 The Cell as a Unit of Health and Disease females. XIST itself escapes X inactivation but forms a Homologous gRNA sequence repressive “cloak” on the X chromosome from which it is transcribed, resulting in gene silencing. Conversely, many Cas9 enhancers are actually sites of lncRNA synthesis. In this protein gRNA case the lncRNAs expand transcription from gene promoters via a variety of mechanisms (see Fig. 1.4). Gene Editing An exciting new development that allows high-fidelity Cleavage genome editing may usher in the next era of the molecular revolution. This advance comes from a wholly unexpected Double-stranded source: the discovery of clustered regularly interspaced short DNA palindromic repeats (CRISPRs) and CRISPR-associated genes Target genomic (Cas), such as the Cas9 nuclease. These are linked genetic sequence elements that endow prokaryotes with a form of acquired immunity to phages and plasmids. Bacteria use the system Double-stranded DNA break 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 NHEJ 20-base guide RNAs (gRNAs) that bind Cas9 and are HDR 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 nonhomolo- gous end joining) or can introduce new genetic material Insertion/ Donor DNA with precision (by homologous recombination). Both the deletion guide sequences and the Cas enzyme, either as a coding DNA with random mutation DNA with specific mutation DNA (cDNA) or a protein, can be easily introduced into Figure 1.5 Gene editing with clustered regularly interspersed short cells. The potential application to genetic engineering, due palindromic repeats (CRISPRs) and the nuclease Cas9. In bacteria, DNA to the impressive specificity of the Cas9 system (up to sequences consisting of CRISPRs are transcribed into guide RNAs (gRNAs) 10,000-fold better than other previous editing systems), has with a constant region and a variable sequence of approximately 20 bases. led to great excitement. Applications include inserting specific The gRNA constant regions bind to Cas9, while the variable regions form mutations in cells and tissues to model cancers and other heteroduplexes with homologous DNA sequences of interest; the Cas9 diseases and rapidly generating transgenic animal models nuclease then cleaves the bound DNA to produce a double-stranded DNA break. In nature, bacteria use the CRISPR/Cas9 system to protect from edited embryonic stem cells. CRISPR also makes it against phages and plasmids; CRISPR sequences from previous assaults are possible to selectively edit mutations that cause hereditable transcribed into gRNA from the bacterial genome. These bind to pathogen disease, or—perhaps more worrisome—to just eliminate nucleotide sequences and form a complex with the Cas9 nuclease that less “desirable” traits. Predictably the technology has inspired leads to cleavage and, ultimately, destruction of the invader’s DNA. a vigorous debate regarding the ethics of its use. 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 double- CELLULAR HOUSEKEEPING stranded break is repaired by nonhomologous end-joining (NHEJ), an error-prone mechanism that typically introduces disruptive insertions or Normal functioning and intracellular homeostasis depend deletions (indels). Conversely, in the presence of homologous “donor” on a variety of fundamental cell housekeeping functions that DNA that spans the region target by the CRISPR/Cas9 complex, cells all differentiated cells must perform to maintain viability instead can use homologous DNA recombination (HDR) to repair the break. HDR is less efficient than NHEJ but has the capacity to introduce and normal activity. These include protection from the precise changes in DNA sequence. Potential applications of CRISPR/Cas9 environment, nutrient acquisition, metabolism, communica- coupled with HDR include repair of inherited genetic diseases and the tion, movement, renewal of senescent molecules, molecular creation of pathogenic mutations in inducible pluripotent stem cells. catabolism, and energy generation. Many of the normal housekeeping functions of the cell are compartmentalized within membrane bound intracel- compartmentalization also allows the creation of unique lular organelles (Fig. 1.6). By isolating certain cellular intracellular environments (e.g., low pH or high calcium) functions within distinct compartments, potentially injurious that permit more efficient functioning of certain enzymes degradative enzymes or toxic metabolites can be kept at or metabolic pathways. usefully high concentrations without risking damage to New proteins destined for the plasma membrane or more delicate intracellular constituents. Moreover, secretion are physically assembled in the rough endoplasmic  Cellular housekeeping 7 Relative volumes of intracellular organelles (hepatocyte) Compartment % total volume number/cell role in the cell Cytosol 54% 1 metabolism, transport, protein translation Mitochondria 22% 1700 energy generation, apoptosis Rough ER 9% 1 synthesis of membrane and export protein Smooth ER, Golgi 6% 1 protein modification, sorting, catabolism Nucleus 6% 1 cell regulation, proliferation, DNA transcription Endosomes 1% 200 intracellular transport and export Lysosomes 1% 300 cellular