Histology_Ross

Histology_Ross. The post-translational modification and sequestration of proteins within the rER is the first step in the exportation of proteins destined to leave the cell. As polypeptide chains are synthesized by the membrane-bound polysomes, the protein is injected into the lumen of the rER cisterna, where it is further modified post-translationally by enzymes. These modifications include core glycosylation, disulfide-bond and internal hydrogen-bond formation, folding of the newly synthesized protein with the help of molecular chaperones, and partial subunit assembly. Proteins are then concentrated within a lumen of neighboring cisternae of rER, or they are carried to another part of the cell in the continuous channels of the rER.


Histology_Ross

Histology_Ross. Except for the few proteins that remain permanent residents of the rER membranes and those proteins secreted by the constitutive pathway, the newly synthesized proteins are normally delivered to the Golgi apparatus within minutes. A few diseases are characterized by an inability of the rER to export a mutated protein to Golgi. For example, in 1antitrypsin defciency, a single amino acid substitution renders the rER unable to export 1-antitrypsin (A1AT). This leads to decreased activity of A1AT in the blood and lungs and abnormal deposition of defective A1AT within the rER of liver hepatocytes, resulting in emphysema (chronic obstructive pulmonary disease) and impaired liver function. In cells in which the constitutive pathway is dominant— namely, plasma cells and activated fibroblasts—newly synthesized proteins may accumulate in the rER cisternae, causing their engorgement and distension.


Histology_Ross

Histology_Ross. The rER also serves as a quality checkpoint in the process of protein production. If the newly synthesized protein is not properly post-translationally modified or misfolded, it is then exported from the rER back to the cytoplasm via the mechanism of retrotranslocation. Defective proteins are here deglycosylated, polyubiquitylated, and degraded within proteasomes (see page 44). The rER is most highly developed in active secretory cells.


Histology_Ross

Histology_Ross. The rER is most highly developed in active secretory cells. The rER is particularly well developed in those cells that synthesize proteins destined to leave the cell (secretory cells) as well as in cells with large amounts of plasma membrane, such as neurons. Secretory cells include glandular cells, activated fibroblasts, plasma cells, odontoblasts, ameloblasts, and osteoblasts. The rER is not limited, however, to secretory cells and neurons. Virtually every cell of the body contains profiles of rER. However, they may be few in number, a reﬂection of the amount of protein secretion, and dispersed so that in the light microscope they are not evident as areas of basophilia.


Histology_Ross

Histology_Ross. The rER is most highly developed in active secretory cells because secretory proteins are synthesized exclusively by the ribosomes of the rER. In all cells, however, the ribosomes of the rER also synthesize proteins that are to become permanent components of the lysosomes, Golgi apparatus, rER, or nuclear envelope (these structures are discussed in the next sections) or integral components of the plasma membrane. Coatomers mediate bidirectional traffic between the rER and Golgi apparatus.


Histology_Ross

Histology_Ross. Coatomers mediate bidirectional traffic between the rER and Golgi apparatus. Experimental data indicate that two classes of coated vesicles are involved in the transport of protein from and to the rER. A protein coat similar to clathrin surrounds vesicles transporting proteins between the rER and the Golgi apparatus (page 35). However, unlike clathrins, which mediate bidirectional transport from and to the plasma membrane, one class of proteins is involved only in anterograde transport from the rER to the cis-Golgi network (CGN), the Golgi cisternae closest to the rER. Another class of proteins mediates retrograde transport from the CGN back to the rER (Fig. 2.28). These two classes of proteins are called coatomers or COPs.  COP-I mediates transport vesicles originating in the CGN back to the rER (Fig. 2.29a). This retrograde proteins mistakenly transferred to the CGN during nor mal anterograde transport. In addition, COP-I is also re the Golgi cisternae.


Histology_Ross

Histology_Ross. CGN back to the rER (Fig. 2.29a). This retrograde proteins mistakenly transferred to the CGN during nor mal anterograde transport. In addition, COP-I is also re the Golgi cisternae. FIGURE 2.28 • Anterograde and retrograde transport between the rER and cis-Golgi network. Two classes of coated vesicles are involved in protein transport to and from the rER. These vesicles are surrounded by COP-I and COP-II protein coat complexes, respectively. COP-II is involved in anterograde transport from the rER to the cis-Golgi network (CGN), and COP-I is involved in retrograde transport from the CGN back to the rER. After a vesicle is formed, the coat components dissociate from the vesicle and are recycled to their site of origin. The COP-I protein coat is also involved in retrograde transport between cisternae within the Golgi apparatus (see Fig. 2.13).


