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Histology_Ross. The membranous organelles include:  the plasma (cell) membrane, a lipid bilayer that forms the cell boundary as well as the boundaries of many organelles within the cell; rough-surfaced endoplasmic reticulum (rER), a region of endoplasmic reticulum associated with ribosomes and the site of protein synthesis and modification of newly synthesized proteins;  smooth-surfaced endoplasmic reticulum (sER), a region of endoplasmic reticulum involved in lipid and steroid synthesis but not associated with ribosomes; Golgi apparatus, a membranous organelle composed of multiple ﬂattened cisternae responsible for modifying, sorting, and packaging proteins and lipids for intracellular or extracellular transport;  endosomes, membrane-bounded compartments interposed within endocytotic pathways that have the major function of sorting proteins delivered to them via endocytotic vesicles and redirecting them to different cellular compartments for their final destination;  lysosomes, small


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Histology_Ross. that have the major function of sorting proteins delivered to them via endocytotic vesicles and redirecting them to different cellular compartments for their final destination;  lysosomes, small organelles containing digestive enzymes that are formed from endosomes by targeted delivery of unique lysosomal membrane proteins and lysosomal enzymes;  transport vesicles—including pinocytotic vesicles, endocytotic vesicles, and coated vesicles—that are involved in both endocytosis and exocytosis and vary in shape and the material that they transport;  mitochondria, organelles that provide most of the energy to the cell by producing adenosine triphosphate (ATP) in the process of oxidative phosphorylation; and  peroxisomes, small organelles involved in the production and degradation of H2O2 and degradation of fatty acids.


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Histology_Ross. The nonmembranous organelles include:  microtubules, which together with actin and intermedi ate filaments form elements of the cytoskeleton and continuously elongate (by adding tubulin dimers) and shorten (by removing tubulin dimers), a property referred to as dynamic instability;  flaments, which are also part of the cytoskeleton and can be classified into two groups—actin flaments, which are ﬂexible chains of actin molecules, and intermediate flaments, which are ropelike fibers formed from a variety of proteins—both groups providing tensile strength to withstand tension and confer resistance to shearing forces;  centrioles, or short, paired cylindrical structures found in the center of the microtubule-organizing center (MTOC) or centrosome and whose derivatives give rise to basal bodies of cilia; and  ribosomes, structures essential for protein synthesis and composed of ribosomal RNA (rRNA) and ribosomal proteins (including proteins attached to membranes of the rER and proteins


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Histology_Ross. of cilia; and  ribosomes, structures essential for protein synthesis and composed of ribosomal RNA (rRNA) and ribosomal proteins (including proteins attached to membranes of the rER and proteins free in the cytoplasm).


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Histology_Ross. An outline of the key features of cellular organelles and inclusions is provided in Table 2.1. The normal function and related pathologies of the organelles are summarized in Table 2.2.


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Histology_Ross. Nucleus 3–10 Largest organelle within the cell Surrounded by two membranes (nuclear with distinct boundary envelope) containing nuclear pore complexes and perinuclear cisternal space Often visible nucleoli and Regions with condensed and diffused chromatin pattern regions chromatin pattern (heterochromatin and euchromatin) Nucleolus 1–2 Roughly circular, basophilic Dense, nonmembranous structure region within the nucleus containing fbrillar and granular material Visible in living cells throughout interphase with interference microscopy Plasma membrane 0.008–0.01 Not visible External membrane and membranes surrounding membranous organelles of cell; two inner and outer electron-dense layers separated by intermediate electron-lucent layer rER Area 5–10 Often observed as basophilic Flattened sheets, sacs, and tubes of region of cytoplasm referred membranes with attached ribosomes to as ergastoplasm sER Throughout Not visible Flattened sheets, sacs, and tubes of cytoplasm Cytoplasm in


Histology_Ross

Histology_Ross. sheets, sacs, and tubes of region of cytoplasm referred membranes with attached ribosomes to as ergastoplasm sER Throughout Not visible Flattened sheets, sacs, and tubes of cytoplasm Cytoplasm in region of sER may membranes without attached ribosomes exhibit distinct eosinophilia Golgi apparatus Area 5–10 Sometimes observed as Stack of fattened membrane sheets, “negative staining” region often adjacent to one side of nucleus Appears as network in heavy metal–stained preparations Visible in living cells with interference microscopy TABLE Review of Organelles and Cytoplasmic Inclusions: A Key to Light Microscopic and Electron Microscopic Identification2.1 Organelle or Inclusion Size (m) Light Microscopic Features Electron Microscopic Features continued next page chapter 2 Cell Cytoplasm MEMBRANOUS ORGANELLES TABLE Review of Organelles and Cytoplasmic Inclusions: A Key to Light Microscopic and Electron Microscopic Identification (Cont.)2.1 Organelle or Inclusion Size (m) Light


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Histology_Ross. MEMBRANOUS ORGANELLES TABLE Review of Organelles and Cytoplasmic Inclusions: A Key to Light Microscopic and Electron Microscopic Identification (Cont.)2.1 Organelle or Inclusion Size (m) Light Microscopic Features Electron Microscopic Features Secretory vesicles 0.050–1.0 Observed only when vesicles Many relatively small, membrane-bounded are very large (e.g., zymogen vesicles of uniform diameter, often granules in pancreas) polarized on one side of cell Mitochondria 0.2–7 Sometimes observed in Two-membrane system: Outer membrane favorable situations (e.g., liver and inner membrane arranged in numerous or nerve cells) as miniscule, dark folds (cristae) dots; visible in living cells stained In steroid-producing cells, inner membrane with vital dyes (e.g., Janus green) arranged in tubular cristae Endosomes 0.02–0.5 Not visible Tubulovesicular structures with subdivided lumen containing electron-lucent material or other smaller vesicles Lysosomes 0.2–0.5 Visible only after special enzyme


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Histology_Ross. Endosomes 0.02–0.5 Not visible Tubulovesicular structures with subdivided lumen containing electron-lucent material or other smaller vesicles Lysosomes 0.2–0.5 Visible only after special enzyme Membrane-bounded vesicles, often electron histochemical staining dense Peroxisomes 0.2–0.5 Visible only after special enzyme Membrane-bounded vesicles, often with histochemical staining electron-dense crystalloid inclusions Cytoskeletal elements 0.006–0.025 Only observed when organized Long, linear staining pattern with width into large structures (e.g., muscle and features characteristic of each fbrils) flament type Ribosomes 0.025 Not visible Minute dark dots, often associated with the rER Glycogen 0.010–0.040 Observed as a “purple haze” Nonmembranous, extremely dense grapelike region of cytoplasm inclusions metachromasia with toluidine blue–stained specimen Lipid droplets 0.2–5, up Readily visible when extremely Nonmembranous inclusions to 80 large (e.g., in adipocytes) Generally appear as a


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Histology_Ross. inclusions metachromasia with toluidine blue–stained specimen Lipid droplets 0.2–5, up Readily visible when extremely Nonmembranous inclusions to 80 large (e.g., in adipocytes) Generally appear as a void in the section Appear as large empty holes in section (lipid itself is usually removed by embedding solvents)


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Histology_Ross. The plasma membrane is a lipid-bilayered structure visible with transmission electron microscopy. The plasma membrane (cell membrane) is a dynamic structure that actively participates in many physiologic and biochemical activities essential to cell function and survival. When the plasma membrane is properly fixed, sectioned, stained, and viewed on edge with the transmission electron microscope (TEM), it appears as two electron-dense layers separated by an intermediate, electron-lucent (nonstaining) layer (Fig. 2.2). The total thickness of the plasma membrane is about 8 to 10 nm. The plasma membrane is composed of an amphipathic lipid layer containing embedded integral membrane proteins with peripheral membrane proteins attached to its surfaces.


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Histology_Ross. The current interpretation of the molecular organization of the plasma membrane is referred to as the modifed fuid–mosaic model (Fig. 2.3). The membrane consists primarily of phospholipid, cholesterol, and protein molecules. The lipid molecules form a lipid bilayer with an amphipathic character (it is both hydrophobic and hydrophilic). The fatty-acid chains of the lipid molecules face each other, making the inner portion of the membrane hydrophobic (i.e., having no affinity for water). The surfaces of the membrane are formed by the polar head groups of the lipid molecules, thereby making the surfaces hydrophilic (i.e., they have an affinity for water). Lipids are distributed asymmetrically between the inner and outer leaﬂets of the lipid bilayer, and their composition varies considerably among different biologic membranes.


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Histology_Ross. In most plasma membranes, protein molecules constitute approximately half of the total membrane mass. Most of the proteins are embedded within the lipid bilayer or pass through the lipid bilayer completely. These proteins are called integral membrane proteins. The other types of protein— peripheral membrane proteins—are not embedded within the lipid bilayer. They are associated with the plasma


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Histology_Ross. TABLE Organelles and Cytoplasmic Inclusions: Functions and Pathologies 2.2 Organelle or Inclusion Function Examples of Associated Pathologies Nucleus Storage and use of genome Inherited genetic diseases; environmentally induced mutations Nucleolus Synthesis of rRNA and partial Werner syndrome (premature aging assembly of ribosomal subunits disease) Involved in regulation of cell cycle Malfunctions of cell cycle leading to cancerogenesis Plasmamembrane Ion and nutrient transport Cystic fbrosis Recognition of environmental signal Intestinal malabsorption syndromes Cell-to-cell and cell-to-extracellular Lactose intolerance matrix adhesions rER Binds ribosomes engaged in translating Pseudoachondroplasia mRNA for proteins destined for secretion Calcium phosphate dihydrate crystal or for membrane insertion deposition disease Also involved in chemical modifcations of proteins and membrane lipid synthesis sER Involved in lipid and steroid metabolism Hepatic endoplasmic reticular storage


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Histology_Ross. insertion deposition disease Also involved in chemical modifcations of proteins and membrane lipid synthesis sER Involved in lipid and steroid metabolism Hepatic endoplasmic reticular storage disease Golgi apparatus Chemical modifcation of proteins I-cell disease Sorting and packaging of molecules Polycystic kidney disease for secretion or transport to other organelles Secretory vesicles Transport and storage of secreted proteins Lewy bodies of Parkinson’s disease to plasma membrane Proinsulin diabetes Mitochondria Aerobic energy supply (oxidative Mitochondrial myopathies such as MERRFa , phosphorylation, ATP) MELASb, Kearns-Sayre syndromes, and Initiation of apoptosis Leber’s hereditary optic atrophy Endosomes Transport of endocytosed material M-6-P receptor defciency Biogenesis of lysosomes Lysosomes Digestion of macromolecules Lysosomal storage diseases (see Folder 2.1, Clinical Correlation: Lysosomal Storage Diseases) Peroxisomes Oxidative digestion (e.g., fatty acids)


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Histology_Ross. of lysosomes Lysosomes Digestion of macromolecules Lysosomal storage diseases (see Folder 2.1, Clinical Correlation: Lysosomal Storage Diseases) Peroxisomes Oxidative digestion (e.g., fatty acids) Zellweger’s syndrome Cytoskeletal elements Various functions, including cell motility, Immotile cilia syndrome, Alzheimer’s cell adhesions, intracellular and disease, epidermolysis bullosa extracellular transport Maintenance of cellular skeleton Ribosomes Synthesis of protein by translating Ribosomal dysfunction in Alzheimer’s protein-coding sequence from disease; Diamond-Blackfan anemia mRNA Many antibiotics act selectively on bacterial ribosomes: e.g., tetracyclines, aminoglycosides (gentamicin, streptomycin) Glycogen Short-term storage of glucose in Several known glycogen-storage diseases, the form of branched polymer including major groups of hepatic-Found in liver, skeletal muscle, hypoglycemic and muscle-energy and adipose tissue pathophysiologies Lipiddroplets Storage of esterified


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Histology_Ross. the form of branched polymer including major groups of hepatic-Found in liver, skeletal muscle, hypoglycemic and muscle-energy and adipose tissue pathophysiologies Lipiddroplets Storage of esterified forms of fatty acids Lipid storage diseases such as Gaucher’s as high-energy storage molecules and Niemann-Pick disease, liver cirrhosis aMyoclonic epilepsy and ragged red fibers syndrome. bMitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes syndrome.


