by the gRNA, Cas9 induces double-strand DNA breaks. #51 | | |
Repair of the resulting highly specific cleavage sites can lead to somewhat random disruptive mutations in the targeted sequences (through nonhomologous end joining [NHEJ]), or the precise introduction of new sequences of interest (by homologous recombination). Both the gRNAs and the Cas9 enzyme can be delivered to cells with a single easy-to-build plasmid (Fig. 1.5). However, the real beauty of the system (and the excitement about its genetic engi- neering potential) comes from its impressive flexibility and specificity, which is substantially better than other previ- ous editing systems. Applications include inserting specific mutations into the genomes of cells to model cancers and other diseases, and rapidly generating transgenic animals from edited embryonic stem cells. On the flip side, it now is feasible to selectively “correct” mutations that cause hered- itable disease, or—perhaps more worrisome—to just elimi- nate less “desirable” traits. Predictably, the technology has inspired a vigorous debate regarding its application. #52 | | |
Fig. 1.5 Gene editing with clustered regularly interspersed short palin- #53 | | |
dromic repeats (CRISPRs)/Cas9. In bacteria, DNA sequences consisting of CRISPRs are transcribed into guide RNAs (gRNAs) with a constant region and a variable sequence of about 20 bases. The constant regions of gRNAs bind to Cas9, permitting the variable regions to form heteroduplexes with homologous host cell DNA sequences. The Cas9 nuclease then cleaves the bound DNA, producing a double-stranded DNA break. To perform gene editing, gRNAs are designed with variable regions that are homologous to a target DNA sequence of interest. Coexpression of the gRNA and Cas9 in cells leads to efficient cleavage of the target sequence. In the absence of homologous DNA, the broken DNA is repaired by non homologous end joining (NHEJ), an error-prone method that often introduces disruptive insertions or deletions (indels). By contrast, in the presence of a homologous “donor” DNA spanning the region targeted by CRISPR/Cas9, cells instead may use homologous DNA recombination (HDR) to repair the DNA break. HDR is less efficient than NHEJ, but has the capacity to introduce precise changes in DNA sequence. Potential applications of CRISPR/Cas9 coupled with HDR include the repair of inherited genetic defects and the creation of pathogenic mutations. #54 | | |
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The viability and normal activity of cells depend on a variety of fundamental housekeeping functions that all dif- ferentiated cells must perform. #56 | | |
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 or stored at high concentrations in specific organelles without risking damage to other cellular constituents. Moreover, compartmentalization allows for the creation of unique intracellular environments (e.g., low pH or high calcium) that are optimal for certain enzymes or metabolic pathways. #57 | | |
New proteins destined for the plasma membrane or secretion are synthesized in the rough endoplasmic reticulum (RER) and physically assembled in the Golgi apparatus; pro- teins intended for the cytosol are synthesized on free ribo- somes. Smooth endoplasmic reticulum (SER) may be abundant in certain cell types such as gonads and liver where it serves as the site of steroid hormone and lipoprotein syn- thesis, as well as the modification of hydrophobic com- pounds such as drugs into water-soluble molecules for export. #58 | | |
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 and renewed. Breakdown of these constituents takes place at three different sites, ultimately serving different functions. #59 | | |
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Proteasomes are “disposal” complexes that degrade denatured or otherwise “tagged” cytosolic proteins and release short peptides. In some cases the peptides so generated are presented in the context of class I major histocompatibility molecules to help drive the adaptive immune response (Chapter 5). In other cases, protea- somal degradation of regulatory proteins or transcrip- tion factors can trigger or shut down cellular signaling pathways. #61 | | |
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Lysosomes are intracellular organelles that contain enzymes that digest a wide range of macromolecules, including proteins, polysaccharides, lipids, and nucleic acids. They are the organelle in which phagocytosed microbes and damaged or unwanted cellular organelles are degraded and eliminated. #63 | | |