Histology_Ross

Histology_Ross.  COP-II is responsible for anterograde transport, forming rER transport vesicles destined for the CGN (Fig. 2.29b). COP-II assists in the physical deformation of rER membranes into sharply curved buds and further separation of vesicles from the rER membrane. Most proteins produced in the rER use COP-II–coated vesicles to reach the CGN. Shortly after formation of COP-I– or COP-II–coated vesicles, the coats dissociate from the newly formed vesicles, allowing the vesicle to fuse with its target. The coat components then recycle to their site of origin. “Free” ribosomes synthesize proteins that will remain in the cell as cytoplasmic structural or functional elements.


Histology_Ross

Histology_Ross. “Free” ribosomes synthesize proteins that will remain in the cell as cytoplasmic structural or functional elements. Proteins targeted to the nucleus, mitochondria, or peroxisomes are synthesized on free ribosomes and then released into the cytosol. In the absence of a signal sequence, proteins that are synthesized on free ribosomes remain in the cytosol. Cytoplasmic basophilia is associated with cells that produce large amounts of protein that will remain in the cell. Such cells and their products include developing red blood cells (hemoglobin), developing muscle cells (the contractile proteins actin and myosin), nerve cells (neurofilaments), and keratinocytes of the skin (keratin). In addition, most enzymes of the mitochondrion are synthesized by free polysomes and transferred into that organelle.


Histology_Ross

Histology_Ross. FIGURE 2.29 • Electron micrograph of COP-I– and COP-II–coated vesicles. a. This image shows COP-I–coated vesicles that initiate retrograde transport from the cis-Golgi network to the rER. In this image, taken with a quick-freeze deep-edge microscope, note the structure of the CGN and emerging vesicles. 27,000. b. Image of COP-II–coated vesicles that are responsible for anterograde transport. Note that the surface coat of these vesicles is different from that of clathrin-coated vesicles. 50,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.)


Histology_Ross

Histology_Ross. 50,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.) Basophilia in these cells was formerly called ergastoplasm and is caused by the presence of large amounts of RNA. In this case, the ribosomes and polysomes are free in the cytoplasm (i.e., they are not attached to membranes of the endoplasmic reticulum). The large basophilic bodies of nerve cells, which are called Nissl bodies, consist of both rER and large numbers of free ribosomes (Fig. 2.30). All ribosomes contain RNA; it is the phosphate groups of the RNA of the ribosomes, not the membranous component of the endoplasmic reticulum, that accounts for basophilic staining of the cytoplasm.


Histology_Ross

Histology_Ross. FIGURE 2.30 • Electron micrograph of a nerve cell body showing the rER. This image shows rER profles as well as numerous free ribosomes located between the membranes of the rER. Collectively, the free ribosomes and membrane-attached ribosomes are responsible for the characteristic cytoplasmic basophilia (Nissl bodies) observed in the light microscope in the perinuclear cytoplasm of neurons. 45,000. The sER consists of short anastomosing tubules that are not associated with ribosomes.


Histology_Ross

Histology_Ross. The sER consists of short anastomosing tubules that are not associated with ribosomes. Cells with large amounts of smooth-surfaced endoplasmic reticulum may exhibit distinct cytoplasmic eosinophilia (acidophilia) when viewed in the light microscope. The sER is structurally similar to the rER but lacks the ribosome-docking proteins. It tends to be tubular rather than sheetlike, and it may be separate from the rER or an extension of it. The sER is abundant in cells that function in lipid metabolism (i.e., cells that synthesize fatty acids and phospholipids), and it proliferates in hepatocytes when animals are challenged with lipophilic drugs. The sER is well developed in cells that synthesize and secrete steroids such as adrenocortical cells and testicular Leydig (interstitial) cells (Fig. 2.31). In skeletal and cardiac muscle, the sER is also called the sarcoplasmic


Histology_Ross

Histology_Ross. FIGURE 2.31 • Electron micrograph of the sER. This image shows numerous profles of sER in an interstitial (Leydig) cell of the testis, a cell that produces steroid hormones. The sER seen here is a complex system of anastomosing tubules. The small, dense objects are glycogen particles. 60,000. reticulum. It sequesters Ca2 , which is essential for the contractile process and is closely apposed to the plasma-membrane invaginations that conduct the contractile impulses to the interior of the cell. The sER is the principal organelle involved in detoxification and conjugation of noxious substances.