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Histology_Ross. FIGURE 2.2 • Electron micrograph of microvilli on the apical surface of an absorptive cell. This electron micrograph shows the apical portion of absorptive cells with microvilli. Note that at this magnifcation the plasma membrane displays its characteristic appearance, showing two electron-dense lines separated by an electron-lucent intermediate layer. The glycoproteins of the glycocalyx can be seen extending from the tips of the microvilli into the lumen. The relationship between the outer plasma-membrane leafet and the glycocalyx is particularly well demonstrated. Glycoproteins of the glycocalyx include terminal digestive enzymes such as dipeptidases and disaccharidases.


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Histology_Ross. 100,000. (Courtesy of Dr. Ray C. Henrikson.) membrane by strong ionic interactions, mainly with integral proteins on both the extracellular and intracellular surfaces of the membrane (see Fig. 2.3). In addition, on the extracellular surface of the plasma membrane, carbohydrates may be attached to proteins, thereby forming glycoproteins; or to lipids of the bilayer, thereby forming glycolipids. These surface molecules constitute a layer at the surface of the cell, referred to as the cell coat or glycocalyx (see Fig. 2.2). They help establish extracellular microenvironments at the membrane surface that have specific functions in metabolism, cell recognition, and cell association and serve as receptor sites for hormones. Microdomains of the plasma membrane, known as lipid rafts, control the movement and distribution of proteins within the lipid bilayer.


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Histology_Ross. The ﬂuidity of the plasma membrane is not revealed in static electron micrographs. Experiments reveal that the membrane behaves as though it were a two-dimensional lipid ﬂuid. For many years it was thought that integral membrane proteins moved freely within the plane of the membrane; this movement was compared to the movement of icebergs ﬂoating in the ocean (see Fig. 2.3). However, recent evidence shows that the distribution and movement of proteins within the lipid bilayer is not as random as once thought. Localized regions within the plasma membrane contain high concentrations of cholesterol and glycosphingolipids. These regions are called lipid rafts. Owing to the high concentration of cholesterol and the presence of longer, highly saturated fatty-acid chains, the lipid raft area is thicker and exhibits less ﬂuidity than the surrounding plasma membrane (Fig. 2.4). Lipid rafts contain a variety of integral and peripheral membrane proteins involved in cell signaling. They can be


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Histology_Ross. thicker and exhibits less ﬂuidity than the surrounding plasma membrane (Fig. 2.4). Lipid rafts contain a variety of integral and peripheral membrane proteins involved in cell signaling. They can be viewed as “signaling platforms” ﬂoating in the ocean of lipids. Each individual raft is equipped with all of the necessary elements (receptors, coupling factors, effector enzymes, and substrates) to receive and convey specific signals. Signal transduction in lipid rafts occurs more rapidly and efficiently because of the close proximity of interacting proteins. In addition, different signaling rafts allow for the separation of specific signaling molecules from each other.


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Histology_Ross. Integral membrane proteins can be visualized with the special tissue preparation technique of freeze fracture. The existence of protein within the substance of the plasma membrane (i.e., integral proteins) was confirmed by a technique called freeze fracture. When tissue is prepared for electron microscopy by the freeze fracture process (Fig. 2.5a), membranes typically split or cleave along the hydrophobic plane (i.e., between the two lipid layers) to expose two interior faces of the membrane, an E-face and a P-face (see Fig. 2.5b). The E-face is backed by extracellular space, whereas the P-face is backed by cytoplasm ( protoplasm). The numerous particles seen on the Eand P-faces with the TEM represent the integral proteins of the membrane. Usually, the P-face displays more particles, thus more protein, than the E-face (see Fig. 2.5c). Integral membrane proteins have important functions in cell metabolism, regulation, and integration.


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Histology_Ross. Integral membrane proteins have important functions in cell metabolism, regulation, and integration. Six broad categories of membrane proteins have been defined in terms of their function: pumps, channels, receptors, linkers,


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Histology_Ross. FIGURE 2.3 • Diagram of a plasma membrane showing the modifed fuid–mosaic model. The plasma membrane is a lipid bilayer consisting primarily of phospholipid molecules, cholesterol, and protein molecules. The hydrophobic fatty-acid chains of phospholipids face each other to form the inner portion of the membrane, whereas the hydrophilic polar heads of the phospholipids form the extracellular and intracellular surfaces of the membrane. Cholesterol molecules are incorporated within the gaps between phospholipids equally on both sides of the membrane. Note the elevated area of the lipid raft that is characterized by the high concentration of glycosphingolipids and cholesterol. It contains large numbers of integral and peripheral membrane proteins. The raft protrudes above the level of asymmetrically distributed phospholipids in the membrane bilayer (indicated by the different colors of the phospholipid heads). Carbohydrate chains attach to both integral and peripheral membrane proteins to


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Histology_Ross. distributed phospholipids in the membrane bilayer (indicated by the different colors of the phospholipid heads). Carbohydrate chains attach to both integral and peripheral membrane proteins to form glycoproteins, as well as to polar phospholipid heads to form glycolipids.


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Histology_Ross. enzymes, and structural proteins (Fig. 2.6). The categories are  Pumps serve to transport certain ions, such as Na , actively not mutually exclusive (e.g., a structural membrane protein across membranes. Pumps also transport metabolic precursors may simultaneously serve as a receptor, an enzyme, a pump, or of macromolecules, such as amino acids and sugars, across any combination of these functions). membranes, either by themselves or linked to the Na pump.


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Histology_Ross. FIGURE 2.4 • An image of lipid rafts obtained with tapping-mode atomic force microscopy (AFM). This image shows a 5-nm-thick lipid bilayer spread on the mica support. The bilayer is composed of dioleoylphosphatidylcholine (dioleoyl-PC), sphingomyelin, and cholesterol. Sphingomyelin and cholesterol together form lipid rafts represented on the image by the pink areas; the blue-purple areas are the nonraft background of the bilayer. Because the sphingomyelin molecules are longer than the dioleoyl-PC molecules, the rafts protrude from the nonraft background by about 0.8 nm, and the AFM is sensitive enough to detect this protrusion. The black regions represent the mica support. The image also shows molecules of the Helicobacter pylori toxin VacA (white particles), which preferentially bind to protein receptors on the raft domains. The area depicted in this image is 800 nm square. (Courtesy of Drs. Nicholas A. Geisse, Timothy L. Cover, Robert M. Henderson, and J. Michael Edwardson.) inner


Histology_Ross

Histology_Ross. protein receptors on the raft domains. The area depicted in this image is 800 nm square. (Courtesy of Drs. Nicholas A. Geisse, Timothy L. Cover, Robert M. Henderson, and J. Michael Edwardson.) inner leaflet of lipid layer


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Histology_Ross. FIGURE 2.5 • Freeze fracture examination of the plasma membrane. a. View of the plasma membrane seen on edge, with arrow indicating the preferential plane of splitting of the lipid bilayer through the hydrophobic portion of the membrane. When the membrane splits, some proteins are carried with the outer leaflet, though most are retained within the inner leaflet. b. View of the plasma membrane with the leaflets separating along the cleavage plane. The surfaces of the cleaved membrane are coated, forming replicas; the replicas are separated from the tissue and examined with the TEM. Proteins appear as bumps. The replica of the inner leaflet is called the P-face; it is backed by cytoplasm (protoplasm). A view of the outer leaflet is called the E-face; it is backed by extracellular space. c. Electron micrograph of a freeze fracture replica shows the E-face of the membrane of one epithelial cell and the P-face of the membrane of the adjoining cell. The cleavage plane has jumped from the


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Histology_Ross. Electron micrograph of a freeze fracture replica shows the E-face of the membrane of one epithelial cell and the P-face of the membrane of the adjoining cell. The cleavage plane has jumped from the membrane of one cell to the membrane of the other cell, as indicated by the clear space (intercellular space) across the middle of the figure. Note the paucity of particles in the E-face compared with the P-face, from which the majority of the integral membrane proteins project. (Courtesy of Dr. Giuseppina d’Elia Raviola.)  Channels allow the passage of small ions, molecules, and include the family of integrins that link cytoplasmic actin water across the plasma membrane in either direction (i.e., filaments to an extracellular matrix protein (fibronectin). passive diffusion). Gap junctions formed by aligned chan- Enzymes have a variety of roles. ATPases have specific nels in the membranes of adjacent cells permit passage of roles in ion pumping: ATP synthase is the major protein of ions


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Histology_Ross. by aligned chan- Enzymes have a variety of roles. ATPases have specific nels in the membranes of adjacent cells permit passage of roles in ion pumping: ATP synthase is the major protein of ions and small molecules from the cytoplasm of one cell to the inner mitochondrial membrane, and digestive enzymes the cytoplasm of the adjacent cells. such as disaccharidases and dipeptidases are integral mem  Receptor proteins allow recognition and localized brane proteins. binding of ligands (molecules that bind to the extracellu- Structural proteins are visualized by the freeze fracture lar surface of the plasma membrane) in processes such as method, especially where they form junctions with neigh-hormonal stimulation, coated-vesicle endocytosis, and boring cells. Often, certain proteins and lipids are concenantibody reactions. trated in localized regions of the plasma membrane to  Linker proteins anchor the intracellular cytoskeleton to carry out specific functions. Examples of such regions


Histology_Ross

Histology_Ross. are concenantibody reactions. trated in localized regions of the plasma membrane to  Linker proteins anchor the intracellular cytoskeleton to carry out specific functions. Examples of such regions can the extracellular matrix. Examples of linker proteins be recognized in polarized cells such as epithelial cells.