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Peroxisomes are specialized cell organelles that contain catalase, peroxidase and other oxidative enzymes. They play a specialized role in the breakdown of very long chain fatty acids, generating hydrogen peroxide in the process. #65 | | |
The contents and position of cellular organelles also are subject to regulation. Endosomal vesicles shuttle inter- nalized material to the appropriate intracellular sites or direct newly synthesized materials to the cell surface or targeted organelle. Movement of both organelles and pro- teins within the cell and of the cell in its environment is orchestrated by the cytoskeleton. These structural proteins also regulate cellular shape and intracellular organiza- tion, requisites for maintaining cell polarity. This is par- ticularly critical in epithelia, in which the top of the cell (apical) and the bottom and side of the cell (basolateral) are often exposed to different environments and have distinct functions. #66 | | |
Most of the adenosine triphosphate (ATP) that powers cells is made through oxidative phosphorylation in the mitochondria. However, mitochondria also serve as an important source of metabolic intermediates that are needed for anabolic metabolism. They also are sites of syn- thesis of certain macromolecules (e.g., heme), and contain important sensors of cell damage that can initiate and regu- late the process of apoptotic cell death. #67 | | |
Cell growth and maintenance require a constant supply of both energy and the building blocks that are needed for synthesis of macromolecules. In growing and dividing cells, all of these organelles have to be replicated (organel- lar biogenesis) and correctly apportioned in daughter cells following mitosis. Moreover, because the macromolecules and organelles have finite life spans (mitochondria, e.g., last only about 10 days), mechanisms also must exist that allow for the recognition and degradation of “worn out” cellular components. The final catabolism occurs in lysosomes. #68 | | |
With this as a primer, we now move on to discuss cellular components and their function in greater detail. #69 | | |
Plasma Membrane: Protection and Nutrient Acquisition #70 | | |
Plasma membranes (and all other organellar membranes) are more than just static lipid sheaths. Rather, they are fluid bilayers of amphipathic phospholipids with hydro- philic head groups that face the aqueous environment and hydrophobic lipid tails that interact with each other to form a barrier to passive diffusion of large or charged molecules (Fig. 1.7A). The bilayer is composed of a hetero- geneous collection of different phospholipids, which are distributed asymmetrically—for example, certain mem- brane lipids preferentially associate with extracellular or cytosolic faces. Asymmetric partitioning of phospholipids is important in several cellular processes: #71 | | |
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Phosphatidylinositol on the inner membrane leaflet can be phosphorylated, serving as an electrostatic scaffold for intracellular proteins; alternatively, polyphosphoinosit- ides can be hydrolyzed by phospholipase C to generate intracellular second signals such as diacylglycerol and inositol trisphosphate. #73 | | |
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Phosphatidylserine is normally restricted to the inner face where it confers a negative charge and is involved in electrostatic interactions with proteins; however, when it flips to the extracellular face, which happens in cells undergoing apoptosis (programmed cell death), it becomes an “eat me” signal for phagocytes. In the special case of platelets, it serves as a cofactor in the clotting of blood. #75 | | |
Glycolipids and sphingomyelin are preferentially expressed on the extracellular face; glycolipids (and particularly gangliosides, with complex sugar linkages and terminal sialic acids that confer negative charges) are important in cell–cell and cell–matrix interactions, including inflam- matory cell recruitment and sperm–egg interactions. #76 | | |
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Certain membrane components associate laterally with each other in the bilayer, leading to distinct domains called lipid rafts. Because inserted membrane proteins have differ- ent intrinsic solubilities in various lipid domains, they tend to accumulate in certain regions of the membrane (e.g., rafts) and to become depleted from others. Such nonran- dom distributions of lipids and membrane proteins impact cell–cell and cell–matrix interactions, as well as intracel- lular signaling and the generation of specialized membrane regions involved in secretory or endocytic pathways. #78 | | |