Histology_Ross

Histology_Ross. The sER is the principal organelle involved in detoxification and conjugation of noxious substances. The sER is particularly well developed in the liver and contains a variety of detoxifying enzymes related to cytochrome P450 that are anchored directly into sER plasma membranes. They modify and detoxify hydrophobic compounds such as pesticides and carcinogens by chemically converting them into water-soluble conjugated products that can be eliminated from the body. The degree to which the liver is involved in detoxification at any given time may be estimated by the amount of sER present in liver cells. The sER is also involved in:  lipid and steroid metabolism,  glycogen metabolism, and  membrane formation and recycling. Because of these widely disparate functions, numerous other enzymes—including hydrolases, methylases, glucose-6phosphatase, ATPases, and lipid oxidases—are associated with the sER, depending on its functional role.


Histology_Ross

Histology_Ross. The Golgi apparatus is well developed in secretory cells and does not stain with hematoxylin or eosin. The Golgi apparatus was described more than 100 years ago by histologist Camillo Golgi. In studies of osmium-impregnated nerve cells, he discovered an organelle that formed networks around the nucleus. It was also described as well developed in secretory cells. Changes in the shape and FIGURE 2.32 • Photomicrograph of plasma cells. This photomicrograph of a plastic-embedded specimen showing the lamina propria of the small intestine is stained with toluidine blue. The plasma cells, where appropriately oriented, exhibit a clear area in the cytoplasm near the nucleus. These negatively stained regions (arrows) represent extensive accumulation of membranous cisternae that belong to the Golgi apparatus. The surrounding cytoplasm is deeply metachromatically stained because of the presence of ribosomes associated with the extensive rER. 1,200.


Histology_Ross

Histology_Ross. location of the Golgi apparatus relative to its secretory state were described even before it was viewed with the electron microscope and before its functional relationship to the rER was established. It is active both in cells that secrete protein by exocytosis and in cells that synthesize large amounts of membrane and membrane-associated proteins such as nerve cells. In the light microscope, secretory cells that have a large Golgi apparatus (e.g., plasma cells, osteoblasts, and cells of the epididymis) typically exhibit a clear area partially surrounded by ergastoplasm (Fig. 2.32). In EM, the Golgi apparatus appears as a series of stacked, ﬂattened, membrane-limited sacs or cisternae and tubular extensions embedded in a network of microtubules near the microtubule-organizing center (see page 65). Small vesicles involved in vesicular transport are seen in association with the cisternae. The Golgi apparatus is polarized both morphologically and functionally. The ﬂattened cisternae


Histology_Ross

Histology_Ross. page 65). Small vesicles involved in vesicular transport are seen in association with the cisternae. The Golgi apparatus is polarized both morphologically and functionally. The ﬂattened cisternae located closest to the rER represent the forming face, or the cis-Golgi network (CGN); the cisternae located away from the rER represent the maturing face, or the trans-Golgi network (TGN); (Figs. 2.33 and 2.34). The cisternae located between the TGN and CGN are commonly referred as the medial-Golgi network.


Histology_Ross

Histology_Ross. The Golgi apparatus functions in the post-translational modification, sorting, and packaging of proteins. Small COP-II coated transport vesicle s carry newly synthesized proteins (both secretory and membrane) from the rER to the CGN. From there, they travel within the transport vesicles from one cisterna to the next. The vesicles bud from one cisterna and fuse with the adjacent cisternae (Fig. 2.35). As proteins and lipids travel through the Golgi stacks, they undergo a series of post-translational modifcations that involve remodeling of N-linked oligosaccharides previously added in the rER.


Histology_Ross

Histology_Ross. FIGURE 2.33 • Electron micrograph of the Golgi apparatus. This electron micrograph shows the extensive Golgi apparatus in an islet cell of the pancreas. The fattened membrane sacs of the Golgi apparatus are arranged in layers. The CGN is represented by the fattened vesicles on the outer convex surface, whereas the fattened vesicles of the inner convex region constitute the TGN. Budding off the TGN are several vesicles (1). These vesicles are released (2) and eventually become secretory vesicles (3). 55,000.