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Histology_Ross. FIGURE 2.6 • Different functions of integral membrane proteins. The six major categories of integral membrane proteins are shown in this diagram: pumps, channels, receptors, linkers, enzymes, and structural proteins. These categories are not mutually exclu-sive. A structural membrane protein involved in cell-to-cell junctions might simultaneously serve as a receptor, enzyme, linker, or a com-bination of these functions. pumps channels receptors linkers enzymes structural proteins actin NaHK Integral membrane proteins move within the lipid bilayer of the membrane. Particles bound to the membrane can move on the surface of a cell; even integral membrane proteins, such as enzymes, may move from one cell surface to another (e.g., from apical to lateral) when barriers to ﬂow, such as cell junctions, are disrupted. The ﬂuidity of the membrane is a function of the types of phospholipids in the membrane and variations in their local concentrations.


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Histology_Ross. As previously mentioned, lipid rafts containing integral membrane proteins may move to a different region of the plasma membrane. The movement of an integral protein anchored on a lipid raft makes signaling more precise and prevents nonspecific interactions. The lateral migration of proteins is often limited by physical connections between membrane proteins and intracellular or extracellular structures. Such connections may exist between:  proteins associated with cytoskeletal elements and portions of the membrane proteins that extend into the adjacent cytoplasm, the cytoplasmic domains of membrane proteins, and  peripheral proteins associated with the extracellular matrix and the integral membrane proteins that extend from the cell surface (i.e., the extracellular domain).


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Histology_Ross. Through these connections, proteins can be localized or restricted to specialized regions of the plasma membrane or act as transmembrane linkers between intracellular and extracellular filaments (see the next section). Cell injury often manifests as morphologic changes in the cell’s plasma membrane, which results in the formation of plasma-membrane blebs. These are dynamic cell protrusions of the plasma membrane that are commonly observed in acute cell injury, in dividing and dying cells, and during cell movement. Blebbing is caused by the detachment of the plasma membrane from underlying actin flaments of the cell cytoskeleton. Cytoskeletal poisons that act on actin flaments such as phalloidin and cytochalasin-B cause extensive membrane blebbing. Substances that enter or leave the cell must traverse the plasma membrane.


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Histology_Ross. Substances that enter or leave the cell must traverse the plasma membrane. Some substances (fat-soluble and small, uncharged molecules) cross the plasma membrane by simple diffusion down their concentration gradient (Fig. 2.7a). All other molecules require membrane transport proteins to provide them with indi vidual passage across the plasma membrane. There are generally two classes of transport proteins:  Carrier proteins transfer small, water-soluble molecules. They are highly selective, often transporting only one type of molecule. After binding to a molecule designated for transport, the carrier protein undergoes a series of conformational changes and releases the molecule on the other side of the membrane (Fig. 2.7b). Some carrier proteins, such as the Na /K pump or H pump, require energy for active transport of molecules against their concentration gradient. Other carrier proteins, such as glucose carriers, do not require energy and participate in passive transport.


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Histology_Ross.  Channel proteins also transfer small, water-soluble molecules. In general, channels are made of transmembrane proteins with several membrane-spanning domains that create hydrophilic channels through the plasma membrane. Usually, channel proteins contain a pore domain that partially penetrates the membrane bilayer and serves as the ionselectivity filter. The pore domain is responsible for exquisite ion selectivity, which is achieved by regulation of its three-dimensional structure (Fig. 2.7c). Channels are ion selective and are regulated on the basis of the cell’s needs. Channel protein transport can be regulated by membrane potentials (e.g., voltage-gated ion channels in neurons), neurotransmitters (e.g., ligand-gated ion channels such as acetylcholine receptors in


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Histology_Ross. FIGURE 2.7 • Movement of molecules through the plasma membrane. Fat-soluble and other small, uncharged molecules (in green) cross the plasma membrane by simple diffusion down their concentration gradient. Other molecules require membrane transport proteins to provide them with individual passage across the plasma membrane. Small water-soluble molecules (in blue) require highly selective carrier proteins to transfer them across the plasma membrane. After binding with a molecule, the carrier protein undergoes a series of conformational changes and releases the molecule on the other side of the membrane. If the process requires energy, it is called active transport (e.g., transport of H ions against their concentration gradient). The process is called passive transport when energy is not required (e.g., glucose transport). Ions and other small charged molecules (in red) are transported through the plasma membrane by ion-selective channel proteins. In neurons, for instance, ion transport


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Histology_Ross. (e.g., glucose transport). Ions and other small charged molecules (in red) are transported through the plasma membrane by ion-selective channel proteins. In neurons, for instance, ion transport is regulated by membrane potentials (voltage-gated ion channels); in skeletal muscle cells, neuromuscular junctions possess ligand-gated ion channels.


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Histology_Ross. muscle cells), or mechanical stress (e.g., mechanically gated ion channels in the internal ear). Vesicular transport maintains the integrity of the plasma membrane and also provides for the transfer of molecules between different cellular compartments. Some substances enter and leave cells by vesicular transport, a process that involves configurational changes in the plasma membrane at localized sites and subsequent formation of vesicles from the membrane or fusion of vesicles with the membrane (Fig. 2.8). The major mechanism by which large molecules enter, leave, and move within the cell is called vesicle budding. Vesicles formed by budding from the plasma membrane of one compartment fuse with the plasma membrane of another compartment. Within the cell, this process ensures intercompartmental transfer of the vesicle contents.


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Histology_Ross. Vesicular transport involving the cell membrane may also be described in more specific terms:  Endocytosis is the general term for processes of vesicular transport in which substances enter the cell.  Exocytosis is the general term for processes of vesicular transport in which substances leave the cell. Both processes can be visualized with the electron microscope. FIGURE 2.8 • Endocytosis and exocytosis are two major forms of vesicular transport. Endocytosis brings molecules and other substances into the cell. In exocytosis, synthesized molecules and other substances leave the cell. Endocytosis is associated with the formation and budding of vesicles from the plasma membrane; exocytosis is associated with the fusion of vesicles originating from intracellular organelles with the plasma membrane, and it is a primary secretory modality. Uptake of ﬂuid and macromolecules during endocytosis depends on three different mechanisms.


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Histology_Ross. Some of the endocytotic mechanisms require special proteins during vesicle formation. The best known protein that interacts with the plasma membrane in vesicle formation is clathrin. Therefore, endocytosis can also be classified as either clathrin dependent or clathrin independent. In general, three mechanisms of endocytosis are recognized in the cell:  Pinocytosis [Gr., cell drinking] is the nonspecific ingestion of ﬂuid and small protein molecules via small vesicles, usually smaller than 150 nm in diameter. Pinocytosis is performed by virtually every cell in the organism, and it is constitutive (i.e., it involves a continuous dynamic formation of small vesicles at the cell surface (Fig. 2.9a). Recent studies indicate that mechanoenzymes such as GTPase (dynamin) are involved in pinocytotic vesicle scission (the process of pinching off from the plasma membrane). Pinocytotic vesicles are visible with the TEM, and they have a smooth surface. These smooth pinocytotic vesicles are


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Histology_Ross. vesicle scission (the process of pinching off from the plasma membrane). Pinocytotic vesicles are visible with the TEM, and they have a smooth surface. These smooth pinocytotic vesicles are especially numerous in the endothelium of blood vessels (Fig. 2.9b) and in smooth muscle cells. Pinocytosis does not require clathrin and


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Histology_Ross. FIGURE 2.9 • Pinocytosis. a. Pinocytosis involves the dynamic formation of small vesicles at the cell surface. First, substances to be pinocytosed (e.g., small soluble proteins, colloidal tracers) make contact with the extracellular surface of the plasma membrane; next, the surface becomes indented; and fnally, the invaginated portion of the membrane pinches off from the membrane to become a pinocytotic vesicle within the cell. b. This electron micrograph shows numerous smooth-surfaced pinocytotic vesicles (arrows) within the cytoplasm of endothelial cells of a blood vessel. 60,000. therefore may be referred to as clathrin-independent endocytosis.


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Histology_Ross.  Phagocytosis [Gr., cell eating ] is the ingestion of large particles such as cell debris, bacteria, and other foreign materials. In this nonselective process, plasma membrane sends out pseudopodia to engulf phagocytosed particles into large vesicles (larger than approximately 250 nm in diameter) called phagosomes. Phagocytosis is performed mainly by a specialized group of cells belonging to the mononuclear phagocytotic system (MPS). Phagocytosis is generally a receptor-mediated process in which receptors on the cell surface recognize non–antigen-binding domains (Fc fragments) of antibodies coating the surface of an invading microorganism or cell (Fig. 2.10a). Phagocytosis is also triggered by recognition of pathogen-associated molecular patterns (PAMPs) that are commonly expressed on pathogen surfaces by Toll-like receptors (page 278). This recognition of PAMPs leads to activation of nuclear factor kappa B (NF-B) transcription factor, which regulates genes that control cell


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Histology_Ross. on pathogen surfaces by Toll-like receptors (page 278). This recognition of PAMPs leads to activation of nuclear factor kappa B (NF-B) transcription factor, which regulates genes that control cell responses in phagocytosis. However, nonbiologic materials such as inhaled carbon particles, inorganic dusts, and asbestos fibers, as well as biologic debris from inﬂammation, wound healing, and dead cells, are sequestered by cells of the MPS without involvement of Fc receptors (Fig. 2.10b). This process does not require clathrin for phagosome formation. However, because of initial pseudopodial extensions of plasma membrane that contribute to the formation of phagosome, the actin cytoskeleton must be rearranged in a process that requires depolymerization and repolymerization of the actin filaments. Thus, phagocytosis is referred to as clathrin-independent but actin-dependent endocytosis.


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Histology_Ross.  Receptor-mediated endocytosis allows entry of specific molecules into the cell. In this mechanism, receptors for specific molecules, called cargo receptors, accumulate in well-defined regions of the cell membrane. These regions, which are represented by the lipid rafts in the plasma membrane, eventually become coated pits (Fig. 2.11a). The name coated pit is derived from these


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Histology_Ross. FIGURE 2.10 • Phagocytosis. a. This drawing shows the steps in the phagocytosis of a large particle, such as a bacterium that has been killed as a result of an immune response. The bacterium is surrounded by antibodies attached to the bacterial surface antigens. Fc receptors on the surface of the plasma membrane of the phagocytotic cells recognize the Fc portion of the antibodies. This interaction triggers rearrangement of actin cytoskeleton. Depolymerizations and repolymerizations of actin flaments produce temporary projections of the plasma membrane called pseudopodia. They surround phagocytosed particle and lead to the formation of phagosomes. By targeted delivery of lysosomal enzymes, a phagosome matures into a lysosome that digests its phagocytosed content. b. Nonbiologic materials such as inhaled carbon particles, inorganic dusts, and asbestos fbers, as well as cellular debris resulting from infammation, are internalized without involvement of antibodies and Fc receptors. These


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Histology_Ross. as inhaled carbon particles, inorganic dusts, and asbestos fbers, as well as cellular debris resulting from infammation, are internalized without involvement of antibodies and Fc receptors. These particles are bound to multiple receptors on the plasma membrane.