The plasma membrane is liberally studded with a variety of proteins and glycoproteins involved in (1) ion and metabolite transport, (2) fluid-phase and receptor- mediated uptake of macromolecules, and (3) cell–ligand, cell–matrix, and cell–cell interactions. Proteins interact with the lipid bilayer by one of four general arrangements (Fig. 1.7B): #79 | | |
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Most proteins are transmembrane (integral) proteins, having one or more relatively hydrophobic α-helical segments that traverse the lipid bilayer. Integral mem- brane proteins typically contain positively charged amino acids in their cytoplasmic domains that anchor the proteins to the negatively charged head groups of membrane phospholipids. #81 | | |
Proteins may be synthesized in the cytosol and post- translationally attached to prenyl groups (e.g., farnesyl, related to cholesterol) or fatty acids (e.g., palmitic or myristic acid) that insert into the cytosolic side of the plasma membrane. #82 | | |
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Attachment to membranes can occur through glyco- sylphosphatidylinositol (GPI) anchors on the extracel- lular face of the membrane. #84 | | |
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Extracellular proteins can noncovalently associate with transmembrane proteins, which serve to anchor them to the cell. #86 | | |
Many plasma membrane proteins function together as larger complexes; these may assemble under the control of chaperone molecules in the RER or by lateral diffusion in the plasma membrane. The latter mechanism is charac- teristic of many protein receptors (e.g., cytokine receptors) that dimerize or trimerize in the presence of ligand to form functional signaling units. Although lipid bilayers are fluid in the two-dimensional plane of the membrane, membrane components can nevertheless be constrained to discrete domains. This can occur by localization to lipid rafts (dis- cussed earlier), or through intercellular protein–protein interactions (e.g., at tight junctions) that establish discrete boundaries; indeed, this strategy is used to maintain cell polarity (e.g., top/apical versus bottom/basolateral) in epi- thelial layers. Alternatively, unique domains can be formed through the interaction of membrane proteins with cyto- skeletal molecules or an extracellular matrix (ECM). #87 | | |
The extracellular face of the plasma membrane is dif- fusely studded with carbohydrates, not only as complex oligosaccharides on glycoproteins and glycolipids, but also as polysaccharide chains attached to integral membrane proteoglycans. This glycocalyx functions as a chemical and mechanical barrier, and is also involved in cell–cell and cell–matrix interactions. #88 | | |
Passive Membrane Diffusion #89 | | |
Small, nonpolar molecules such as O2 and CO2 readily dis- #90 | | |
solve in lipid bilayers and therefore rapidly diffuse across them, as do hydrophobic molecules (e.g., steroid-based molecules such as estradiol or vitamin D). Similarly, small polar molecules (<75 daltons in mass, such as water, ethanol, and urea) readily cross membranes. In contrast, the lipid bilayer is an effective barrier to the passage of larger polar molecules, even those only slightly greater than 75 daltons, such as glucose. Lipid bilayers also are impermeant to ions, no matter how small, because of their charge and high degree of hydration. We will next discuss specialized mechanisms that regulate traffic across plasma membranes. #91 | | |
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For each of the larger polar molecules that must cross #93 | | |
membranes to support normal cellular functions (e.g., for nutrient uptake and waste disposal), unique plasma mem- brane protein complexes are typically required. For #94 | | |
Cellular Housekeeping 9 low-molecular-weight species (ions and small molecules #95 | | |
up to approximately 1000 daltons), channel proteins and carrier proteins may be used (although this discussion focuses on plasma membranes, it should be noted that similar pores and channels are needed for transport across organellar membranes). Each transported molecule (e.g., ion, sugar, nucleotide) requires a transporter that is typi- cally highly specific (e.g., glucose but not galactose): #96 | | |
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Channel proteins create hydrophilic pores that, when open, permit rapid movement of solutes (usually restricted by size and charge; Fig. 1.8). #98 | | |
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Carrier proteins bind their specific solute and undergo a series of conformational changes to transfer the ligand across the membrane; their transport is relatively slow. #100 | | |
