Robbins Basic Pathology - Kumar, 10E PDF

See Targeted Therapy available online at studentconsult.com C H A P T E R The Cell as a Unit of Health and Disease...

See Targeted Therapy available online at studentconsult.com C H A P T E R The Cell as a Unit of Health and Disease 1 CHAPTER OUTLINE The Genome 1 Waste Disposal: Lysosomes and Extracellular Matrix 21 Noncoding DNA 1 Proteasomes 13 Components of the Extracellular Histone Organization 3 Cellular Metabolism and Mitochondrial Matrix 22 Micro-RNA and Long Noncoding RNA 4 Function 13 Maintaining Cell Populations 24 Cellular Housekeeping 6 Cellular Activation 16 Proliferation and the Cell Cycle 24 Plasma Membrane: Protection and Nutrient Cell Signaling 16 Stem Cells 25 Acquisition 8 Signal Transduction Pathways 16 Concluding Remarks 28 Cytoskeleton 11 Modular Signaling Proteins, Hubs, and Cell-Cell Interactions 12 Nodes 18 Biosynthetic Machinery: Endoplasmic Reticulum Transcription Factors 19 and Golgi Apparatus 12 Growth Factors and Receptors 19 " PROTEIN " Genome " Before " Rodolph "Virchow ↳ "" " "" " "" Suffering " " " " " cellular dist But now study of die. but Karan dinn mono Pathology literally translates to the study of suffering (Greek the genome. The potential for these new powerful tools pathos = suffering, logos = study); as applied to modern to expand our understanding of pathogenesis and drive molecules medicine, it is the study of disease. I Virchow was certainly therapeutic innovation excites and inspires scientists and - genes correct in asserting that disease originates at the cellular the lay public alike. DNA base 3.2 billion pairs level, but we now realize that cellular disturbances arise Human genome - - proteins within the genome - 20,000 Protein coding genes ↳ from alterations in molecules (genes, proteins, and others) Noncoding DNA 1.5 % of the genome that influence the survival and behavior of cells. Thus, the foundation of modern pathology is understanding the cel- - The human genome contains about 3.2 billion DNA base lular and molecular abnormalities that give rise to diseases. , pairs. Yet, within the genome there are only roughly 20,000 It is helpful to consider these abnormalities in the context protein-encoding genes, comprising just 1.5% of the _ of normal cellular structure and function, which is the genome. The proteins encoded by these genes are the fun- Proteins in theme of this introductory chapter. damental constituents of cells, functioning as enzymes, the genes It is unrealistic (and even undesirable) to condense the structural elements, and signaling molecules. Although are e- vast and fascinating field of cell biology into a single 20,000 underestimates the actual number of proteins Fundamental chapter. Consequently, rather than attempting a compre- encoded (many genes produce multiple RNA transcripts constituents hensive review, the goal here is to survey basic principles that encode distinct protein isoforms), it is nevertheless Of cells. Functions : and highlight recent advances that are relevant to the startling that worms composed of fewer than 1000 cells— signaling mechanisms of disease that are emphasized throughout the and with genomes 30-fold smaller—are also assembled - molecules rest of the book. from roughly 20,000 protein-encoding genes. Perhaps even - structural more unsettling is that many of these proteins are recogniz- elements sequence of human genome able homologs of molecules expressed in humans. What - e mymet THE GENOME is e- landmark of Biomedical science. then separates humans from worms? The answer is not completely known, but evidence sup- The sequencing of the human genome at the beginning of ports the assertion that the difference lies in the 98.5% of the 21st century represented a landmark achievement of the human genome that does not encode proteins. The biomedical science. Since then, the rapidly dropping cost function of such long stretches of DNA (which has been of sequencing and the computational capacity to analyze called the “dark matter” of the genome) was mysterious for vast amounts of data promise to revolutionize our under- many years. However, it is now clear that more than 85% of standing of health and disease. At the same time, the the human genome is ultimately transcribed, with almost emerging information has also revealed a breathtaking 80% being devoted to the regulation of gene expression. It level of complexity far beyond the linear sequencing of follows that whereas proteins provide the building blocks 85% of c- human genome is transcribe 1 fur regulation 804. of gene expression  2 CHAPTER 1 The Cell as a Unit of Health and Disease dictates only euchromatin Heterochromatin Nucleolus Heterochromatin qEuchromatingene expression 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 Fig. 1.1 The organization of nuclear DNA. At the light microscopic level, the nuclear genetic material is organized into dispersed, transcriptionally active euchromatin or densely packed, transcriptionally inactive heterochromatin; chromatin can also be mechanically connected with the nuclear membrane, and nuclear membrane perturbation can thus influence transcription. Chromosomes (as shown) can only be visualized by light microscopy during cell division. 