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Histology_Ross. regions’ appearance in the electron microscope (EM) as an accumulation of electron-dense material that represents aggregation of clathrin molecules on the cytoplasmic surface of the plasma membrane. Cargo receptors recognize and bind to specific molecules that come in contact with the plasma membrane. Clathrin molecules then assemble into a basketlike cage that helps change the shape of the plasma membrane into a vesiclelike invagination (Fig. 2.11b). Clathrin interacts with the cargo receptor via another coating-protein complex, adaptin, which is instrumental in selecting appropriate cargo molecules for transport into the cells. Thus, selected cargo proteins and their receptors are pulled from the extracellular space into the lumen of a forming vesicle. The large (100kilodalton) mechanoenzyme GTPase called dynamin mediates the liberation of forming clathrin-coated vesicles from the plasma membrane during receptor-mediated endocytosis. The type of vesicle formed as a result of


Histology_Ross

Histology_Ross. GTPase called dynamin mediates the liberation of forming clathrin-coated vesicles from the plasma membrane during receptor-mediated endocytosis. The type of vesicle formed as a result of receptor-mediated endocytosis is referred to as a coated vesicle, and the process itself is known as clathrindependent endocytosis. Clathrin-coated vesicles are also involved in the movement of the cargo material from the plasma membrane to early endosomes and from the Golgi apparatus to the early and late endosomes.


Histology_Ross

Histology_Ross. Exocytosis is the process by which a vesicle moves from the cytoplasm to the plasma membrane, where it discharges its contents to the extracellular space.


Histology_Ross

Histology_Ross. A variety of molecules produced by the cell for export are initially delivered from the site of their formation to the Golgi apparatus. The next step involves sorting and packaging the secretory product into transport vesicles that are destined to fuse with the plasma membrane in a process known as exocytosis. Intracellular traffic of these vesicles is achieved by the presence of specific proteins on their surface (coatomers such as COP-I and COP-II) that mediate their movements (see page 48). The molecules that travel this route are often chemically modified (e.g., glycosylated, sulfated) as they pass through different cellular compartments. The membrane that is added to the plasma membrane by exocytosis is recovered into the cytoplasmic compartment by an endocytotic process. There are two general pathways of exocytosis:  In the constitutive pathway, substances designated for export are continuously delivered in transport vesicles to the plasma membrane. Proteins that leave the


Histology_Ross

Histology_Ross. two general pathways of exocytosis:  In the constitutive pathway, substances designated for export are continuously delivered in transport vesicles to the plasma membrane. Proteins that leave the cell by this process are secreted immediately after their synthesis and exit from the Golgi apparatus, as seen in the secretion of


Histology_Ross

Histology_Ross. FIGURE 2.11 • Receptor-mediated endocytosis. a. This diagram shows the steps in receptor-mediated endocytosis, a transport mechanism that allows selected molecules to enter the cell. Cargo receptors recognize and bind specifc molecules that come in contact with the plasma membrane. Cargo receptor–molecule complexes are recognized by adaptin, a protein that helps select and gather appropriate complexes in specifc areas of the plasma membrane for transport into cells. Clathrin molecules then bind to the adaptin–cargo receptor–molecule complex to assemble into a shallow basketlike cage and form a coated pit. Clathrin interactions then assist the plasma membrane to change shape to form a deep depression, a fully formed coated pit that becomes pinched off from the plasma membrane by the protein complex dynamin as a coated vesicle (i.e., budding from the membrane). Selected cargo proteins and their receptors are thus pulled from the extracellular space into the lumen of a forming coated


Histology_Ross

Histology_Ross. complex dynamin as a coated vesicle (i.e., budding from the membrane). Selected cargo proteins and their receptors are thus pulled from the extracellular space into the lumen of a forming coated vesicle. After budding and internalization of the vesicle, the coat proteins are removed and recycled for further use. The uncoated vesicle travels to its destination to fuse with a cytoplasmic organelle. b. Electron micrograph of the cytoplasmic surface of the plasma membrane of A431 cells prepared by the quick-freeze deep-etch technique. This image shows coated pits and clathrin-coated vesicles in different stages of their formation. Note that the coated pits and clathrin-coated vesicles are formed in areas devoid of actin flaments. The small uniform pinocytotic vesicles do not have a clathrin coat and are located in close proximity to actin flaments. 200,000. (Courtesy of Dr. John E. Heuser,


Histology_Ross

Histology_Ross. Washington University School of Medicine.) immunoglobulins by plasma cells and of procollagen by fibroblasts. This pathway is present to some degree in all cells. The TEM reveals that these cells lack secretory granules.  In the regulated secretory pathway, specialized cells, such as endocrine and exocrine cells and neurons, concentrate secretory proteins and transiently store them in secretory vesicles within the cytoplasm (Fig. 2.12). In this case, a regulatory event (hormonal or neural stimulus) must be activated for secretion to occur, as in the release of secretory vesicles by chief cells of the gastric mucosa and by acinar cells of the pancreas. The signaling stimulus causes a transient inﬂux of Ca2 into the cytoplasm, which in turn stimulates secretory vesicles to fuse with the plasma membrane and discharge their contents (Fig. 2.13). In the past, secretory vesicle containing inactive precursor (zymogen) were called zymogen granules.


Histology_Ross

Histology_Ross. In addition to excretory pathways, proteins can be transported between the Golgi apparatus and other organelles along endosomal pathways. These pathways are used for delivery of organelle-specific proteins, such as lysosomal structural proteins, into the appropriate organelles. The precise targeting of vesicles to the appropriate cellular compartment is initially controlled by docking proteins, and specificity is ensured by interactions between soluble NSF attachment receptor (SNARE) proteins.


Histology_Ross

Histology_Ross. As discussed previously, newly formed vesicles that bud off from the donor membrane (such as cell membrane or Golgi cisternae) can fuse with a number of possible target membranes within the cell. Shortly after budding and shedding its clathrin coat, a vesicle must be targeted to the appropriate cellular compartment. A targeting mechanism can be likened to a taxi driver in a large city who successfully delivers a passenger to the proper street address. In the cell, the street address is recognized by Rab-GTPase bound to the membrane of the traveling vesicle. Rab-GTPase interacts with tethering proteins located on the target membrane. This initial interaction provides recognition of the vesicle and recruits the necessary number of tethering proteins to dock the incoming vesicle. The docking complex between Rab-GT-Pase and its receptor immobilizes the vesicle near the target membrane (Fig. 2.14). To ensure accurate targeting, each vesicle contains a vesicle-specifc membrane protein


Histology_Ross

Histology_Ross. complex between Rab-GT-Pase and its receptor immobilizes the vesicle near the target membrane (Fig. 2.14). To ensure accurate targeting, each vesicle contains a vesicle-specifc membrane protein called a v-SNARE. The target membrane also contains a specific membrane protein, t-SNARE, that interacts with v-SNARE to form the cis-SNARE complex. SNAREs are a family of transmembrane proteins that were originally grouped according to their location within the vesicle (v-SNARE) or target membrane (t-SNARE). They guarantee the specificity of interaction between a particular vesicle and its target membrane and also promote membrane fusion that follows immediately after the cis-SNARE complexes are formed. After fusion, the SNARE complexes are dismantled with the help of the NSF/ -SNAP protein complex and recycled for use in another round of vesicle fusion.


Histology_Ross

Histology_Ross. FIGURE 2.12 • Photomicrograph of secretory cells of the pancreas. Note that secretory vesicles containing protein ready for secretion fll the apical portion of the cells. This process re-quires an external signaling mechanism for the cell to discharge the accumulated granules. 860. The TEM reveals the presence in the cytoplasm of membrane-enclosed compartments associated with all the endocytotic pathways described above (Fig. 2.15). These compartments, called early endosomes, are restricted to a portion of the


Histology_Ross

Histology_Ross. FIGURE 2.13 • Diagram showing two pathways for exocytosis. Newly synthesized proteins are synthesized in the rough endoplasmic reticulum (rER). After their initial post-translational modifcation, they are delivered in COP-II–coated vesicles to the Golgi apparatus. After additional modifcation in the Golgi apparatus, sorting, and packaging, the fnal secretory product is transported to the plasma membrane in vesicles that form from the trans-Golgi network (TGN). Note that retrograde transport is present between Golgi cisternae and is mediated by the COP-I–coated vesicle. Two distinct pathways are recognized. Blue arrows indicate the constitutive pathway in which proteins leave the cell immediately after their synthesis. In cells using this pathway, almost no secretory product accumulates, and thus few secretory vesicles are present in the cytoplasm. Red arrows indicate the regulated secretory pathway in which protein secretion is regulated by hormonal or neural stimuli. In cells using


Histology_Ross

Histology_Ross. and thus few secretory vesicles are present in the cytoplasm. Red arrows indicate the regulated secretory pathway in which protein secretion is regulated by hormonal or neural stimuli. In cells using this pathway, such as the pancreatic acinar cells in Figure 2.12, secretory proteins are concentrated and transiently stored in secretory vesicles within the cytoplasm. After appropriate stimulation, the secretory vesicles fuse with the plasma membrane and discharge their contents.


Histology_Ross

Histology_Ross. cytoplasm near the cell membrane where vesicles originating from the cell membrane fuse. From here, many vesicles return to the plasma membrane. However, large numbers of vesicles originating in early endosomes travel to deeper structures in the cytoplasm called late endosomes. The latter typically mature into lysosomes.


Histology_Ross

Histology_Ross. FIGURE 2.14 • Steps in formation, targeting, docking, and fusion of transport vesicles with the target membrane. (1) Lipid raft with cargo receptors ready to interact with cargo protein. Note the presence of the specifc targeting protein v-SNARE. (2) Initial step in vesicle formation: The binding of the adaptin complex and clathrin forms a coated pit. (3) Formation (budding) of fully assembled coated vesicle. (5) Disassembly of clathrin coat. Note the expression of Rab-GTPase activity. (6) Tethering of the vesicle to the target membrane by the interaction between Rab-GTPase and tethering proteins. (7) Beginning of the docking process (recruitment of tethering proteins). (8) Formation of the docking complex between Rab-GTPase and its protein in the target membrane: v-SNAREs on the immobilized vesicle interact with t-SNAREs on the target membrane to form the cis-SNARE complex. (9) Fusion of the vesicle to the target membrane. (10) Discharge of the cargo protein into the early endosomal


Histology_Ross

Histology_Ross. vesicle interact with t-SNAREs on the target membrane to form the cis-SNARE complex. (9) Fusion of the vesicle to the target membrane. (10) Discharge of the cargo protein into the early endosomal compartment and disassembly of the cis complex by the interaction of the NSF/ -SNAP protein complex. (11) Recycling of v-SNAREs in the transport vesicles for use in another round of vesicle targeting and fusion.