catabolism Peroxisomes 1% 400 very long-chain fatty acid metabolism Rough Free endoplasmic ribosomes reticulum Nucleolus Golgi Nucleus apparatus Lysosome Mitochondrion Endosome Smooth Cytoskeleton endoplasmic Plasma reticulum membrane Peroxisome Centrioles Microtubules 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 degradation of regulatory proteins or transcription factors cytosol are synthesized on free ribosomes. Smooth endoplasmic can trigger initiation or suppression of signaling pathways. reticulum (SER) is used for steroid hormone and lipoprotein Lysosomes are intracellular organelles containing degrada- synthesis and modification of hydrophobic compounds into tive enzymes that permit digestion of a wide range of water-soluble molecules for export. macromolecules, including proteins, polysaccharides, Cells catabolize the wide variety of molecules that they lipids, and nucleic acids. They are the site of senescent endocytose, as well as the entire repertoire of their own intracellular organelle breakdown (a process called proteins and organelles—all of which are constantly being autophagy) and where phagocytosed microbes are killed degraded and renewed. Breakdown of these constituents and catabolized. takes place at three different sites, ultimately serving different Peroxisomes contain catalase, peroxidase, and other oxida- functions. tive enzymes; they play a specialized role in the break- Proteasomes are “disposal” complexes that degrade down of very long-chain fatty acids, generating hydrogen denatured or otherwise “tagged” cytosolic proteins. In peroxide in the process. antigen-presenting cells, the resulting short peptides are presented in the context of class I or class II major The contents and location of cellular organelles are also histocompatibility molecules to help drive the adaptive highly regulated. Endosomal vesicles shuttle internalized immune response (Chapter 6). In other cases, proteasomal material to the appropriate intracellular site(s), while other 8 CHAPTER 1 The Cell as a Unit of Health and Disease membrane-bound vesicles direct newly synthesized materials about 10 days), mechanisms must also exist that allow for to the cell surface or specific organelles. Movement—of both the recognition and degradation of “worn-out” cellular organelles and proteins within the cell, as well as the entire components. cell in its environment—is accomplished by the cytoskeleton, With this as a primer, we will now move on to discuss which is composed of filamentous actin (microfilaments), cellular components and their function in greater detail. keratins (intermediate filaments), and microtubules. These structural proteins also maintain cellular shape and intracel- Plasma Membrane: Protection and lular organization, which are essential to generation and Nutrient Acquisition maintenance of cell polarity. This is particularly important in epithelium where the top of the cell (apical) and the bottom Plasma membranes (and all other organellar membranes for and sides of the cell (basolateral) are exposed to different that matter) are more than just static lipid sheaths. Rather, environments and have distinct functions. Loss of polarity they are fluid bilayers of amphipathic phospholipids— could, for example, disrupt vectorial transcellular transport hydrophilic head groups that face the aqueous environment in the intestine or renal tubule. and hydrophobic lipid tails that interact with each other Cell growth and maintenance require a constant supply to form a barrier to passive diffusion of large or charged of both energy and the building blocks that are needed for molecules (Fig. 1.7). The bilayer has a remarkably heteroge- synthesis of macromolecules. Most of the adenosine tri- neous composition of different phospholipids that vary by phosphate (ATP) that powers cells is generated via mito- location and are also asymmetric—that is, membrane lipids chondrial oxidative phosphorylation. Mitochondria also serve preferentially associate with extracellular or cytosolic faces. as an important source of metabolic intermediates needed Proper localization of these molecules is important for cell for anabolic metabolism, are sites of synthesis of certain health. For example, specific phospholipids interact with macromolecules (e.g., heme), and contain important sensors particular membrane proteins and modify their distributions of cell damage that can initiate and regulate programmed and functions. cell death (e.g., apoptosis). Phosphatidylinositol on the inner membrane leaflet can be In growing and dividing cells, all of these organelles phosphorylated, serving as an electrostatic scaffold for have to be replicated (organellar biogenesis) and correctly intracellular proteins; alternatively, polyphosphoinositides apportioned in daughter cells following mitosis. More- can be hydrolyzed by phospholipase C to generate over, because the macromolecules and organelles have intracellular second signals like diacylglycerol and inositol finite lifespans (mitochondria, for example, last only trisphosphate. Extracellular Glycosylphosphatidylinositol Outside protein (GPI) linked protein Glycolipids Phosphatidyl- Sphingo- Lipid choline myelin