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 loss of DNA at the chromosome 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 composed 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 are regulatory elements that can modulate gene expression across distances of 100 kB or more by looping back onto promoters and recruiting additional factors that are needed to drive the expression of pre-mRNA species. The intronic sequences are subsequently spliced out of the pre-mRNA to produce the definitive message that includes exons that are translated into protein and 3′- and 5′-untranslated regions (UTR) that may have regulatory functions. In addition to the enhancer, promoter, and UTR sequences, noncoding elements are found throughout the genome; these include short repeats, regulatory factor binding regions, noncoding regulatory RNAs, and transposons. Retains MM Functional Non Protein coding DNA noncoding telomere 's centromere Promoter dn enhances chromatin structure Mobile genetic elements and machinery required for assembling cells, tissues, and regulation may prove to be more important in disease cau- organisms, it is the noncoding regions of the genome that sation than structural changes in specific proteins. Another provide the critical “architectural planning.” surprise that emerged from genome sequencing is that any The major classes of functional non–protein-coding DNA two humans are typically >99.5% DNA-identical (and are sequences found in the human genome include (Fig. 1.1): 99% sequence-identical with chimpanzees)! Thus, individ- Promoter and enhancer regions that bind protein tran- ual variation, including differential susceptibility to dis- scription factors eases and environmental exposures, is encoded in 200 inactive heterochromatin and (2) histochemically dis- nucleotides in length.µ Post-transcriptional silencing of gene expression. persed and transcriptionally active euchromatin. Because Micro-RNAs (miRNAs) are relatively short RNAs (22 only euchromatin permits gene expression and thereby nucleotides on average) that function primarily to dictates cellular identity and activity, there are a host of modulate the translation of target mRNAs into their mechanisms that tightly regulate the state of chromatin corresponding proteins. Posttranscriptional silencing (described below). of gene expression by miRNA is a fundamental and DNA methylation. High levels of DNA methylation in - evolutionarily conserved mechanism of gene regula- gene regulatory elements typically result in chroma- tion present in all eukaryotes (plants and animals). tin condensation and transcriptional silencing. Like Even bacteria have a primitive version of the same histone modifications (see later), DNA methylation is general machinery that they use to protect themselves tightly regulated by methyltransferases, demethylating against foreign DNA (e.g., from phages and viruses). enzymes, and methylated-DNA-binding proteins. The human genome contains almost 6000 miRNA genes, Histone modifying factors. Nucleosomes are highly only 3.5-fold less than the number of protein-coding dynamic structures regulated by an array of nuclear genes. Moreover, individual miRNAs appear to regu- proteins and post-translational modifications: late multiple protein-coding genes, allowing each Chromatin remodeling complexes can reposition nucleo- miRNA to coregulate entire programs of gene expres- somes on DNA, exposing (or obscuring) gene regula- sion. Transcription of miRNA genes produces a primary tory elements such as promoters. transcript (pri-miRNA) that is processed into progres- “Chromatin writer” complexes carry out more than sively smaller segments, including trimming by the 70 different covalent histone modifications generi- enzyme Dicer. This generates mature single-stranded cally denoted as marks. These include methylation, miRNAs of 21 to 30 nucleotides that associate with a acetylation, and phosphorylation of specific histone multiprotein aggregate called RNA-induced silencing amino acid residues: Histone methylation of lysines complex (RISC; Fig. 1.3). Subsequent base pairing and arginines is accomplished by specific writer between the miRNA strand and its