Histology_Ross

Histology_Ross. FIGURE 2.15 • Electron micrograph of an early endosome. This deep-etch electron micrograph shows the structure of an early endosome in Dictyostelium. Early endosomes are located near the plasma membrane and, as in many other sorting compartments, have a typical tubulovesicle structure. The tubular portions contain the majority of integral membrane proteins destined for membrane recycling, whereas the luminal portions collect secretory cargo proteins. The lumen of the endosome is subdivided into multiple compartments, or cisternae, by the invagination of its membrane and undergoes frequent changes in shape. 15,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.) Endosomes can be viewed either as stable cytoplasmic organelles or as transient structures formed as the result of endocytosis.


Histology_Ross

Histology_Ross. Endosomes can be viewed either as stable cytoplasmic organelles or as transient structures formed as the result of endocytosis. Recent experimental observations of endocytotic pathways conducted in vitro and in vivo suggest two different models that explain the origin and formation of the endosomal compartments in the cell:  The stable compartment model describes early and late endosomes as stable cellular organelles connected by vesicular transport with the external environment of the cell and with the Golgi apparatus. Coated vesicles formed at the plasma membrane fuse only with early endosomes because of their expression of specific surface receptors. The receptor remains a resident component of the early endosomal membrane.


Histology_Ross

Histology_Ross.  In the maturation model, early endosomes are formed de novo from endocytotic vesicles originating from the plasma membrane. Therefore, the composition of the early endosomal membrane changes progressively as some components are recycled between the cell surface and the Golgi apparatus. This maturation process leads to formation of late endosomes and then to lysosomes. Specific receptors present on early endosomes (e.g., for coated vesicles) are removed by recycling, degradation, or inactivation as this compartment matures. Both models actually complement rather than contradict each other in describing, identifying, and studying the pathways of internalized molecules. Endosomes destined to become lysosomes receive newly synthesized lysosomal enzymes that are targeted via the mannose-6-phosphate receptor.


Histology_Ross

Histology_Ross. Some endosomes also communicate with the vesicular transport system of the rER. This pathway provides constant delivery of newly synthesized lysosomal enzymes, or hydrolases. A hydrolase is synthesized in the rER as an enzymatically inactive precursor called a prohydrolase. This heavily glycosylated protein then folds in a specific way so that a signal patch is formed and exposed on its surface. This recognition signal is created when specific amino acids are brought into close proximity by the three-dimensional folding of the protein. The signal patch on a protein destined for a lysosome is then modified by several enzymes that attach mannose-6phosphate (M-6-P) to the prohydrolase surface. M-6-P acts as a target for proteins possessing an M-6-P receptor. M-6-P receptors are present in early and late endosomes, lysosomes, and the Golgi apparatus, which is involved in sorting and retrieving secreted prohydrolases destined for transport to endosomes (Fig. 2.16). The acidic environment


Histology_Ross

Histology_Ross. and late endosomes, lysosomes, and the Golgi apparatus, which is involved in sorting and retrieving secreted prohydrolases destined for transport to endosomes (Fig. 2.16). The acidic environment of late endosomes causes the release of prohydrolases from the M-6-P receptors. Prohydrolases are next activated by cleavage and by removing phosphate groups from the mannose residues.


Histology_Ross

Histology_Ross. Early and late endosomes differ in their cellular localization, morphology, and state of acidification and function. Early and late endosomes are localized in different areas of the cell. Early endosomes can be found in the more peripheral cytoplasm, whereas late endosomes are often positioned near FIGURE 2.16 • Pathways for delivery of newly synthesized lysosomal enzymes. Lysosomal enzymes (such as lysosomal hydrolases) are synthesized and glycosylated within the rough endoplasmic reticulum (rER). The enzymes then fold in a specifc way so that a signal patch is formed, which allows for further modifcation by the addition of M-6-P, which allows the enzyme to be targeted to specifc proteins that possess M-6-P receptor activity. M-6-P receptors are present in the TGN of the Golgi apparatus, where the lysosomal enzymes are sorted and packaged into vesicles later transported to the early or late endosomes.


Histology_Ross

Histology_Ross. the Golgi apparatus and the nucleus. An early endosome has a tubulovesicular structure: The lumen is subdivided into cisternae that are separated by invagination of its membrane. It exhibits only a slightly more acidic environment (pH 6.2 to 6.5) than the cytoplasm of the cell. In contrast, late endosomes have a more complex structure and often exhibit onionlike internal membranes. Their pH is more acidic, averaging 5.5. TEM studies reveal specific vesicles that transport substances between early and late endosomes. These vesicles, called multivesicular bodies (MVBs), are highly selective transporters. Within early endosomes, proteins destined to be transported to late endosomes are sorted and separated from proteins destined for recycling and packaging into MVBs (Fig. 2.17). In general, substances transported to late endosomes are eventually degraded in lysosomes in a default process that does not require any additional signals. Because late endosomes mature into lysosomes, they are


Histology_Ross

Histology_Ross. substances transported to late endosomes are eventually degraded in lysosomes in a default process that does not require any additional signals. Because late endosomes mature into lysosomes, they are also called prelysosomes. Advances in videomicroscopy now allow researchers to observe the complex behavior of these organelles; late lysosomes may fuse with each other or with mature lysosomes.


Histology_Ross

Histology_Ross. The major function of early endosomes is to sort and recycle proteins internalized by endocytotic pathways. MVBHpH 6.2 pH 5.5 pH 4.7


Histology_Ross

Histology_Ross. MVBHpH 6.2 pH 5.5 pH 4.7 FIGURE 2.17 • Schematic diagram of endosomal compartments of the cell. This diagram shows the fate of protein (red circles) endocytosed from the cell surface and destined for lysosomal destruction. Proteins are frst found in endocytotic (coated) vesicles that deliver them to early endosomes, which are located in the peripheral part of cytoplasm. Because of the sorting capability of the early endosomes, receptors are usually recycled to the plasma membrane, and endocytosed proteins are transported via multi-vesicular bodies (MVB) to late endosomes positioned near the Golgi apparatus and the nucleus. The proteins transported to late endosomes eventually will be degraded in lysosomes. Note the acidifcation scale (left) that illustrates changes of pH from early endosomes to lysosomes. The acidifcation is accomplished by the active transport of protons into endosomal compartments.


Histology_Ross

Histology_Ross. Early endosomes sort proteins that have been internalized by endocytotic processes. The morphologic shape and geometry of the tubules and vesicles emerging from the early endosome create an environment in which localized changes in pH constitute the basis of the sorting mechanism. This mechanism includes dissociation of ligands from their receptor protein; thus, in the past, early endosomes were referred to as compartments of uncoupling receptors and ligands (CURLs). In addition, the narrow diameter of the tubules and vesicles may also aid in the sorting of large molecules, which can be mechanically prevented from entering specific sorting compartments. After sorting, most of the protein is rapidly recycled, and the excess membrane is returned to the plasma membrane. The fate of the internalized ligand–receptor complex depends on the sorting and recycling ability of the early endosome.


Histology_Ross

Histology_Ross. The following pathways for processing internalized ligand–receptor complexes are present in the cell:  The receptor is recycled and the ligand is degraded. Surface receptors allow the cell to bring in substances selectively through the process of endocytosis. This pathway occurs most often in the cell; it is important because it allows surface receptors to be recycled. Most ligand–receptor complexes dissociate in the acidic pH of the early endosome. The receptor, most likely an integral membrane protein (see page 29), is recycled to the surface via vesicles that bud off the ends of narrow-diameter tubules of the early endosome. Ligands are usually sequestered in the spherical vacuolar part of the endosome that will later form MVBs, which will transport the ligand to late endosomes for further degradation in the lysosome (Fig. 2.18a). This pathway is described for the low-density lipoprotein (LDL)–receptor complex, insulin–glucose transporter (GLUT) receptor complex, and a variety


Histology_Ross

Histology_Ross. further degradation in the lysosome (Fig. 2.18a). This pathway is described for the low-density lipoprotein (LDL)–receptor complex, insulin–glucose transporter (GLUT) receptor complex, and a variety of peptide hormones and their receptors.


Histology_Ross

Histology_Ross.  Both receptor and ligand are recycled. Ligand–receptor complex dissociation does not always accompany receptor recycling. For example, the low pH of the endosome dissociates iron from the iron-carrier protein transferrin, but transferrin remains associated with its receptor. Once the transferrin–receptor complex returns to the cell surface, however, transferrin is released. At neutral extracellular pH, transferrin must again bind iron to be recognized by and bound to its receptor. A similar pathway is recognized for major histocompatibility complex (MHC) I and II molecules, which are recycled to the cell surface with a foreign antigen protein attached to them (Fig. 2.18b).


Histology_Ross

Histology_Ross.  Both receptor and ligand are degraded. This pathway has been identified for epidermal growth factor (EGF) and its receptor. Like many other proteins, EGF binds to its receptor on the cell surface. The complex is internalized and carried to the early endosomes. Here EGF dissociates from its receptor, and both are sorted, packaged in separate MVBs, and transferred to the late endosome. From there, both ligand and receptor are transferred to lysosomes, where they are degraded (Fig. 2.18c).  Both receptor and ligand are transported through the cell. This pathway is used for secretion of immunoglobulins (secretory IgA) into the saliva and human milk. During this process, commonly referred to as transcytosis, substances can be altered as they are transported across the epithelial cell (Fig. 2.18d). Transport of maternal IgG across the placental barrier into the fetus also follows a similar pathway.


Histology_Ross

Histology_Ross. Lysosomes are digestive organelles that were recognized only after histochemical procedures were used to demonstrate lysosomal enzymes. Lysosomes are organelles rich in hydrolytic enzymes such as proteases, nucleases, glycosidases, lipases, and phospholipases.