raft P (outer mostly) (outer mostly) P Phosphatidyl- Phosphatidyl- Phosphatidyl- Cholesterol ethanolamine serine inositol (both faces) (inner mostly) (inner mostly) (both faces) Transmembrane proteins Cytosolic Cytoplasm protein Lipid-linked protein A B 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 9 Phosphatidylserine is normally restricted to the inner face composition. The latter strategy is used to maintain cell where it confers a negative charge involved in electrostatic polarity (e.g., top/apical/free vs. bottom/basolateral/ protein interactions; however, when flipped to the bound to extracellular matrix [ECM]) in epithelial cells. extracellular leaflet, it becomes a potent “eat me” signal Interactions of other membrane and cytosolic proteins with during programmed cell death (e.g., apoptosis). In one another and the cytoskeleton also contributes to cell platelets, phosphatidylserine is also a cofactor in blood polarity. clotting. The extracellular face of the plasma membrane is dif- Glycolipids and sphingomyelin are preferentially located fusely decorated by carbohydrates, not only as complex on the extracellular face; glycolipids, including ganglio- oligosaccharides on glycoproteins and glycolipids, but also sides with complex sugar linkages and terminal sialic as polysaccharide chains attached to integral membrane acids that confer negative charges, support charge-based proteoglycans. This glycocalyx can form a chemical and interactions that contribute to including inflammatory mechanical barrier. cell recruitment and sperm-egg fusion. Membrane Transport Despite substantial lateral fluidity, some membrane Although the barrier provided by plasma membranes is constituents concentrate into specialized domains (e.g., lipid critical, transport of selected molecules across the lipid bilayer rafts) that are enriched in glycosphingolipids and cholesterol. or to intracellular sites via vesicular transport is essential. Since inserted membrane proteins have different intrinsic Several mechanisms contribute to this transport. solubilities in domains with distinct lipid compositions, this membrane organization also impacts protein distribution. Passive Diffusion. Small, nonpolar molecules like O2 and This geographic organization of plasma membrane compo- CO2 readily dissolve in lipid bilayers and therefore rapidly nents impacts cell-cell and cell-matrix interactions, intracel- diffuse across them. Larger hydrophobic molecules, (e.g., lular signaling, and the specialized sites of vesicle budding steroid-based molecules like estradiol or vitamin D) can or fusion. also cross lipid bilayers with relative impunity. While The plasma membrane is liberally studded with a small polar molecules such as water (18 Da) can also diffuse variety of proteins and glycoproteins involved in (1) ion across membranes at low rates, in tissues responsible for and metabolite transport; (2) fluid-phase and receptor- significant water movement (e.g., renal tubular epithelium), mediated uptake of macromolecules; and (3) cell-ligand, special integral membrane proteins called aquaporins form cell-matrix, and cell-cell interactions. The means by which transmembrane channels for water, H2O2, and other small these proteins associate with membranes frequently reflects molecules. In contrast, the lipid bilayer is an effective barrier function. For example, multiple transmembrane-spanning to the passage of larger polar molecules (>75 Da); at 180 Da, proteins are often pores or molecular transporters, while for example, glucose is effectively excluded. Lipid bilay- proteins that are superficially attached to the membrane via ers are also impermeant to ions due to their charge and labile linkages are more likely to participate in signaling. hydration. In general, proteins associate with the lipid bilayer by one of four mechanisms. Carriers and Channels (Fig. 1.8). Plasma membrane Most proteins are integral or transmembrane proteins, transport proteins are required for uptake and secretion of having one or more relatively hydrophobic α-helical ions and larger molecules that are required for cellular segments that traverse the lipid bilayer. function (e.g., nutrient uptake and waste disposal). Ions Proteins synthesized on free ribosomes in the cytosol and small molecules can be transported by channel proteins may be modified posttranslationally by addition of prenyl and carrier proteins. Similar pores and channels also mediate groups (e.g., farnesyl, related to cholesterol) or fatty acids transport across organellar membranes. These transporters (e.g., palmitic or myristic acid) that insert into the cytosolic that move ions, sugars, nucleotides, etc., frequently have side of the plasma membrane. exquisite specificities, and can be either active or passive Proteins on the extracellular face of the membrane may (see below). For example, some transporters accommodate be anchored by glycosylphosphatidylinositol (GPI) tails glucose but reject galactose. that are added posttranslationally. Channel proteins create hydrophilic pores, which, when Peripheral membrane proteins may noncovalently associ- open, permit rapid movement of solutes (usually restricted ate with true