Histology_Ross

Histology_Ross. In general, glycoproteins and glycolipids have their oligosaccharides trimmed and translocated. Glycosylation of proteins and lipids uses several carbohydrate-processing enzymes that add, remove, and modify sugar moieties of oligosaccharide chains. M-6-P is added to those proteins destined to travel to late endosomes and lysosomes (see page 37). In addition, glycoproteins are phosphorylated and sulfated. The proteolytic cleavage of certain proteins is also initiated within the cisternae. Four major pathways of protein secretion from the Golgi apparatus disperse proteins to various cell destinations. As noted, proteins exit the Golgi apparatus from the TGN. This network and the associated tubulovesicular array serve as


Histology_Ross

Histology_Ross. As noted, proteins exit the Golgi apparatus from the TGN. This network and the associated tubulovesicular array serve as FIGURE 2.34 • Electron micrograph of Golgi cisternae. a. This transmission electron micrograph shows a quick-frozen isolated Golgi apparatus replica from a cultured Chinese hamster ovary (CHO) cell line. The trans-Golgi cisternae are in the process of coated vesicle formation. b. Incubation of the trans-Golgi cisternae with the coatomer-depleted cytosol shows a decrease in vesicle formation activity. Note the lack of vesicles and the fenestrated shape of the trans-Golgi cisternae. 85,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.)


Histology_Ross

Histology_Ross. FIGURE 2.35 • The Golgi apparatus and vesicular traffcking. The Golgi apparatus contains several stacks of fattened cisternae with dilated edges. The Golgi cisternae form separate functional compartments. The closest compartment to the rER represents the CGN, to which COP-II–coated transport vesicles originating from the rER fuse and deliver newly synthesized pro teins. Retrograde transport from CGN to rER, as well retrograde transport between Golgi cisternae, is mediated by COP-I–coated vesicles. Once proteins have been modifed within the CGN, the transport vesicles bud off dilated ends of this compartment, and proteins are trans ferred into medial-Golgi cisternae. The process continues; in the same fashion, proteins are translocated into the trans-Golgi cisternae and the sorting station for shuttling vesicles that deliver proteins to the following locations (see Fig. 2.36).


Histology_Ross

Histology_Ross.  Apical plasma membrane. Many extracellular and membrane proteins are delivered to this site. This constitutive pathway most likely uses non–clathrin-coated vesicles. In most cells, secretory proteins destined for the apical plasma membrane have specific sorting signals that guide their sorting process in the TGN. Proteins are then delivered to the apical cell surface.


Histology_Ross

Histology_Ross.  Basolateral plasma membrane. Proteins targeted to the basolateral domain also have a specific sorting signal attached to them by the TGN. This constitutive pathway uses vesicles coated with an as yet unidentified protein associated with an epithelium-specific adaptor protein. The transported membrane proteins are continuously incorporated into the basolateral cell surface. This type of targeting is present in most polarized epithelial cells. In liver hepatocytes, however, the process of protein sorting into the basolateral and apical domains is quite different. All integral plasma-membrane proteins that are destined for both apical and basolateral domains are first transported from the TGN to the basolateral plasma membrane. From here, both proteins are endocytosed and sorted into early endo somal compartments. Basolateral proteins are recycled back into the basolateral membrane, whereas apical pro teins are transported across the cytoplasm to the apical cell membrane via


Histology_Ross

Histology_Ross. into early endo somal compartments. Basolateral proteins are recycled back into the basolateral membrane, whereas apical pro teins are transported across the cytoplasm to the apical cell membrane via transcytosis.


Histology_Ross

Histology_Ross.  Endosomes or lysosomes. Most proteins destined for organelles bear specific signal sequences. They are sorted in the TGN and delivered to specific organelles. However, TGN sorting mechanisms are never completely accurate. For instance, about 10% of lysosomal integral membrane proteins (limps) instead travel directly into early or late endosomes, take an extended route traveling via the apical plasma membrane (see Fig. 2.20), and from there move back into the endosomal pathways. Enzymes destined for lysosomes using M-6-P markers (see page 37) are delivered into early or late endosomes as they develop into mature lysosomes.


