Ribosomes

Ribosomes are RNA-protein complexes (nucleoproteins) that are synthesized in the nucleolus and secreted into the cytoplasm through pores in the nuclear envelope called nuclear pore complexes (NPCs).² These tiny ribosomes may float free in the cytoplasm or attach themselves to the outer membranes of the endoplasmic reticulum (see Figure 1-1, A). Their chief function is to provide sites for cellular protein synthesis. Newly formed ribosomes synthesize a “recognition sequence,” or signal, like an address on a letter. Signal recognition particles (SRPs) in the cytosol bind to the ribosome after recognizing the SRP. Ribophorins, receiver proteins found on the rough sections of the endoplasmic reticulum (ER), act as the “address” site or binding site. The developing protein threads its way through the ER membrane into the lumen. The SRP is removed and the new protein chain is folded into its final conformation.

Endoplasmic Reticulum

The endoplasmic reticulum (ER) (endo = within; plasma = cytoplasm; reticulum = network) is a membrane factory that specializes in the synthesis and transport of the protein and lipid components of most of the cell’s organelles. It consists of a network of tubular or saclike channels (cisternae) that extend throughout the cytoplasm and are continuous with the outer nuclear membrane (Figure 1-3). The folded membranes that form the cisternae of the endoplasmic reticulum may be rough (granular) or smooth (agranular). The rough endoplasmic reticulum (rER) is rough because ribosomes and ribonucleoprotein particles are attached to it (see Figure 1-3). Some of the proteins synthesized by these ribosomes remain in the ER, and others are used to construct membranes of other organelles (the Golgi complex, lysosomes, peroxisomes, and nucleus) and of the cell itself. Importantly, the ER is responsible for much of a cell’s protein synthesis and folding, and a new role is sensing cellular stress (see What’s New? Endoplasmic Reticulum, Protein Folding, and ER Stress). Understanding mechanisms of cellular stress will aid diagnosis and treatment of disease.

WHAT’S NEW?

Endoplasmic Reticulum, Protein Folding, and ER Stress

Protein folding in the endoplasmic reticulum (ER) is critical for us. As the biologic workhorses, proteins perform vital functions in every cell. To do these tasks proteins must fold into complex three-dimensional structures (see figure at right). Most secreted proteins fold and are modified in an error-free manner, but ER or cell stress, mutations, or random (stochastic) errors during protein synthesis can decrease the folding amount or the rate of folding. Pathophysiologic processes, such as viral infections, environmental toxins, and mutant protein expression, can perturb the sensitive ER environment. Natural processes also can perturb the environment, such as the large protein-synthesizing load placed on the ER. These perturbations cause the accumulation of immature and abnormal proteins in cells, leading to ER stress. Fortunately, the ER is loaded with protective ways to help folding, for example, protein so-called chaperones that facilitate folding and prevent the formation of off-pathway types. Because specialized cells produce large amounts of secreted proteins, the movement or flux through the ER is tremendous. Therefore, misfolded proteins not repaired in the ER are observed in some diseases and can initiate apoptosis or cell death. It has recently been shown that the endoplasmic reticulum mediates intracellular signaling pathways in response to the accumulation of unfolded or misfolded proteins; collectively, the pathways are known as the unfolded-protein response (UPR). Investigators are studying UPR-associated inflammation and how the UPR is coupled to inflammation in health and disease. Specific diseases include Alzheimer disease, Parkinson disease, prion disease, amyotrophic lateral sclerosis, and diabetes mellitus. Additionally being studied is ER stress and how it may accelerate age-related dysfunction.

Data from Brodsky J, Skach WR: Curr Opin Cell Biol 23:464-475, 2011; Jäger R et al: Biol Cell, Jan 23, 2012, [Epub ahead of print]; Ron D, Walter P: Nat Rev Mol Cell Biol 8:519–529, 2007.