transmembrane proteins. by size and charge). Carrier proteins bind their specific solute and undergo a Many plasma membrane proteins function as large series of conformational changes to transfer the ligand complexes; these may be aggregated either under the control across the membrane; their transport is relatively slow. of chaperone molecules in the RER or by lateral diffusion in the plasma membrane, followed by complex formation Solute transport across the plasma membrane is frequently in situ. For example, many protein receptors (e.g., cytokine driven by a concentration and/or electrical gradient between receptors) dimerize or trimerize in the presence of ligand the inside and outside of the cell via passive transport (virtually to form functional signaling units. Although lipid bilayers all plasma membranes have an electrical potential difference are fluid within the plane of the membrane, components across them, with the inside negative relative to the outside). can be confined to discrete domains. This can occur by In other cases, active transport of certain solutes (against a localization to lipid rafts (discussed earlier) or through concentration gradient) is accomplished by carrier molecules intercellular protein-protein interactions (e.g., tight junctions) (never channels) at the expense of ATP hydrolysis or a that establish discrete boundaries and also have unique lipid coupled ion gradient. For example, most apical nutrient 10 CHAPTER 1 The Cell as a Unit of Health and Disease Extracellular Carrier Channel Endocytosis Exocytosis Phagocytosis Transcytosis Energy Caveolae- Receptors Receptor- Microbe mediated mediated Membrane Cytosol Coated pit Receptor Caveolin recycling Lysosome Coated vesicle Phagosome Early endosome (low pH) Reconstitution Phagolysosome Late endosome Undigested Lysosome-late residual material endosome fusion vesicle 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 counterions that increase intracellular osmolarity. Thus to extracellular to intracellular Na+ gradient to allow absorption prevent overhydration, cells must constantly pump out small even when intracellular nutrient concentrations exceed inorganic ions (e.g., Na+)—typically through the activity of extracellular concentrations. This form of active transport the membrane ion-exchanging ATPase. Loss of the ability does not use ATP directly, but depends on the Na+ gradient to generate energy (e.g., in a cell injured by toxins or generated by Na+-Ka+ ATPase. Other transporters are ischemia) therefore results in osmotic swelling and eventual ATPases. One example is the multidrug resistance (MDR) cell rupture. Similar transport mechanisms also regulate protein, which pumps polar compounds (e.g., chemothera- concentrations of other ions (e.g., Ca2+ and H+). This is critical peutic drugs) out of cells and may render cancer cells resistant to many processes. For example, cytosolic enzymes are most to treatment. active at pH 7.4 and are often regulated by Ca2+, whereas Water movement into or out of cells is passive and lysosomal enzymes function best at pH 5 or less. directed by solute concentrations. Thus extracellular salt in Uptake of fluids or macromolecules by the cell is called excess of that in the cytoplasm (hypertonicity) causes net endocytosis. Depending on the size of the vesicle, endocytosis movement of water out of cells, while hypotonicity causes may be denoted pinocytosis (“cellular drinking”) or phago- net movement of water into cells. Conversely, the charged cytosis (“cellular eating”). Generally, phagocytosis is metabolites and proteins within the cytoplasm attract charged restricted to certain cell types (e.g., macrophages and  Cellular housekeeping 11 neutrophils) whose role is to specifically ingest invading signaling. Defects in receptor-mediated transport of LDL organisms or dead cell fragments. underlie familial hypercholesterolemia, as described in Chapter 5. Receptor-Mediated and Fluid-Phase Uptake (see Fig. 1.8) Endocytosis requires recycling of internalized vesicles Certain small molecules—including some vitamins—bind back to the plasma membrane (exocytosis) for another round to cell-surface receptors and are taken up through invagi- of ingestion. This is critical, as a cell will typically ingest nations of the plasma membrane called caveolae. Uptake from the extracellular space the equivalent of 10% to 20% can also occur through membrane invaginations coated by of its own cell volume each hour—amounting to 1% to 2% an intracellular matrix of clathrin proteins that spontane- of its plasma membrane each minute! Without recycling, ously assemble into a basket-like lattice which helps drive the plasma membrane would be rapidly depleted. Endo- endocytosis (discussed more later). In both cases, activity cytosis and exocytosis must therefore be tightly coupled to of the “pinchase” dynamin is required for vesicle release. avoid large changes in plasma membrane area. Macromolecules can also be exported from cells by exocytosis. In this process, proteins synthesized and packaged Cytoskeleton within the RER and Golgi apparatus are concentrated in secretory vesicles, which then fuse with the plasma mem- The ability of cells to