target mRNA directs enzymes; methylation of histone lysine residues the RISC to either induce mRNA cleavage or to repress can lead to transcriptional activation or repression, its translation. In this way, the target mRNA is posttran- depending on which histone residue is “marked.” scriptionally silenced. Histone acetylation of lysine residues (occurring through histone acetyl transferases) tends to open Taking advantage of the same pathway, small interfering up chromatin and increase transcription; histone RNAs (siRNAs) are short RNA sequences that can be intro- deacetylases (HDAC) reverse this process, leading duced into cells. These serve as substrates for Dicer and to chromatin condensation. Histone phosphorylation interact with the RISC complex in a manner analogous to of serine residues can variably open or condense endogenous miRNAs. Synthetic siRNAs that can target chromatin, to increase or decrease transcription, specific mRNA species are therefore powerful laboratory respectively. tools to study gene function (so-called knockdown technol- Histone marks are reversible through the activity of ogy); they also are promising as therapeutic agents to “chromatin erasers.” Other proteins function as “chro- silence pathogenic genes, e.g., oncogenes involved in neo- matin readers,” binding histones that bear particular plastic transformation. marks and thereby regulating gene expression. Long noncoding RNA (lncRNA). The human genome also contains a very large number of lncRNAs—at least The mechanisms involved in the cell-specific epigenetic 30,000, with the total number potentially exceeding regulation of genomic organization and gene expression coding mRNAs by 10- to 20-fold. lncRNAs modulate are undeniably complex. Despite the intricacies, learning gene expression in many ways (Fig. 1.4); for example, to manipulate these processes will likely bear significant they can bind to regions of chromatin, restricting RNA therapeutic benefits because many diseases are associated polymerase access to coding genes within the region. with inherited or acquired epigenetic alterations, and The best-known example of a repressive function dysregulation of the “epigenome” has a central role in the involves XIST, which is transcribed from the X chromo- genesis of benign and malignant neoplasms (Chapter 6). some and plays an essential role in physiologic X chro- Moreover—unlike genetic changes—epigenetic alterations mosome inactivation. XIST itself escapes X inactivation, (e.g., histone acetylation and DNA methylation) are readily but forms a repressive “cloak” on the X chromosome reversible and are therefore amenable to intervention; from which it is transcribed, resulting in gene silencing. indeed, HDAC inhibitors and DNA methylation inhibitors Conversely, it has been appreciated that many enhanc- are already being used in the treatment of various forms ers are sites of lncRNA synthesis, with the lncRNAs of cancer. expanding transcription from gene promoters through ?⃝ ?⃝  The Genome 5 A. Gene activation Ribonucleoprotein miRNA gene transcription complex lncRNA Gene activation pri-miRNA Decoy lncRNA Target gene B. Gene suppression pre-miRNA Gene suppression Export protein pre-miRNA C. Promote chromatin modification Dicer Methylation, acetylation Target mRNA miRNA D. Assembly of protein complexes Act on chromatin structure Unwinding of miRNA duplex RISC Multi-subunit complex complex Fig. 1.4 Roles of long noncoding RNAs (lncRNAs). (A) Long noncoding RNAs (lncRNAs) can facilitate transcription factor binding and thus promote Imperfect Perfect gene activation. (B) Conversely, lncRNAs can preemptively bind transcription match match factors and thus prevent gene transcription. (C) Histone and DNA modifica- Target tion by acetylases or methylases (or deacetylases and demethylases) may be mRNA directed by the binding of lncRNAs. (D) In other instances lncRNAs may act Translational mRNA as scaffolding to stabilize secondary or tertiary structures and/or multisub- repression cleavage unit complexes that influence general chromatin architecture or gene activity. (Adapted from Wang KC, Chang HY: Molecular mechanisms of long noncoding RNAs, Mol Cell 43:904, 2011.) a variety of mechanisms (Fig. 1.4). Ongoing studies are exploring the role of lncRNAs in diseases like athero- Ribosome sclerosis and cancer. GENE SILENCING Gene Editing Exciting new developments that permit exquisitely specific Fig. 1.3 Generation of microRNAs (miRNA) and their mode of action in genome editing stand to usher in an era of molecular revo- regulating gene function. miRNA genes are transcribed to produce a primary lution. These advances come from a wholly unexpected miRNA (pri-miRNA), which is processed within