Smooth endoplasmic reticulum (sER) does not contain ribosomes or ribonucleoprotein particles (see Figure 1-1). Rather, membranous surfaces of the smooth endoplasmic reticulum contain enzymes involved in the synthesis of steroid hormones and are responsible for a variety of reactions required to remove toxic substances from the cell. The endoplasmic reticulum communicates with the Golgi complex and interacts with other organelles, particularly lysosomes and peroxisomes.

Golgi Complex

The Golgi complex (or Golgi apparatus) is a network of flattened, smooth membranes and vesicles frequently located near the nucleus of the cell (Figure 1-4). Proteins from the endoplasmic reticulum are processed and packaged into small membrane-bound sacs or vesicles called secretory vesicles, which collect at the end of the membranous folds of the Golgi bodies—called cisternae. The secretory vesicles then break off from the Golgi complex and migrate to a variety of intracellular and extracellular destinations, including the plasma membrane. The vesicles fuse with the plasma membrane, and their contents are released from the cell. The best known vesicles are those that have coats made largely of the protein clathrin and are called clathrin-coated vesicles. They bud from the Golgi complex on the outward secretory pathway and from the plasma membrane on the inward endocytotic pathway (see p. 33). Many molecules, including lipids, proteins, glycoproteins, and enzymes of lysosomes, pass through the Golgi complex at some stage in their maturation. The Golgi complex is a refining plant and directs traffic (e.g., protein, polynucleotide, polysaccharide molecules) in the cell¹ (Figure 1-5).

Lysosomes

Lysosomes (lyso = dissolution; soma = body) are saclike structures that originate from the Golgi complex (see Figure 1-1, A). They contain more than 40 digestive enzymes called hydrolases, which catalyze bonds in proteins, lipids, nucleic acids, and carbohydrates. Lysosomes function as the intracellular digestive system (Figure 1-6). Lysosomal enzymes are capable of digesting most cellular constituents completely to their basic components, such as amino acids, fatty acids, and carbohydrates.

The lysosomal membrane acts as a protective shield between the powerful digestive enzymes within the lysosome and the cytoplasm, preventing their leakage into the cytoplasmic matrix. Disruption of the membrane by various treatments or cellular injury leads to a release of the lysosomal enzymes, which can then react with their specific substrates, causing cellular self-digestion. Lysosomal abnormalities are involved in a number of conditions that involve cellular injury and death.

Lysosomal storage diseases may be the result of a genetic defect or lack of one or more lysosomal enzymes. For example, the lack of lysosomal α-1,4-glucosidase leads to an accumulation of glycogen in lysosomes known as Pompe disease. Tay-Sachs disease is characterized by an accumulation of GM2 ganglioside (a lipid) in lysosomes as a result of the deficiency or absence of lysosomal hexosaminidase A. In gout, undigested uric acid accumulates within lysosomes, damaging the lysosomal membrane. Subsequent enzyme leakage results in cell death and tissue injury.

Lysosomes are necessary for normal digestion of cellular nutrients, intracellular debris, and potentially harmful extracellular substances that must be removed from the body. Extracellular substances are taken into the cell and encapsulated in a membrane-bound vesicle (see discussion on endocytosis, p. 33). Lysosomes merge with the vesicle to form a digestive vacuole. Lysosomes remain fully active by maintaining a low internal pH. They do this by pumping hydrogen ions into their interiors. The hydrolytic enzymes are only maximally active at acid pH values. Lysosomes that are not active do not maintain such an acid internal pH. Lysosomes in this “holding pattern” are called primary lysosomes. When a primary lysosome fuses with a vacuole or other organelle, its pH falls and the hydrolytic enzymes become activated. When it becomes active, it is called a secondary lysosome, or heterophagosome.

As cells complete their life span and die, lysosomes digest the resultant cellular debris. Lysosomes involved in this process, which is called autodigestion, are called autolysosomes, or autophagosomes. In living cells, cellular debris is encapsulated within a vesicle that reacts with a lysosome to complete its degradation. The degradation process, called autophagy, promotes homeostasis because it involves continuous biosynthesis and cell turnover. Recent data indicate that autophagy plays a crucial role in health and disease3,4 (see Chapter 2).