adopt a particular shape, maintain brane to expel their contents. Common examples include polarity, organize intracellular organelles, and migrate peptide hormones (e.g., insulin) and cytokines. depends on an intracellular scaffold of structural proteins Transcytosis is the movement of endocytosed vesicles that form the cytoskeleton (Fig. 1.9). Although the cyto- between the apical and basolateral compartments of cells. skeleton can have an appearance similar to the beams and This is a mechanism for transferring large amounts of intact supports of a building, cytoskeletal structures are constantly proteins across epithelial barriers (e.g., ingested antibodies elongating and shrinking; microfilaments and microtubules in maternal milk) or for rapid movement of large solute are most active, and their assembly and disassembly can volumes. drive cell migration. We now return to the specifics of endocytosis (see Fig. 1.8). In eukaryotic cells, there are three major classes of Caveolae-mediated endocytosis. Caveolae (“little caves”) are cytoskeletal proteins. noncoated plasma membrane invaginations associated Actin microfilaments are 5- to 9-nm diameter fibrils formed with GPI-linked molecules, cyclic adenosine monophos- from the globular protein actin (G-actin), the most phate (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. Internal- ization of caveolae along with bound molecules and Microvilli associated extracellular fluid is called potocytosis—literally “cellular sipping.” In addition to supporting transmem- brane delivery of some molecules (e.g., folate), caveolae Microtubules regulate transmembrane signaling and cellular adhesion Tight junction via internalization of receptors and integrins. Receptor-mediated endocytosis. Macromolecules bound to Actin microfilaments membrane receptors (such as transferrin or low-density lipoprotein [LDL] receptors) are taken up at specialized Adherins junction regions of the plasma membrane called clathrin-coated pits. The receptors are efficiently internalized by mem- Desmosome brane invaginations driven by the associated clathrin matrix, eventually pinching off to form clathrin-coated Gap junctions vesicles. Trapped within these vesicles is also a gulp of the extracellular milieu (fluid-phase pinocytosis). The vesicles Intermediate then rapidly lose their clathrin coating and fuse with an filaments acidic intracellular structure called the early endosome; the endosomal vesicles undergo progressive maturation Hemi- to late endosomes, ultimately fusing with lysosomes. In desmosome the acidic environment of the endosomes, LDL and transferrin receptors release their cargo (cholesterol and Basement membrane Integrins iron, respectively), which is then transported into the Figure 1.9 Cytoskeletal elements and cell-cell interactions. Interepithelial cytosol. adhesion involves several different surface protein interactions at tight junctions, adherens junctions, and desmosomes; adhesion to the After release of bound ligand, some receptors recycle extracellular matrix involves cellular integrins (and associated proteins) to the plasma membrane and are reused (e.g., transferrin within hemidesmosomes. The various adhesion proteins within the plasma membrane associate with actin microfilaments and intermediate filaments and LDL receptors), while others are degraded within to provide a mechanical matrix for cell structure and signaling. Gap lysosomes (e.g., epidermal growth factor receptor). In junctions do not impart structural integrity but allow cell-cell the latter case, degradation after internalization results communication by the movement of small molecular weight and/or in receptor downregulation that limits receptor-mediated charged species. See text for details. 12 CHAPTER 1 The Cell as a Unit of Health and Disease abundant cytosolic protein in cells. G-actin monomers Mediate sister chromatid segregation during mitosis. noncovalently polymerize into long filaments (F-actin) Form the core of primary cilia, single nonmotile projec- that intertwine to form double-stranded helices with a tions on many nucleated cells that contribute to the defined polarity. Although the details are (as always) regulation of cellular proliferation and differentia- more nuanced, new subunits are typically added at tion (mutations in the proteins of the primary cilia the “positive” end of the strand and removed from the complex lead to forms of polycystic kidney disease; see “negative” end—a process referred to as actin treadmill- Chapter 20). ing. Actin nucleating, binding, and regulatory proteins Can be adapted to form the core of motile cilia (e.g., organize polymerization, bundling, and branching to in bronchial epithelium) or flagella (in sperm). form networks that control cell shape and movement. This complex and its association with motor proteins Cell-Cell Interactions (e.g., myosin) is so precisely arrayed in skeletal and cardiac muscle that a banding pattern is apparent by Cells connect and communicate with each other via light microscopy. ATP hydrolysis by myosin slides the junctional complexes that form mechanical links and actin filaments relative to one another to cause muscle facilitate receptor-ligand