the nucleus to form pre- source: the discovery of clustered regularly interspaced miRNA composed of a single RNA strand with secondary hairpin loop short palindromic repeats (CRISPRs) and Cas (or CRISPR- structures that form stretches of double-stranded RNA. After this pre- miRNA is exported out of the nucleus via specific transporter proteins, the associated genes). These are linked genetic elements that cytoplasmic enzyme Dicer trims the pre-miRNA to generate mature double- endow prokaryotes with a form of acquired immunity to stranded miRNAs of 21 to 30 nucleotides.The miRNA subsequently unwinds, phages and plasmids. Bacteria use this system to sample and the resulting single strands are incorporated into the multiprotein RISC. the DNA of infecting agents, incorporating it into the host Base pairing between the single-stranded miRNA and its target mRNA genome as CRISPRs. CRISPRs are transcribed and pro- directs RISC to either cleave the mRNA target or to repress its translation. cessed into an RNA sequence that binds and directs the In either case, the target mRNA gene is silenced posttranscriptionally. nuclease Cas9 to a sequences (e.g., a phage), leading to its cleavage and the destruction of the phage. Gene editing repurposes this process by using artificial guide RNAs (gRNAs) that bind Cas9 and are complementary to a DNA 6 CHAPTER 1 The Cell as a Unit of Health and Disease sequence of interest. Once directed to the target sequence of the system (and the excitement about its genetic engi- by the gRNA, Cas9 induces double-strand DNA breaks. neering potential) comes from its impressive flexibility and Repair of the resulting highly specific cleavage sites specificity, which is substantially better than other previ- can lead to somewhat random disruptive mutations in the ous editing systems. Applications include inserting specific targeted sequences (through nonhomologous end joining mutations into the genomes of cells to model cancers and [NHEJ]), or the precise introduction of new sequences of other diseases, and rapidly generating transgenic animals interest (by homologous recombination). Both the gRNAs from edited embryonic stem cells. On the flip side, it now is and the Cas9 enzyme can be delivered to cells with a single feasible to selectively “correct” mutations that cause hered- easy-to-build plasmid (Fig. 1.5). However, the real beauty itable disease, or—perhaps more worrisome—to just elimi- nate less “desirable” traits. Predictably, the technology has inspired a vigorous debate regarding its application. Homologous gRNA sequence CELLULAR HOUSEKEEPING The viability and normal activity of cells depend on a Cas9 protein gRNA variety of fundamental housekeeping functions that all dif- ferentiated cells must perform. Many normal housekeeping functions are compart- mentalized within membrane-bound intracellular organ- elles (Fig. 1.6). By isolating certain cellular functions within distinct compartments, potentially injurious degradative enzymes or reactive metabolites can be concentrated Cleavage or stored at high concentrations in specific organelles Double-stranded without risking damage to other cellular constituents. DNA Moreover, compartmentalization allows for the creation Target genomic of unique intracellular environments (e.g., low pH or high sequence calcium) that are optimal for certain enzymes or metabolic pathways. New proteins destined for the plasma membrane or Double-stranded DNA break secretion are synthesized in the rough endoplasmic reticulum - process (RER) and physically assembled in the Golgi apparatus; pro- packed teins intended for the cytosol are synthesized on free ribo- secrete somes. Smooth endoplasmic reticulum (SER) may be abundant modified all products in certain cell types such as gonads and liver where it serves as the site of steroid hormone and lipoprotein syn- NHEJ thesis, as well as the modification of hydrophobic com- pounds such as drugs into water-soluble molecules for HDR export. Cells catabolize the wide variety of molecules that they endocytose, as well as their own repertoire of proteins and organelles—all of which are constantly being degraded Insertion/ Donor DNA and renewed. Breakdown of these constituents takes deletion place at three different sites, ultimately serving different DNA with random mutation DNA with specific mutation functions. Proteasomes are “disposal” complexes that degrade Fig. 1.5 Gene editing with clustered regularly interspersed short palin- dromic repeats (CRISPRs)/Cas9. In bacteria, DNA sequences consisting of denatured or otherwise “tagged” cytosolic proteins and CRISPRs are transcribed into guide RNAs (gRNAs) with a constant region release short peptides. In some cases the