Products of autophagy (and of phagocytosis, the ingestion of harmful foreign substances; see Chapter 7) pass out of the lysosome and are reused by the cell. Indigestible material is stored in vesicles called residual bodies, whose contents are actively expelled from the cell (see Figure 1-6). High concentrations of lipids may accumulate within the residual bodies and remain there for a long time. The lipids are eventually oxidized, and a pigmented substance containing polyunsaturated fatty acids and proteins accumulates in the cell. This pigmented substance, termed lipofuscin, is often called “age pigment” or “age spots,” and is noted in older individuals (see Chapter 2).

Peroxisomes

Peroxisomes (microbodies) are membrane-bound organelles that contain several oxidative enzymes such as catalase and urate oxidase. These oxidative enzymes can detoxify compounds and fatty acids. Similar to lysosomes in microscopic appearance, peroxisomes are larger and oval or irregular in shape. Like mitochondria, peroxisomes are major sites of oxygen utilization. Peroxisomes are so named because they usually contain enzymes that use oxygen to remove hydrogen atoms from specific substrates in an oxidative reaction that produces hydrogen peroxide (H₂O₂). Hydrogen peroxide is a powerful oxidant, potentially destructive if it accumulates or escapes from peroxisomes. Catalase, an antioxidant enzyme, uses the H₂O₂ to oxidize a variety of other substrates—phenols, formic acid, formaldehyde, and alcohol—by the peroxidative reaction:

H2O2+R1H2→R1+2H2O

Thus the breakdown of H₂O₂ yields H₂O and O₂ (see discussion of free radicals in Chapter 2). Peroxisomes also have an important role in the synthesis of specialized phospholipids necessary for nerve cell myelination. Such reactions are important in detoxifying various wastes within the cell or foreign components that enter the cell, such as ethanol. Impairment of peroxisomes can lead to disease.

Mitochondria

Mitochondria (mito = thread; chondros = granule), organelles found in large numbers in most cells, are responsible for cellular respiration and energy production (see p. XX). These cytoplasmic organelles appear as spheres, rods, or filamentous bodies that are bound by a double membrane (Figure 1-7). The outer membrane is smooth and surrounds the mitochondrion itself; the inner membrane is convoluted in the mitochondrial matrix to form partitions called cristae. The inner membrane contains the enzymes of the respiratory chain—the name given to the electron-transport chain. These enzymes are essential to the process of oxidative phosphorylation that generates most of the cell’s ATP. Metabolic pathways involved in the metabolism of carbohydrates, lipids, and amino acids and special pathways involving urea and heme synthesis are located in the mitochondrial matrix.

The outer membrane is permeable (passable) to many substances, but the inner membrane is highly selective and contains many transmembranous transport systems. The inner membrane contains a transporter to move electrically charged calcium (calcium ions). (Membrane transport is discussed on p. 25.) Mitochondria contain their own DNA that codes for enzymes needed for oxidative phosphorylation.

Vaults

Vaults are cytoplasmic organelles, also called ribonucleoproteins, that are much larger than ribosomes and shaped like octagonal barrels (Figure 1-8). Their name comes from their multiple arches, which reminded their discoverers of vaulted or cathedral ceilings. A single cell can contain thousands of vaults. The function of vaults is not fully understood and may be related to their octagonal shape. Vaults have a large interior volume and can encapsulate proteins.⁵ Similarly, the pores in the membrane surrounding the nucleus (see Figure 1-2, B) are also octagonally shaped and the same size as vaults, leading to speculation that vaults may be cellular “trucks.” Further, vaults would dock at nuclear pores, pick up molecules synthesized in the nucleus, and deliver their load elsewhere in the cell. Because at any given time about 5% of the vaults are localized near the nuclear pores, it is thought that vaults may be carrying messenger RNA (mRNA) from the nucleus to the ribosomal sites of protein synthesis within the cytoplasm. Investigators suggest that vaults transport several copies of untranslated RNA and that they are transported along cytoskeletal-based cellular tracks—much like an assembly line.⁶ Vault complexes are being exploited for the delivery of drugs to the inside of the cell, for example, in kidney diseases.