interactions. Similar complexes contraction. Although less coordinated, myosins, of which also mediate interaction with the ECM. Cell-cell junctions there are many, are responsible for other functions that are organized into three basic types (see Fig. 1.9): depend on actin contraction including vesicular transport, Occluding junctions (tight junctions) seal adjacent epithelial epithelial barrier regulation, and cell migration. cells together to create a continuous barrier that restricts Intermediate filaments are 10-nm diameter fibrils that the paracellular (between cells) movement of ions and comprise a large and heterogeneous family that includes other molecules. Occluding junctions form a tight meshlike keratin proteins and nuclear lamins. Intermediate fila- network (when viewed en face by freeze-fracture electron ments predominantly form ropelike polymers and do not microscopy) of macromolecular contacts between neigh- usually actively reorganize like actin and microtubules. boring cells; the complexes that mediate the cell-cell This allows intermediate filaments to provide tensile interactions are composed of transmembrane proteins strength so that cells can bear mechanical stress, e.g., in including the tetraspanning claudin and tight junction– epithelia where intermediate filaments link desmosomes associated MARVEL protein (TAMP) families. These connect and hemidesmosomes (see Fig. 1.9). Individual intermedi- to a host of intracellular adaptor and scaffolding proteins, ate filament proteins have characteristic tissue-specific including the three members of the zonula occludens protein patterns of expression that can be useful for assigning a family (ZO-1, ZO-2, ZO-3) and cingulin. Besides forming cell of origin for poorly differentiated tumors. Examples a selectively permeable barrier that seals the space include: between cells (i.e., the paracellular space), this zone also Vimentin, in mesenchymal cells (fibroblasts, represents the boundary that separates apical and baso- endothelium). lateral membrane domains and helps to maintain cellular Desmin in muscle cells forms the scaffold on which polarity. Nevertheless, these junctions are dynamic actin and myosin contract. structures that can be modified to facilitate epithelial Neurofilaments are critical for neuronal axon structure healing and inflammatory cell migration across epithelial- and confer both strength and rigidity. lined mucosal surfaces. Glial fibrillary acidic protein is expressed in glial cells. Anchoring junctions (adherens junctions and desmosomes) Cytokeratins are expressed in epithelial cells. There are mechanically attach cells—and their cytoskeletons—to at least 30 distinct different cytokeratins that are other cells or the ECM. Adherens junctions are often closely expressed in different cell lineages (e.g., lung vs. associated with and beneath tight junctions. Desmosomes gastrointestinal epithelium). are more basal and form several types of junctions. When Lamins are intermediate filament proteins that form desmosomes attach the cell to the extracellular matrix the nuclear lamina, define nuclear shape, and can (ECM) they are referred to as hemidesmosomes (half a regulate transcription. desmosome), since the other half of the desmosome is Microtubules are 25-nm-thick fibrils composed of nonco- not present within the ECM. Both adherens junctions valently polymerized α- and β-tubulin dimers organized and desmosomes are formed by homotypic extracellular into hollow tubes. These fibrils are extremely dynamic interactions between transmembrane glycoproteins called and polarized, with “+” and “−” ends. The “−” end is cadherins, on adjacent cells. typically embedded in a microtubule organizing center In adherens junctions the transmembrane adhesion (MTOC or centrosome) near the nucleus, where it is associ- molecules are associated with intracellular actin ated with paired centrioles; the “+” end elongates or microfilaments through which they can also influence recedes in response to various stimuli by the addition cell shape and/or motility. Loss of the epithelial or subtraction of tubulin dimers. Microtubules: adherens junction protein E-cadherin explains the Serve as mooring lines for molecular motor proteins discohesive invasion pattern of some gastric cancers that use ATP to translocate vesicles, organelles, or and lobular carcinomas of the breast (Chapters 17 other molecules around cells. There are two varieties and 23). of these motor proteins, kinesins and dyneins, that In desmosomes the cadherins are linked to intracellular typically (but not exclusively) transport cargo in intermediate filaments, allowing extracellular forces anterograde (− to +) or retrograde (+ to −) directions, to be mechanically communicated (and dissipated) respectively. over multiple cells.  