peptides so and a variable sequence of about 20 bases. The constant regions of gRNAs generated are presented in the context of class I major bind to Cas9, permitting the variable regions to form heteroduplexes with histocompatibility molecules to help drive the adaptive homologous host cell DNA sequences. The Cas9 nuclease then cleaves the immune response (Chapter 5). In other cases, protea- bound DNA, producing a double-stranded DNA break. To perform gene somal degradation of regulatory proteins or transcrip- editing, gRNAs are designed with variable regions that are homologous to a target DNA sequence of interest. Coexpression of the gRNA and Cas9 in tion factors can trigger or shut down cellular signaling cells leads to efficient cleavage of the target sequence. In the absence of pathways. homologous DNA, the broken DNA is repaired by nonhomologous end Lysosomes are intracellular organelles that contain joining (NHEJ), an error-prone method that often introduces disruptive enzymes that digest a wide range of macromolecules, insertions or deletions (indels). By contrast, in the presence of a homologous including proteins, polysaccharides, lipids, and nucleic “donor” DNA spanning the region targeted by CRISPR/Cas9, cells instead acids. They are the organelle in which phagocytosed may use homologous DNA recombination (HDR) to repair the DNA break. HDR is less efficient than NHEJ, but has the capacity to introduce precise microbes and damaged or unwanted cellular organelles changes in DNA sequence. Potential applications of CRISPR/Cas9 coupled are degraded and eliminated. with HDR include the repair of inherited genetic defects and the creation Peroxisomes are specialized cell organelles that contain of pathogenic mutations. [ catalase, peroxidase and other oxidative enzymes. They contains catalase RER SEK - - w/ ribosomes , synthesize w/out Ribosomes synthesize , Chon lipid molecules  Cellular Housekeeping 7 units Machine 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 secreted proteins Smooth ER, Golgi 6% 1* = protein modification,O - sorting, catabolism - Nucleus DNA transcription6% 1 cell regulation, proliferation, DNA transcription " " Endosomes 1% 200 intracellular transport and export, ingestion of extracellular substances Lysosomes 1% 300 cellular catabolism I Peroxisomes cellular catabolism → 1% FINAL CATABOLISM 400 Rough very long-chain fatty acid metabolism ↳ breakdown ↳ Breakdown Free endoplasmic ↳ Lyse ribosomes reticulum Nucleolus Golgi Nucleus apparatus Lysosome Mitochondrion Endosome Smooth Cytoskeleton endoplasmic Plasma reticulum membrane Peroxisome Centrioles Microtubules Fig. 1.6 Basic subcellular constituents of cells. The table presents the number of 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. *Rough and smooth ER form a single compartment; the Golgi apparatus is organized as a set of discrete stacked cisternae interconnected by transport vesicles. (Adapted 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.) ATP made by mitochondria THROUG OXIDATIVE PHOPORYUAT / M. play a specialized role in the breakdown of very long Most of the adenosine triphosphate (ATP) that powers chain fatty acids, generating hydrogen peroxide in the cells is made through oxidative phosphorylation in the process. mitochondria. However, mitochondria also serve as an important source of metabolic intermediates that are The contents and position of cellular organelles also needed for anabolic metabolism. They also are sites of syn- are subject to regulation. Endosomal vesicles shuttle inter- thesis of certain macromolecules (e.g., heme), and contain nalized material to the appropriate intracellular sites or important sensors of cell damage that can initiate and regu- direct newly synthesized materials to the cell surface or late the process of apoptotic cell death. targeted organelle. Movement of both organelles and pro- Cell growth and maintenance require a constant supply teins within the cell and of the cell in its environment is of both energy and the building blocks that are needed orchestrated by the cytoskeleton. These structural proteins for synthesis of macromolecules. In growing and dividing also regulate cellular shape and intracellular organiza- cells, all of these organelles have to be replicated (organel- tion, requisites for maintaining cell polarity. This is par- lar biogenesis) and correctly apportioned in daughter cells ticularly critical in epithelia, in which the top of the cell following mitosis. Moreover, because the macromolecules (apical) and the bottom and side of the cell (basolateral) are and organelles have finite life spans (mitochondria, e.g., often exposed to different environments and have distinct last only about 