Cytosol

Cytosol is the gelatinous, semiliquid portion of the cytoplasm accounting for about 55% of the total cell volume. Functions of the cytosol include intermediary metabolism involving enzymatic biochemical reactions; ribosomal protein synthesis; and storage of carbohydrates, fat, and secretory vesicles.

Intermediary metabolism refers to the intracellular chemical reactions that include synthesis, degradation, and transformation of small organic molecules (e.g., simple sugars, fatty acids, and amino acids). All intermediary metabolism occurs in the cytoplasm or that portion of the cell interior not occupied by the nucleus—with most of the metabolism being accomplished in the cytosol. These reactions enable energy to be used for managing cellular activities and for providing substrates to maintain cell integrity.

Ribosomal protein synthesis takes place in free ribosomes in the cytosol. Cytosolic ribosomes that synthesize identical proteins are collected together in “factories” known as polyribosomes.

Excess stored nutrients not immediately used for ATP production are converted in the cytosol into storage forms; for example, excess glucose is stored as glycogen. These temporary masses are known as inclusions (see Chapter 2). Secretory vesicles that have been processed and packaged by the endoplasmic reticulum and Golgi complex also remain in the cytosol. By means of signaling, the vesicles transport and empty their contents outside the cell.

Cytoskeleton

All eukaryotic cells contain elaborate and specialized internal structures in the cytosol that provide the “bones and muscles” of the cell—the cytoskeleton. The cytoskeleton maintains the cell’s shape and internal organization, and it permits movement of substances within the cell and movement of external projections (cilia or microvilli; flagella in sperm) outside the plasma membrane. The cytoskeleton is involved with the extracellular matrix and nuclear interior in force transmission (mechanical forces) called mechanotransduction. Mechanotransduction describes the cellular processes that translate mechanical stimuli into biochemical signals, allowing cells to adapt to their surroundings. Cell stresses, however, that involve alterations of mechanotransduction are associated with several diseases (see Chapter 2).^7,8 The internal skeleton is composed of a network of protein filaments; two of the most important are microtubules and actin filaments, or microfilaments.

Microtubules are small, hollow, cylindrical, unbranched tubules made of protein. When found together, microtubules exhibit rigidity, unlike the rest of the cytoplasm. Microtubules thus add strength to the cell’s structure (Figure 1-9, A, B). Within the cell, microtubules support and move organelles from one part of the cytoplasm to another, facilitate transport of impulses along nerve cells, and have roles in the inflammatory and immune responses and hormone secretion (Figure 1-9, C). Microtubules are also involved in the external movement, or motility, of some cells.

Microtubules are arranged in the thickened base, or basal body, of a protrusion from the cell’s plasma membrane. This arrangement occurs in the basal bodies of sperm flagella and the cilia of certain other cells. The long, whiplike flagella enable the movement of sperm cells. Cilia usually move substances past the cell, which remains stationary. For example, cilia on cells lining the respiratory tract move together to “beat” mucus toward the throat so it can be removed by coughing.

While the cell is not in the process of division, only a few microtubules are assembled; cellular division (mitosis) or defense (phagocytosis) does, however, induce a cycle of rapid assembly and disassembly. Microtubules involved in cellular division are arranged in a centriole. Centrioles always consist of nine bundles containing three microtubules each. During division the pairs of centrioles split and migrate to opposite poles of the cell (see p. 37).