Cellular housekeeping 13 In hemidesmosomes the transmembrane connector or oligomerize, it is retained and degraded within the proteins are called integrins; like desmosomal cadherins, ER. A good example of this is the most common mutation these attach to intermediate filaments and link the of the CFTR protein in cystic fibrosis. In mutant CFTR, cytoskeleton to the ECM. Focal adhesion complexes are a codon deletion leads to the absence of a single amino composed of >100 proteins and localize at hemides- acid (Phe508) which results in its misfolding, ER retention mosomes. Their component proteins can generate and catabolism and therefore reduced surface expression. intracellular signals when cells are subjected to shear Moreover, excess accumulation of misfolded proteins— stress (e.g., endothelium in the bloodstream or cardiac exceeding the capacity of the ER to edit and degrade myocytes in a failing heart). them—leads to the ER stress response (also called the Communicating junctions (gap junctions) permit the diffusion unfolded protein response [UPR]) (Fig. 1.10B). Detection of of chemical or electrical signals from one cell to another. excess abnormally folded proteins leads to a reduction The junction consists of a dense planar array of 1.5- to in protein synthesis overall with a concurrent increase 2-nm pores (called connexons) formed by a pair of hexam- in chaperone proteins; failure to correct the overload can ers (one on each cell) of transmembrane connexin proteins. trigger cell death through apoptosis (Chapter 2). These form pores that permit passage of ions, nucleotides, Golgi apparatus: From the RER, proteins and lipids sugars, amino acids, vitamins, and other small molecules; destined for other organelles or extracellular export are permeability of the junction is rapidly reduced by lowered shuttled into the Golgi apparatus. This consists of stacked intracellular pH or increased intracellular calcium. Gap cisternae that progressively modify proteins in an orderly junctions play a critical role in cell-cell communication. fashion from cis (near the ER) to trans (near the plasma For example, gap junctions in cardiac myocytes allow membrane). Cisternal progression, i.e., movement of cell-to-cell calcium fluxes that allow the many cells of cis-face cisternae to the trans aspect of the Golgi, akin the myocardium to behave as a functional syncytium to steps on an escalator, allows sequential processing of with coordinated waves of contraction. newly synthesized proteins and can be facilitated by small membrane-bound vesicles. Similar vesicles shuttle Golgi- Biosynthetic Machinery: Endoplasmic Reticulum resident enzymes from trans to cis in order to maintain the and Golgi Apparatus different cisternal contents along this assembly line. As cisternae mature, the N-linked oligosaccharides originally All cellular constituents—including structural proteins, added in the ER are pruned and extended in a stepwise enzymes, transcription factors, and even the phospholipid fashion; O-linked oligosaccharides (sugar moieties membranes—are constantly renewed in an ongoing process linked to serine or threonine) are also appended. Some balancing synthesis and degradation. The endoplasmic of this glycosylation is important in sorting molecules reticulum (ER) is the site for synthesis of all transmembrane to lysosomes (via the mannose-6-phosphate receptor); proteins and lipids for plasma membrane and cellular other glycosylation adducts are important for cell-cell organelles, including the ER itself. It is also the initial site or cell-matrix interactions or for clearing senescent of synthesis for secreted proteins. The ER is organized into cells (e.g., platelets and erythrocytes). In the trans Golgi a meshlike interconnected maze of branching tubes and network, proteins and lipids are sorted and dispatched flattened lamellae, forming a continuous sheet around a to other organelles, plasma membrane, or secretory single lumen that is topologically equivalent to the extracel- vesicles. The Golgi complex is especially prominent in lular environment. ER is composed of contiguous but distinct cells specialized for secretion, including goblet cells of domains that are distinguished by the presence (RER) or the intestinal or bronchial epithelium, which secrete large absence (SER) of ribosomes (see Fig. 1.6). amounts of polysaccharide-rich mucus. In plasma cells Rough endoplasmic reticulum (RER): Membrane-bound that secrete antibodies, the Golgi can be recognized as a ribosomes on the cytosolic face of RER translate mRNA perinuclear hoff on simple hematoxylin and eosin stains into proteins that are extruded into the ER lumen or become (Chapter 6). integrated into the ER membrane. This process is directed Smooth endoplasmic reticulum (SER): In most cells, the SER is by specific signal sequences on the N-termini of nascent relatively sparse and primarily exists as the transition zone proteins; synthesis of proteins with signal peptides is extending from RER to generate transport vesicles that initiated on free ribosomes, but the complex then becomes carry newly synthesized proteins to the Golgi apparatus. attached to the ER membrane, and the protein is inserted The SER may, however, be particularly conspicuous into or passed across the ER membrane as it is translated. in cells that synthesize steroid hormones (e.g., within For proteins lacking a signal sequence, translation remains the gonads or adrenals) or that catabolize lipid-soluble on free ribosomes in the cytosol, forming polyribosomes molecules (e.g., hepatocytes). Indeed, repeated exposure