10 days), mechanisms also must exist functions. that allow for the recognition and degradation of “worn 8 CHAPTER 1 The Cell as a Unit of Health and Disease out” cellular components. The final catabolism occurs in cells undergoing apoptosis (programmed cell death), it lysosomes. becomes an “eat me” signal for phagocytes. In the With this as a primer, we now move on to discuss cel- special case of platelets, it serves as a cofactor in the lular components and their function in greater detail. clotting of blood. Glycolipids and sphingomyelin are preferentially expressed Plasma Membrane: Protection and on the extracellular face; glycolipids (and particularly Nutrient Acquisition gangliosides, with complex sugar linkages and terminal sialic acids that confer negative charges) are important Plasma membranes (and all other organellar membranes) in cell–cell and cell–matrix interactions, including inflam- are more than just static lipid sheaths. Rather, they are matory cell recruitment and sperm–egg interactions. fluid bilayers of amphipathic phospholipids with hydro- philic head groups that face the aqueous environment and Certain membrane components associate laterally with hydrophobic lipid tails that interact with each other to each other in the bilayer, leading to distinct domains called form a barrier to passive diffusion of large or charged lipid rafts. Because inserted membrane proteins have differ- molecules (Fig. 1.7A). The bilayer is composed of a hetero- ent intrinsic solubilities in various lipid domains, they tend geneous collection of different phospholipids, which are to accumulate in certain regions of the membrane (e.g., distributed asymmetrically—for example, certain mem- rafts) and to become depleted from others. Such nonran- brane lipids preferentially associate with extracellular or dom distributions of lipids and membrane proteins impact cytosolic faces. Asymmetric partitioning of phospholipids cell–cell and cell–matrix interactions, as well as intracel- is important in several cellular processes: lular signaling and the generation of specialized membrane Phosphatidylinositol on the inner membrane leaflet can be regions involved in secretory or endocytic pathways. phosphorylated, serving as an electrostatic scaffold for The plasma membrane is liberally studded with a intracellular proteins; alternatively, polyphosphoinosit- variety of proteins and glycoproteins involved in (1) ion ides can be hydrolyzed by phospholipase C to generate and metabolite transport, (2) fluid-phase and receptor- intracellular second signals such as diacylglycerol and mediated uptake of macromolecules, and (3) cell–ligand, inositol trisphosphate. cell–matrix, and cell–cell interactions. Proteins interact Phosphatidylserine is normally restricted to the inner with the lipid bilayer by one of four general arrangements face where it confers a negative charge and is involved (Fig. 1.7B): in electrostatic interactions with proteins; however, Most proteins are transmembrane (integral) proteins, when it flips to the extracellular face, which happens in having one or more relatively hydrophobic α-helical 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 Fig. 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; phosphatidylcholine and sphingomyelin are overrepresented in the outer leaflet, and phosphatidyl- serine (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. Non-random partitioning of certain membrane components such as cholesterol creates membrane domains known as lipid rafts. (B) Membrane-associated proteins may traverse the membrane (singly or multiply) via α-helical hydrophobic amino acid sequences; depending on the sequence and hydrophobicity of these domain, such proteins may be enriched or excluded from lipid rafts and other membrane domain. Proteins on the cytosolic face may associate with membranes through posttranslational modifications, for example, farnesylation or addition of palmitic acid. Proteins on the extracytoplasmic face may associate with the membrane via glycosyl phosphatidyl inositol linkages. Besides protein–protein interactions within the mem- brane, membrane proteins can also associate with extracellular and/or intracytoplasmic proteins to generate large, relatively stable complexes (e.g., the focal adhesion complex). Transmembrane proteins can translate mechanical forces (e.g., from the cytoskeleton or ECM) as well as chemical signals across the mem- brane. It is worth remembering that a similar organization of lipids and associated proteins also occurs within the various organellar membranes.  