Histology_Ross

Histology_Ross. FIGURE 2.18 • Fate of receptor and ligand in receptor-mediated endocytosis. This diagram shows four major pathways along which the fate of internalized ligand–receptor complexes is determined. a. The internalized ligand–receptor complex dissociates, the receptor is recycled to the cell surface, and the ligand is directed to late endosomes and eventually degraded within lysosomes. This processing pathway is used by the LDL–receptor complex, insulin–GLUT receptor complex, and a variety of peptide hormone– receptor complexes. MVB, multivesicular bodies. b. Both internalized receptor and ligand are recycled. Ligand–receptor complex dissociation does not occur, and the entire complex is recycled to the surface. An example is the iron–transferrin–transferrin receptor complex that uses this processing pathway. Once iron (Fe) is released in the endosome, the transferrin–transferrin receptor complex returns to the cell surface, where transferrin is released. c. The internalized ligand–receptor


Histology_Ross

Histology_Ross. pathway. Once iron (Fe) is released in the endosome, the transferrin–transferrin receptor complex returns to the cell surface, where transferrin is released. c. The internalized ligand–receptor complex dissociates in the early endosome. The free ligand and the receptor are directed to the late endosomal compartment for further degradation. This pathway is used by many growth factors (i.e., the EGF–receptor complex). d. The internalized ligand–receptor complex is transported through the cell. Dissociation does not occur, and the entire complex undergoes transcytosis and release at a different site of the cell surface. This pathway is used during secretion of immunoglobulins (secretory IgA) into saliva. The antibody IgA–receptor complex is internalized at the basal surface of the secretory cells in the salivary gland and released at the apical surface.


Histology_Ross

Histology_Ross. A lysosome represents a major digestive compartment in the cell that degrades macromolecules derived from endocytotic pathways as well as from the cell itself in a process known as autophagy (removal of cytoplasmic components, particularly membrane-bounded organelles, by digesting them within lysosomes). For more information about autophagy, see page 41.


Histology_Ross

Histology_Ross. The first hypothesis for lysosomal biogenesis, formulated almost a half century ago, postulated that lysosomes arise as complete and functional organelles budding from the Golgi apparatus. These newly formed lysosomes were termed primary lysosomes in contrast to secondary lysosomes, which had already fused with incoming endosomes. However, the primary and secondary lysosome hypothesis has proved to have little validity as new research data allow a better understanding of the details of protein secretory pathways and the fate of endocytotic vesicles. It is now widely accepted that lysosomes are formed in a complex series of pathways that converge at the late endosomes, transforming them into lysosomes. These pathways are responsible for a targeted delivery of newly synthesized lysosomal enzymes and structural lysosomal membrane proteins into the late endosomes. As stated earlier, lysosomal enzymes are synthesized in the rER and sorted in the Golgi apparatus based on their binding


Histology_Ross

Histology_Ross. enzymes and structural lysosomal membrane proteins into the late endosomes. As stated earlier, lysosomal enzymes are synthesized in the rER and sorted in the Golgi apparatus based on their binding ability to M-6-P receptors (see page 37).


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Histology_Ross. Lysosomes have a unique membrane that is resistant to the hydrolytic digestion occurring in their lumen.


Histology_Ross

Histology_Ross. Lysosomes contain a collection of hydrolytic enzymes and are surrounded by a unique membrane that resists hydrolysis by their own enzymes (Fig. 2.19). The lysosomal membrane has an unusual phospholipid structure that contains cholesterol and a unique lipid called lysobisphosphatidic acid. Most of the structural lysosomal membrane proteins are classified into lysosome-associated membrane proteins (lamps), lysosomal membrane glycoproteins (lgps), and lysosomal integral membrane proteins (limps). The lamps, lgps, and limps represent more than 50% of the total membrane proteins in lysosomes and are highly glycosylated on the luminal surface. Sugar molecules cover almost the entire luminal surface of these proteins, thus protecting them from digestion by hydrolytic enzymes. Lysobisphosphatidic acids within the lysosomal membrane may play an important role in restricting the activity of hydrolytic enzymes directed against the membrane. The same family of membrane proteins is also detected


Histology_Ross

Histology_Ross. acids within the lysosomal membrane may play an important role in restricting the activity of hydrolytic enzymes directed against the membrane. The same family of membrane proteins is also detected in late


Histology_Ross

Histology_Ross. FIGURE 2.19 • Schematic diagram of a lysosome. This diagram shows a few selected lysosomal enzymes residing inside the lysosome and their respective substrates. The major lysosomal membrane-specifc proteins, as well as a few other proteins associated H membrane impermeable to enzymes; contains specific lysosomal proteins, lamps, limps, and lgps with membrane transport, are also shown. endosomes. In addition, lysosomes and late endosomes contain proton (H ) pumps that transport H ions into the lysosomal lumen, maintaining a low pH (4.7). The lysosomal membrane also contains transport proteins that transport the final products of digestion (amino acids, sugars, nucleotides) to the cytoplasm, where they are used in the synthetic processes of the cell or are exocytosed.


Histology_Ross

Histology_Ross. Certain drugs can affect lysosomal function. For example, chloroquine, an agent used in the treatment and prevention of malaria, is a lysosomotropic agent that accumulates in the lysosomes. It raises the pH of the lysosomal content, thereby inactivating many lysosomal enzymes. The action of chloroquine on lysosomes accounts for its antimalarial activity; the drug concentrates in the acidic food vacuole of the malaria parasite (Plasmodium falciparum) and interferes with its digestive processes, eventually killing the parasite. Lysosomal membrane proteins are synthesized in the rER and have specific lysosomal targeting signal.


Histology_Ross

Histology_Ross. Lysosomal membrane proteins are synthesized in the rER and have specific lysosomal targeting signal. As mentioned previously, the intracellular trafficking leading to the delivery of many soluble lysosomal enzymes to late endosomes and lysosomes involves the M-6-P signal and its receptor. All membrane proteins destined for lysosomes (and late endosomes) are synthesized in the rER and transported to and sorted in the Golgi apparatus. However, they do not contain the M-6-P signals and must be targeted to lysosomes by a different mechanism. The targeting signal for integral membrane proteins is represented by a short cytoplasmic C-terminus domain, which is recognized by adaptin protein complexes and packaged into clathrin-coated vesicles. These proteins reach their destination by one of two pathways:  In the constitutive secretory pathway, limps exit the


Histology_Ross

Histology_Ross. Golgi apparatus in coated vesicles and are delivered to the cell surface. From here, they are endocytosed and, via the early and late endosomal compartments, finally reach lysosomes (Fig. 2.20).  In the Golgi-derived coated vesicle secretory pathway, limps, after sorting and packaging, exit the Golgi apparatus in clathrin-coated vesicles (see Fig. 2.20). These transport vesicles travel and fuse with late endosomes as a result of interaction between endosome-specific components of v-SNARE and t-SNARE docking proteins (see page 34). Three different pathways deliver material for intracellular digestion in lysosomes.


Histology_Ross

Histology_Ross. Three different pathways deliver material for intracellular digestion in lysosomes. Depending on the nature of the digested material, different pathways deliver material for digestion within the lysosomes (Fig. 2.21). In the digestion process, most of the digested material comes from endocytotic processes; however, the cell also uses lysosomes to digest its own obsolete parts, nonfunctional organelles, and unnecessary molecules. Three pathways for digestion exist:  Extracellular large particles such as bacteria, cell debris, and other foreign materials are engulfed in the process of phagocytosis. A phagosome, formed as the material is internalized within the cytoplasm, subsequently receives hydrolytic enzymes to become a late endosome, which matures into a lysosome.


Histology_Ross

Histology_Ross.  Extracellular small particles such as extracellular proteins, plasma-membrane proteins, and ligand–receptor complexes are internalized by pinocytosis and receptor-mediated endocytosis. These particles follow the endocytotic pathway through early and late endosomal compartments and are finally degraded in lysosomes.  Intracellular particles such as entire organelles, cytoplasmic proteins, and other cellular components are isolated from the cytoplasmic matrix by endoplasmic reticulum membranes, transported to lysosomes, and degraded. This process is called autophagy (see page 41).


Histology_Ross

Histology_Ross. FIGURE 2.20 • Lysosome biogenesis. This diagram shows regulated and constitutive pathways for delivery of lysosomal specifc membrane proteins into early and late endosomes. The lysosomal membrane possesses highly glycosylated specifc membrane proteins that protect the membrane from digestion by lysosomal enzymes. These lysosome-specifc proteins are synthesized in the rough endoplasmic reticulum, transported to the Golgi apparatus, and reach their destination by two pathways. Blue arrows indicate the constitutive secretory pathway in which certain lysosomal membrane proteins exit the Golgi apparatus and are delivered to the cell surface. From there they are endocytosed and, via the early and late endosomal compartments, fnally reach lysosomes. Green arrows indicate the endosomal Golgiderived coated vesicle secretory pathway. Here, other lysosomal proteins, after sorting and packaging, exit the Golgi apparatus in clathrin-coated vesicles to fuse with early and late endosomes.


Histology_Ross

Histology_Ross. In addition, some cells (e.g., osteoclasts involved in bone resorption and neutrophils involved in acute infammation) may release lysosomal enzymes directly into the extracellular space to digest components of the extracellular matrix. Lysosomes in some cells are recognizable in the light microscope because of their number, size, or contents. The numerous azurophilic granules of neutrophils (white blood cells) represent lysosomes and are recognized in aggregate by their specific staining. Lysosomes that contain phagocytized bacteria and fragments of damaged cells are often recognized in macrophages. Hydrolytic breakdown of the contents of lysosomes often produces a debris-filled vacuole called a residual body that may remain for the entire life of the cell. For example, in neurons, residual bodies are called age pig-


Histology_Ross

Histology_Ross. FIGURE 2.21 • Pathways of delivery of materials for digestion in lysosomes. Most of the small extracellular particles are internalized by both receptor-mediated endocytosis and pinocytosis. These two endocytic patways are labeled with red arrows. Large extracellular particles such as bacteria and cellular debris are delivered for cellular digestion via the phagocytotic pathway (blue arrows). The cell also uses lysosomes to digest its own organelles and other intracellular proteins via the autophagic pathway (green arrows). Intracellular particles are isolated from the cytoplasmic matrix by the isolation membrane of the sER, transported to lysosomes, and subsequently degraded. ment or lipofuscin granules. Residual bodies are a normal feature of cell aging. The absence of certain lysosomal enzymes can cause the pathologic accumulation of undigested substrate in residual bodies. This can lead to several disorders collectively termed lysosomal storage diseases (see Folder 2.1).


Histology_Ross

Histology_Ross. Autophagy represents the major cellular pathway in which a number of cytoplasmic proteins, organelles, and other cellular structures are degraded in the lysosomal compartment (Fig. 2.22). This important process maintains a well-controlled balance between anabolic and catabolic cell functions and permits the cell to eliminate unwanted or unnecessary organelles. Digested components of organelles are recycled and reused for normal cell growth and development.