as multiple ribosomes attach to the mRNA; such transcribed to compounds that are metabolized by the SER (e.g., proteins remain within the cytoplasm. phenobarbital, which is catabolized by the cytochrome Proteins inserted into the ER fold into their active P-450 system) can lead to SER hyperplasia. The SER is conformation and can form polypeptide complexes also responsible for sequestering intracellular calcium, (oligomerize); in addition, disulfide bonds are formed, which, when released into the cytosol, can mediate a and N-linked oligosaccharides (sugar moieties attached number of responses to extracellular signals (including to asparagine residues) are added. Chaperone molecules apoptotic cell death). In muscle cells, specialized SER assist in folding and retaining proteins in the ER until called sarcoplasmic reticulum is responsible for the cyclic the modifications are complete and the proper conforma- release and sequestration of calcium ions that regulate tion is achieved. If a protein fails to appropriately fold muscle contraction and relaxation, respectively. 14 CHAPTER 1 The Cell as a Unit of Health and Disease A LYSOSOMAL DEGRADATION Endoplasmic reticulum Nucleus Endocytosis Endosome Senescent organs Phagocytosis Denatured proteins Lysosomes LC3 Phagosome AUTOPHAGY HETEROPHAGY Autophagosome Phagolysosome Exocytosis B PROTEASOMAL DEGRADATION Age, UV, heat, CYTOSOL reactive oxygen species Multiple Chaperones Folded Senescent or ubiquitins Nascent protein denatured peptide protein chains E1, E2, E3 ligases Peptide Proteasome fragments Free ubiquitin Metabolic alterations (e.g., pH) “ER stress” Genetic mutations (unfolded protein APOPTOSIS Viral infections response) Excess Protein synthesis ENDOPLASMIC RETICULUM misfolded protein 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 (microtubule- associated 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 The latter are responsible for catabolism of long-chained fatty acids. Although cells rely primarily on lysosomes to digest internal- Lysosomes are membrane-bound organelles containing ized material and accumulated internal waste, there are roughly 40 different acid hydrolases (i.e., that function multiple other routes to degrade intracellular macromolecules best at pH ≤5); these include proteases, nucleases, lipases, (see Fig. 1.10). These include proteosomes and peroxisomes. glycosidases, phosphatases, and sulfatases. Lysosomal  Cellular metabolism and mitochondrial function 15 enzymes are initially synthesized in the ER lumen and for example, mitochondria initiate protein synthesis with then tagged with mannose-6-phosphate (M6P) within N-formylmethionine and are sensitive to some antibacterial the Golgi apparatus. These M6P-modified proteins are antibiotics. Since mitochondria biogenesis requires a genetic subsequently delivered to lysosomes through trans Golgi contribution from preexisting mitochondria and the ovum vesicles that express M6P receptors. The other macromol- contributes the vast majority of cytoplasmic organelles in ecules destined for catabolism in the lysosomes arrive the fertilized zygote, mitochondrial DNA is almost entirely by one of three pathways (see Fig. 1.10). maternally inherited. Nevertheless, because the protein Material internalized by fluid-phase or receptor-mediated constituents of mitochondria are encoded by both nuclear endocytosis passes from plasma membrane to early and mitochondrial DNA, mitochondrial disorders may be and then late endosomes and ultimately arrives at the X-linked, autosomal, or maternally inherited. lysosome. These compartments are progressively Mitochondria are impressively dynamic, constantly acidified such that proteolytic enzymes become active undergoing fission and fusion with other newly synthesized in late endosome and lysosomes. mitochondria; this supports their renewal and defends Senescent organelles and/or large, denatured protein against degenerative changes that occur through ongoing complexes can be ferried into lysosomes by a process oxygen free radical damage. Even so, mitochondria are called autophagy (Chapter 2). Through a mechanism short-lived—being degraded through autophagy (a process involving the products of a number of autophagy- called mitophagy)—with estimated half-lives of 1 to 10 days, related (Atg) genes, obsolete organelles are corralled depending on the tissue, nutritional status, metabolic by a double membrane derived from the ER. The mem- demands, and intercurrent injury. brane progressively expands to encircle a collection Besides providing the enzymatic machinery for oxidative of organelles and cytosolic constituents, forming the phosphorylation (and thus the relatively efficient generation definitive autophagosome; these structures are targeted of energy from glucose and fatty acid substrates), mito- for eventual destruction by fusion with lysosomes. In chondria play a fundamental role in regulating apoptosis addition to facilitating turnover of aged and/or defunct (Fig. 1.11). Details of mitochondrial functions follow: structures, autophagy can be used to preserve viability Energy generation. Each mitochondrion has two separate during nutrient depletion; is involved in protective