Cellular Housekeeping 9 segments that traverse the lipid bilayer. Integral mem- low-molecular-weight species (ions and small molecules brane proteins typically contain positively charged up to approximately 1000 daltons), channel proteins and amino acids in their cytoplasmic domains that anchor carrier proteins may be used (although this discussion the proteins to the negatively charged head groups of focuses on plasma membranes, it should be noted that membrane phospholipids. similar pores and channels are needed for transport across Proteins may be synthesized in the cytosol and post- organellar membranes). Each transported molecule (e.g., translationally attached to prenyl groups (e.g., farnesyl, ion, sugar, nucleotide) requires a transporter that is typi- related to cholesterol) or fatty acids (e.g., palmitic or cally highly specific (e.g., glucose but not galactose): myristic acid) that insert into the cytosolic side of the Channel proteins create hydrophilic pores that, when plasma membrane. open, permit rapid movement of solutes (usually Attachment to membranes can occur through glyco- restricted by size and charge; Fig. 1.8). sylphosphatidylinositol (GPI) anchors on the extracel- Carrier proteins bind their specific solute and undergo a lular face of the membrane. series of conformational changes to transfer the ligand Extracellular proteins can noncovalently associate with across the membrane; their transport is relatively slow. transmembrane proteins, which serve to anchor them to the cell. In many cases, a concentration and/or electrical gradi- ent between the inside and outside of the cell drives solute Many plasma membrane proteins function together as movement via passive transport (virtually all plasma mem- larger complexes; these may assemble under the control branes have an electrical potential difference across them, of chaperone molecules in the RER or by lateral diffusion with the inside negative relative to the outside). In other in the plasma membrane. The latter mechanism is charac- cases, active transport of certain solutes against a concentra- teristic of many protein receptors (e.g., cytokine receptors) tion gradient is accomplished by carrier molecules (not that dimerize or trimerize in the presence of ligand to form channels) using energy released by ATP hydrolysis or a functional signaling units. Although lipid bilayers are fluid coupled ion gradient. Transporter ATPases include the in the two-dimensional plane of the membrane, membrane notorious multidrug resistance (MDR) protein, which pumps components can nevertheless be constrained to discrete polar compounds (e.g., chemotherapeutic drugs) out of domains. This can occur by localization to lipid rafts (dis- cells and may render cancer cells resistant to treatment. cussed earlier), or through intercellular protein–protein Because membranes are freely permeable to water, it interactions (e.g., at tight junctions) that establish discrete moves into and out of cells by osmosis, depending on rela- boundaries; indeed, this strategy is used to maintain cell tive solute concentrations. Thus, extracellular salt in excess polarity (e.g., top/apical versus bottom/basolateral) in epi- of that in the cytosol (hypertonicity) causes a net movement thelial layers. Alternatively, unique domains can be formed of water out of cells, whereas hypotonicity causes a net through the interaction of membrane proteins with cyto- movement of water into cells. The cytosol is rich in charged skeletal molecules or an extracellular matrix (ECM). metabolites and protein species, which attract a large The extracellular face of the plasma membrane is dif- number of counterions that tend to increase the intracel- fusely studded with carbohydrates, not only as complex lular osmolarity. As a consequence, to prevent overhydra- oligosaccharides on glycoproteins and glycolipids, but also tion cells must constantly pump out small inorganic ions as polysaccharide chains attached to integral membrane (e.g., Na+)—typically through the activity of membrane proteoglycans. This glycocalyx functions as a chemical and ion-exchanging ATPases. Loss of the ability to generate mechanical barrier, and is also involved in cell–cell and energy (e.g., in a cell injured by toxins or ischemia) there- cell–matrix interactions. fore results in osmotic swelling and eventual rupture of cells. Similar transport mechanisms also regulate intracel- Passive Membrane Diffusion lular and intraorganellar pH; most cytosolic enzymes Small, nonpolar molecules such as O2 and CO2 readily dis- prefer to work at pH 7.4, whereas lysosomal enzymes func- solve in lipid bilayers and therefore rapidly diffuse across tion best at pH 5 or less. them, as do hydrophobic molecules (e.g., steroid-based molecules such as estradiol or vitamin D). Similarly, small Receptor-Mediated and polar molecules (