Histology_Ross

Histology_Ross. Many genetic disorders have been identifed in individuals who have mutations in a gene that encodes lysosomal pro-teins. These diseases are termed lysosomal storage diseases (LSDs) and are characterized by dysfunctional lysosomes. The defective protein in most cases is a hy-drolytic enzyme or its cofactor; less commonly, lysosomal membrane proteins or proteins that are involved in sorting, targeting, and transporting lysosomal proteins are defec-tive. The result is an accumulation in cells of the specifc products that lysosomal enzymes normally use as sub-strates in their reactions. These undigested, accumulated products disrupt the normal function of the cell and lead to its death. Currently, 49 disorders are known LSDs with a collec-tive incidence of about 1 in 7,000 live births. The life ex-pectancy across the entire group of people with these disorders is 15 years. The frst LSD was described in 1881 by British ophthalmologist Warren Tay, who reported symp-toms of retinal


Histology_Ross

Histology_Ross. The life ex-pectancy across the entire group of people with these disorders is 15 years. The frst LSD was described in 1881 by British ophthalmologist Warren Tay, who reported symp-toms of retinal abnormalities in a 12-month-old infant with severe neuromuscular symptoms. In 1896, U.S. neurologist Bernard Sachs described a patient with similar eye symp-toms found earlier by Tay. This disease is now known as Tay-Sachs disease. It is caused by the absence of one enzyme, a lysosomal galactosidase ( -hexosaminidase) that catalyzes a step in lysosomal breakdown of ganglio-sides in neurons. The resulting accumulation of the GM2 ganglioside that is found within concentric lamellated struc-tures in residual bodies of neurons interferes with normal cell function. Children born with LSDs usually appear normal at birth; however, they soon show clinical signs of the disease. They often experience slower growth, show changes in fa-cial features, and develop bone and joint deformities that lead to


Histology_Ross

Histology_Ross. normal at birth; however, they soon show clinical signs of the disease. They often experience slower growth, show changes in fa-cial features, and develop bone and joint deformities that lead to signifcant restrictions of limb movement. They may lose already attained skills such as speech and learning ability. Behavioral problems may occur, as well as mental retardation. They are prone to frequent lung infections and heart disease. Some children have enlarged internal or-gans such as the liver and spleen (hepatosplenomegaly). The most common LSDs in children are Gaucher disease, Hurler syndrome (MPS I), Hunter syndrome (MPS II), and Pompe disease. Not long ago, LSDs were seen as neurodegenerative disorders without any potential treatment. In the last two decades, there has been limited success in treating the symptoms of LSDs. Considerable effort has been devoted to genetic research and fnding methods to replace the missing enzymes that cause various forms of LSD. Enzyme-replacement


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Histology_Ross. in treating the symptoms of LSDs. Considerable effort has been devoted to genetic research and fnding methods to replace the missing enzymes that cause various forms of LSD. Enzyme-replacement therapy, which requires the cellu-lar delivery of a manufactured recombinant enzyme, is avail-able for some LSDs such as cystinosis and Gaucher disease. Enzymes have also been supplied by transplanta-tion of bone marrow containing normal genes from an un-affected person. Success of the enzyme-replacement therapy is often limited by insuffcient biodistribution of re-combinant enzymes and high costs. Recently emerging strategies for the treatment of LSDs include pharmaco-logical chaperone therapy in which chaperone molecules are delivered to affected cells. In some cases, synthetic chaparones can assist in folding of mutated en-zymes to improve their stability and advance their lysoso-mal delivery. In the future, the combination of different therapies such as enzyme replacement, pharmacological


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Histology_Ross. in folding of mutated en-zymes to improve their stability and advance their lysoso-mal delivery. In the future, the combination of different therapies such as enzyme replacement, pharmacological chaperone, and gene-transfer therapies with the devel-opment of newborn screening tests will enable early detec-tion and improve clinical outcome of patients with LSDs.


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Histology_Ross. FOLDER 2.1 Clinical Correlation: Lysosomal Storage Diseases


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Histology_Ross. Summary of Common Lysosomal Storage Diseases (LSDs) Accumulating Product Disease Protein Deficiency (or Defective Process) Gaucher disease Glucocerebrosidase Glucosylceramide Tay-Sachs disease -hexosaminidase, -subunit GM2 ganglioside Sandhoff disease -hexosaminidase, -subunit GM2 ganglioside, oligosaccharides Krabbe disease Galactosylceramidase Gal-ceramide, gal-sphingosine Niemann-Pick disease A,B Sphingomyelinase Sphingomyelin Disorders of Glycoprotein Degradation Aspartylglycosaminuria Aspartylglycosaminidase N-linked oligosaccharides -Mannosidosis -Mannosidase -Mannosides Disorders of Sphingolipid Degradation chapter 2 Disorders of Glycosaminoglycan Degradation Hurler syndrome (mucopolysaccharidosis I, -L-iduronidase Dermatan sulfate, heparan sulfate MPS I) Hunter syndrome (MPS II) L-Iduronate sulfatase Dermatan sulfate, heparan sulfate Maroteaux-Lamy syndrome (MPS IV) GalNAc 4-sulfatase/arylsulfatase B Dermatan sulfate Other Disorders of Single Enzyme Deficiency Pompe disease


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Histology_Ross. L-Iduronate sulfatase Dermatan sulfate, heparan sulfate Maroteaux-Lamy syndrome (MPS IV) GalNAc 4-sulfatase/arylsulfatase B Dermatan sulfate Other Disorders of Single Enzyme Deficiency Pompe disease (glycogenosis II) -1,4-Glucosidase Glycogen Wolman disease (familial xanthomatosis) Acid lipase Cholesterol esters, triglycerides Canavan disease (aspartoacylase Aspartoacylase N-acetylaspartic acid deficiency) Disorders of Lysosomal Biogenesis Inclusion-cell (I-cell) disease, GlcNAc-1-phosphotransferase Lysosomal hydrolyses are not mucolipidosis II (GlcNAcPTase) present in lysosomes Leads to defective sorting of most soluble hydrolytic lysosomal enzymes Disorders of the Lysosomal Membrane Danon disease lamp2 Presence of autophagic vacuoles Cystinosis Cystinosin (cystine transporter) Cystine Summary of Common Lysosomal Storage Diseases (LSDs) (Cont.) Accumulating Product Disease Protein Deficiency (or Defective Process)


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Histology_Ross. Cytoplasmic proteins and organelles are substrates for lysosomal degradation in the process of autophagy.


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Histology_Ross. Autophagy plays an essential role during starvation, cellular differentiation, cell death, and cell aging. In the last few years, applying genetic-screening tests originally developed for yeasts, researchers uncovered several autophagy-related genes (Atg genes) in mammalian cell genome. The presence of adequate nutrients and growth factors stimulates enzymatic activity of a serine/threonine kinase known as mammalian target of rapamycin (mTOR). High mTOR activity exerts an inhibitory effect on autophagy. The opposite is found in nutrient starvation, hypoxia, and high temperature, where lack of mTOR activity causes activation of Atg genes. This results in formation of an Atg1 proteinkinase autophagy–regulatory complex that initiates process of autophagy. Generally, autophagy can be divided into three well-characterized pathways:  Macroautophagy, or simply autophagy, is a nonspecific process in which a portion of the cytoplasm or an entire organelle is first surrounded by a double or


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Histology_Ross. into three well-characterized pathways:  Macroautophagy, or simply autophagy, is a nonspecific process in which a portion of the cytoplasm or an entire organelle is first surrounded by a double or multilamellar intracellular membrane of endoplasmic reticulum, called isolation membrane, to form a vacuole called an autophagosome. This process is aided by proteins encoded by several Atg genes. At first, the complex containing Atg12–Atg5–Atg16L proteins attaches to a part of endoplastic reticulum and localizes the isolation membrane. Subsequently, Atg8 is recruited and bound to the membrane. Together they change the shape of the isolation membrane, which bends to enclose and seal an organelle destined for digestion within the lumen of the autophagosome. Once the autophagosome is completed, the Atg12–Atg5–Atg16L complex and Atg8 dissociate from this structure. After targeted delivery of lysosomal enzymes autophagosome matures into a lysosome. The isolation membrane disintegrates within


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Histology_Ross. the Atg12–Atg5–Atg16L complex and Atg8 dissociate from this structure. After targeted delivery of lysosomal enzymes autophagosome matures into a lysosome. The isolation membrane disintegrates within the hydrolytic compartment of a lysosome. Macroautophagy occurs in the liver during the first stages of starvation (Fig. 2.23).


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Histology_Ross.  Microautophagy is also a nonspecific process in which cytoplasmic proteins are degraded in a slow, continuous process under normal physiologic conditions. In microautophagy, small cytoplasmic soluble proteins are internalized into the lysosomes by invagination of the lysosomal membrane.  Chaperone-mediated autophagy is the only selective process of protein degradation and requires assistance from specific cytosolic chaperones such as heat-shock chaperone protein called hsc73. This process is activated during nutrient deprivation and requires the presence of targeting signals on the degraded proteins and a specific receptor on


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Histology_Ross. FIGURE 2.22 • Three autophagic pathways for degradation of cytoplasmic constituents. In macroautophagy, a portion of the cytoplasm or an entire organelle is surrounded by an intracellular membrane of the endoplasmic reticulum to form a double-membraned autophagosome vacuole. After fusion with a lysosome, the inner membrane and the contents of the vacuole are degraded. In microautophagy, cytoplasmic proteins are internalized into lysosomes by invagination of the lysosomal membrane. Chaperone-mediated autophagy to lysosomes is the most selective process for degradation of specifc cytoplasmic proteins. It requires assistance of proteins called chaperones. The chaperone protein—that is, hsc73—binds to the protein and helps transport it into the lysosomal lumen, where it is fnally degraded.


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Histology_Ross. the lysosomal membrane. Chaperone-mediated direct transport resembles the process of protein import to various other cellular organelles: hsc73 binds to the protein and assists in its transport through the lysosomal membrane into the lumen, where it is finally degraded. Chaperone-mediated autophagy is responsible for the degradation of approximately 30% of cytoplasmic proteins in organs such as the liver and kidney. In addition to the lysosomal pathway of protein degradation, cells have an ability to destroy proteins without involvement of lysosomes. Such process occurs within large cytoplasmic or nuclear protein complexes called proteasomes. They represent FIGURE 2.23 • Electron micrograph of autophagosomes in a hepatocyte. This electron micrograph shows several autophagosomes containing degenerating mitochondria. Note the surrounding lysosomes that have been stained with acid phosphatase. 12,600. (Courtesy of Dr. William A. Dunn, Jr.)