Alterations of microtubular function are implicated in disease processes. For example, alterations in actin microfilaments act as a driving force for cell extension during the dissemination of cancer.⁹

Actin filaments (microfilaments) are smaller fibrils that generally occur in bundles rather than singly (Figure 1-9, B). Like microtubules, actin filaments are associated with cellular locomotion and maintenance of cell and tissue shape.⁹ Actin filaments link the interior of the cell to adjacent cells through cell junctions, for example, zonula adherens and zonula occludens (see p. 19).

In addition, microfilaments are necessary for regulating cell growth.¹⁰ Cellular locomotion depends on contractile properties that involve both microtubules and actin filaments. The actin cytoskeleton in motile cells has recently been described as a “wave of excitation” that may account for the spontaneous migration of cells.¹¹ Anesthetic drugs can affect both microtubules and actin filaments, disrupting intracellular movement and cellular motility.

Membrane Composition

Historically, the plasma membrane was described as a fluid lipid bilayer composed of a uniform lipid distribution with inserted moving proteins.¹² It now appears that the lipid bilayer is a much more complex structure where lipids and proteins are not uniformly distributed but can separate into discrete units called microdomains, differing in their protein and lipid compositions¹³ (see What’s New? From the Fluid Mosaic Model to New Understandings about Biologic Membranes). Different membranes have different percentages of lipids and proteins. Intracellular membranes may have a higher percentage of proteins than do plasma membranes, presumably because most enzymatic activity occurs within organelles. The membrane organization is achieved through noncovalent bonds that allow different physical states called phases. The lipid bilayer can be structured into three main phases: a solid gel phase, a fluid liquid-crystalline (or liquid-disordered) phase, and a liquid-ordered phase (see Figure 1-13, p. 14). These phases can change under physiologic factors such as temperature and pressure alterations. The structure of a plasma membrane is shown in Figure 1-12 and Figure 1-13. Intracellular membranes have a higher percentage of proteins than do plasma membranes, presumably because most enzymatic activity occurs within organelles. Carbohydrates are mainly associated with plasma membranes, where they are chemically combined with lipids to form glycolipids and with proteins to form glycoproteins.

The outer surface of the plasma membrane in many types of cells, especially endothelial and adipocytes, is not smooth but dimpled with cavelike indentations known as caveolae (“tiny caves”). Caveolae and caveolae-like domains are structurally heterogeneous.¹³ Caveolae were considered to be functionally insignificant until the mid-1990s, when evidence suggested that they (1) serve as a repository for some receptors, (2) provide a new route for transport into the cell, and (3) act as the initiator for relaying signals from several extracellular chemical messengers into the cell’s interior¹⁴ (see p. 20).

Proteolytic Cascades

Proteases are enzymes that cause the breakdown of proteins. Certain proteases can be tethered to cell membranes. Proteases are involved in the physiologic regulation of essential processes by participating in a tightly orchestrated sequence of events termed a proteolytic cascade. Four major proteolytic cascades with disease relevance are candidates for treatment modalities, including (1) cell death or caspase-mediated apoptosis, (2) blood coagulation cascade, (3) degrading membrane enzymes or matrix metalloproteinase cascade, and (4) the complement cascade. Some proteases within a proteolytic cascade act as initiators; others are involved in amplification and propagation and execution (Figure 1-14). Understanding the various steps involved is crucial for designing drug interventions. Dysregulation of proteases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders.^19–21

Cell-Adhesion Molecules (CAMs)

Cell adhesion molecules (CAMs) are cell-surface proteins that bind the cell to an adjacent cell and to components of the extracellular matrix (ECM). CAMs include four protein families: the integrins, the cadherins, the selectins, and Ig (immunoglobulin) superfamily. Integrins are a major class of receptors within the ECM and regulate cell-ECM interactions with collagen, fibronectin, vitronectin, and fibrinogen. Cadherins are Ca⁺² –dependent glycoproteins and have a unique pattern of tissue distribution, for example, epithelial (E-cadherin). Selectins are a family of proteins that bind certain carbohydrates, for example mucins. The immunoglobulin superfamily CAMs (IgSF CAMs) bind integrins or other IgSF CAMs.