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Histology_Ross. 12,600. (Courtesy of Dr. William A. Dunn, Jr.) ATP-dependent protease complexes that destroy proteins that have been specifically tagged for this pathway. Proteasomemediated degradation is used by cells to destroy abnormal proteins that are misfolded, denaturated, or contain abnormal amino acids. This pathway also degrades normal short-lived regulatory proteins that need to be rapidly inactivated and degraded such as mitotic cyclins that regulate cell-cycle progression, transcriptional factors, tumor suppressors, or tumor promoters. Proteins destined for proteasome-mediated degradation need to be recognized and specifically tagged by the polyubiquitin chain.


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Histology_Ross. Proteins destined for proteasome-mediated degradation need to be recognized and specifically tagged by the polyubiquitin chain. Degradation of a protein in the proteasome-mediated pathway involves two successive steps:  Polyubiquitination, in which proteins targeted for destruction are repeatedly tagged by covalent attachments of a small (8.5-kilodalton) protein called ubiquitin. The tagging reaction is catalyzed by three ubiquitin ligases called ubiquitin-activating enzymes E1, E2, and E3. In a cascade of enzymatic reactions, the targeted protein is first marked by a single ubiquitin molecule. This creates a signal for consecutive attachment of several other ubiquitin molecules, resulting in a linear chain of ubiquitin conjugates. A protein target for destruction within the proteasome must be labeled with at least four ubiquitin molecules in the form of a polyubiquitin chain that serves as a degradation signal for proteasome complex.


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Histology_Ross.  Degradation of the tagged protein by the 26S proteasome complex. Each proteasome consists of a hollow cylinder, shaped like a barrel, containing a 20S core particle (CP) that facilitates the multicatalytic protease activity in which polyubiquitinated proteins are degraded into small polypeptides and amino acids. On both ends of the CP cylinder are two 19S regulatory particles (RPs); one RP that forms the lid of the barrel recognizes polyubiquitin tags, unfolds the protein, and regulates its entry into the destruction chamber. The RP on the opposite side (on the base) of the barrel releases short peptides and amino acids after degradation of the protein is completed. Free ubiquitin molecules are released by de-ubiquitinating (DUB) enzymes and recycled. (Fig. 2.24).


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Histology_Ross. Two groups of pathologic conditions are associated with the malfunction of proteasome-mediated degradation. The frst group of diseases results from a loss of proteasome function because of mutations in the system of ubiquitinactivating enzymes. This leads to a decrease in protein degradation and their subsequent accumulation in the cell cytoplasm (e.g., in Angelman syndrome and Alzheimer’s disease). The second group of diseases results from an accelerated degradation of proteins by overexpressed proteins involved in this system (e.g., infections with human papilloma virus). The recent discovery of specifc proteasome inhibitors holds promise for treatment of cancers and certain viral infections. The protein synthetic system of the cell consists of the rough endoplasmic reticulum and ribosomes.


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Histology_Ross. The protein synthetic system of the cell consists of the rough endoplasmic reticulum and ribosomes. The cytoplasm of a variety of cells engaged chieﬂy in protein synthesis stains intensely with basic dyes. The basophilic staining is caused by the presence of RNA. That portion of the cytoplasm that stains with the basic dye is called ergastoplasm. The ergastoplasm in secretory cells (e.g., pancreatic acinar cells) is the light microscopic image of the organelle called the rough endoplasmic reticulum (rER).


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Histology_Ross. With the TEM, the rER appears as a series of interconnected, membrane-limited, ﬂattened sacs called cisternae, with particles studding the exterior surface of the membrane (Fig. 2.25). These particles, called ribosomes , are attached to the membrane of the rER by ribosomal docking proteins. Ribosomes measure 15 to 20 nm in diameter and consist of a small and large subunit. Each subunit contains ribosomal RNA (rRNA) of different length as well as numerous different proteins. In many instances, the rER is continuous with the outer membrane of the nuclear envelope (see the next section). Groups of ribosomes form short spiral arrays called polyribosomes or polysomes (Fig. 2.26) in which many ribosomes are attached to a thread of messenger RNA (mRNA). Protein synthesis involves transcription and translation.


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Histology_Ross. Protein synthesis involves transcription and translation. The production of proteins by the cell begins within the nucleus with transcription, in which the genetic code for a protein is transcribed from DNA to pre-mRNA. After posttranscriptional modifications of the pre-mRNA molecule— which includes RNA cleavage, excision of introns, rejoining of exons, and capping by addition of poly(A) tracks at the


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Histology_Ross. FIGURE 2.24 • Proteasome-mediated degradation. This degradation pathway involves tagging proteins destined for destruction by a polyubiquitin chain and its subsequent degradation in proteasome complex with the release of free reusable ubiquitin molecules. Ubiquitin in the presence of ATP is activated by a complex of three ubiquitin-activating enzymes (E1, E2, and E3) to form a single polyubiquitin chain that serves as the degradation signal for the 26S proteasome complex. The regulatory particle (19S RP) that forms the lid of the main protein destruction chamber (20S core particle) recognizes polyubiquitin tags, unfolds the protein, and inserts and regulates its entry into the destruction chamber. The regulatory particle on the opposite side of the chamber releases short peptides and amino acids after degradation of the protein is completed. Free ubiquitin molecules are released by de-ubiquitinating enzymes (DUBs) and recycled.


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Histology_Ross. 3 end, and a methylguanosine cap [M(7) GPPP] at the 5 end—the resulting mRNA molecule leaves the nucleus and migrates into the cytoplasm (Fig. 2.27). Transcription is followed by translation in which the coded message contained in the mRNA is read by ribosomal complexes to form a polypeptide. A typical single cytoplasmic mRNA molecule binds to many ribosomes spaced as close as 80 nucleotides apart, thus forming a polyribosome complex, or FIGURE 2.25 • Electron micrograph of the rER. This image of the rER in a chief cell of the stomach shows the membranous cisternae (C) closely packed in parallel arrays. Polyribosomes are present on the cytoplasmic surface of the membrane surrounding the cisternae. The image of a ribosome-studded membrane is the origin of the term rough endoplasmic reticulum. A few ribosomes are free in the cytoplasm. M, mitochondrion. 50,000.


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Histology_Ross. polysome. A polysome attached to the cytoplasmic surface of the rER can translate a single mRNA molecule and simultaneously produce many copies of a particular protein. In contrast, free ribosomes reside within the cytoplasm. They are not associated with any intracellular membranes and are structurally and functionally identical to polysomes of the rER. The differences between the structure of prokaryotic (bacterial) and eukaryotic ribosomes were exploited by researchers, who discovered chemical compounds (antibiotics) that bind to bacterial ribosomes, thereby destroying a bacterial infection without harming the cells of the infected individual. Several types of antibiotics, such as aminoglycosides (streptomycin), macrolides (erythromycin), lincosamides (clindamycin), tetracyclines, and chloramphenicol inhibit protein synthesis by binding to different portions of bacterial ribosomes. Signal peptides direct the post-translational transport of a protein.


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Histology_Ross. Signal peptides direct the post-translational transport of a protein. FIGURE 2.26 • Electron micrograph of the rER and polyribosome complexes. This image shows a small section of the rER adjacent to the nucleus sectioned in two planes. The reticulum has turned within the section. Thus, in the upper right and left, the membranes of the reticulum have been cut at a right angle to their surface. In the center, the reticulum has twisted and is shown as in an aerial view (from above the membrane). The large spiral cytoplasmic assemblies (arrows) are chains of ribosomes that form polyribosomes that are actively engaged in translation of the mRNA molecule. 38,000.


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Histology_Ross. Most proteins that are synthesized for export or to become a part of specific organelles (such as the plasma membrane, mitochondrial matrix, endoplasmic reticulum, or nucleus) require sorting signals that direct proteins to their correct destinations. These signal sequences (signal peptides) are often found in the sequence of the first group of 15 to 60 amino acids on the amino-terminus of a newly synthesized protein. For instance, almost all proteins that are transported to the endoplasmic reticulum have a signal sequence consisting of five to ten hydrophobic amino acids on their amino-termini. The signal sequence of the nascent peptide interacts with a signal-recognition particle (SRP), which arrests further growth of the polypeptide chain. The complex containing the SRP–polyribosome complex with arrested polypeptide synthesis is then relocated toward the rER excision of introns release of translational arrest


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Histology_Ross. FIGURE 2.27 • Summary of events during protein synthesis. Proteins synthesis begins within the nucleus with transcription, during which the genetic code for a protein is transcribed from DNA to mRNA precursors. After post-transcriptional modifcations of the premRNA molecule—which include RNA cleavage, excision of introns, rejoining of exons, and capping by addition of poly(A) tracks at the 3 end and methylguanosine cap at the 5 end— the resulting mRNA molecule leaves the nucleus into the cytoplasm. In the cytoplasm, the mRNA sequence is read by the ribosomal complex in the process of translation to form a polypeptide chain. The frst group of 15 to 60 amino acids on the amino-terminus of a newly synthesized polypeptide forms a signal sequence (signal peptide) that directs protein to its destination (i.e., lumen of rER). The signal peptide interacts with a signal-recognition particle (SRP), which arrests further growth of the polypeptide chain until its relocation toward the rER


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Histology_Ross. its destination (i.e., lumen of rER). The signal peptide interacts with a signal-recognition particle (SRP), which arrests further growth of the polypeptide chain until its relocation toward the rER membrane. Binding of THE SRP to a docking protein on the cytoplasmic surface of the rER aligns ribosome with the translocator protein. Binding of the ribosome to the translocator causes dissociation of the SRP–docking protein complex away from the ribosome, and protein synthesis is resumed. The translocator protein guides the polypeptide chain into the lumen of the rER cisterna. The signal sequence is cleaved from the polypeptide by signal peptidase and is subsequently digested by signal peptide peptidases. On completion of protein synthesis, the ribosome detaches from the translocator protein.


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Histology_Ross. membrane. Binding of SRP to a docking protein on the cytoplasmic surface of rER aligns the ribosome with the translocator, an integral membrane protein of the rER. Binding of the ribosome to the protein translocator causes dissociation of the SRP–docking protein complex away from the ribosome and rER membrane, releasing the translational block and allowing the ribosome to resume protein synthesis (see Fig. 2.27). The translocator protein inserts the polypeptide chain into its aqueous pore, allowing newly formed protein to be discharged into the lumen of the rER cisterna. For simple secretory proteins, the polypeptide continues to be inserted by the translocator into the lumen as it is synthesized. The signal sequence is cleaved from the polypeptide by signal peptidase residing on the cisternal face of the rER membrane, even before the synthesis of the entire chain is completed. For integral membrane proteins, sequences along the polypeptide may instruct the forming protein to pass


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Histology_Ross. face of the rER membrane, even before the synthesis of the entire chain is completed. For integral membrane proteins, sequences along the polypeptide may instruct the forming protein to pass back and forth through the membrane, creating the functional domains that the protein will exhibit at its final membrane. On completion of protein synthesis, the ribosome detaches from the translocator protein and is again free in the cytoplasm.