Histology_Ross

Histology_Ross.  Apical cytoplasm. Proteins that were aggregated or crystallized in the TGN as a result of changes in pH and Ca2 concentration are stored in large secretory vesicles. These vesicles undergo a maturation process in which secretory proteins are retained within the vesicle. All other nonsecretory proteins are recycled into the endosomal compartment or TGN in clathrin-coated vesicles


Histology_Ross

Histology_Ross. FIGURE 2.36 • Summary of events in protein traffcking from the TGN. The tubulovesicular array of the TGN serves as the sorting station for transporting vesicles that deliver proteins to the following destinations: (1) apical plasma membrane (i.e., epithelial cells); (2) apical region of the cell cytoplasm where proteins are stored in secretory vesicles (i.e., secretory cells); (3) early or late endosomal compartment; (4) selected proteins containing lysosomal signals, which are targeted to lysosomes; (5) lateral plasma membrane (i.e., epithelial cells); (6) basal plasma membrane (i.e., epithelial cells); (7) proteins destined for apical, basal, and lateral surfaces of plasma membrane, which are delivered to the basal plasma membrane (i.e., in hepatocytes); (8) all proteins endocytosed and sorted in early endosomes; (9) apical plasma membrane from early endosomes; (10) lateral plasma membrane; and (11) basal plasma membrane. Note two targeting mechanisms of proteins to different


Histology_Ross

Histology_Ross. and sorted in early endosomes; (9) apical plasma membrane from early endosomes; (10) lateral plasma membrane; and (11) basal plasma membrane. Note two targeting mechanisms of proteins to different surfaces of plasma membrane. In epithelial cells, proteins are directly targeted from the TGN into the appropriate cell surface as shown in steps (1), (5), and (6). In hepatocytes, all proteins are secreted frst to the basal cell surface, and then they are distributed to the appropriate cell surface via the endosomal compartment as shown in steps (7) to (11).


Histology_Ross

Histology_Ross. (see Fig. 2.35). Mature secretory vesicles eventually fuse with the plasma membrane to release the secretory product by exocytosis. This type of secretion is characteristic of highly specialized secretory cells found in exocrine glands. Sorting and packaging of proteins into transport vesicles occurs in the trans-Golgi network. Proteins that arrive in the TGN are distributed to different intercellular locations within transport vesicles. The intercellular destination of each protein depends on the sorting signals that are incorporated within the polypeptide chain of the protein.


Histology_Ross

Histology_Ross. The actual sorting and packaging of proteins in TGN is primarily based on sorting signals and physical properties.  Sorting signals are represented by the linear array of amino acid or associated carbohydrate molecules. This type of signal is recognized by the sorting machinery, which directs the protein into the appropriately coated transport vesicle.  Physical properties are important for packaging func tionally associated protein complexes. These groups of proteins are first partitioned into separate lipid rafts that targeted organelle. Mitochondria are abundant in cells that generate and expend large amounts of energy.


Histology_Ross

Histology_Ross. Mitochondria are abundant in cells that generate and expend large amounts of energy. Mitochondria were also known to early cytologists who observed them in cells vitally stained with Janus green B. It is now evident that mitochondria increase in number by division throughout interphase, and their divisions are not synchronized with the cell cycle. Videomicroscopy confirms that mitochondria can both change their location and undergo transient changes in shape. They may therefore be compared to mobile power generators as they migrate from one area of the cell to another to supply needed energy.


Histology_Ross

Histology_Ross. Because mitochondria generate ATP, they are more numerous in cells that use large amounts of energy such as striated muscle cells and cells engaged in ﬂuid and electrolyte transport. Mitochondria also localize at sites in the cell where energy is needed, as in the middle piece of the sperm, the intermyofibrillar spaces in striated muscle cells, and adjacent to the basolateral plasma-membrane infoldings in the cells of the proximal convoluted tubule of the kidney. Mitochondria evolved from aerobic bacteria that were engulfed by eukaryotic cells.


Histology_Ross

Histology_Ross. Mitochondria evolved from aerobic bacteria that were engulfed by eukaryotic cells. Mitochondria are believed to have evolved from an aerobic prokaryote (Eubacterium) that lived symbiotically within primitive eukaryotic cells. This hypothesis received support with the demonstration that mitochondria possess their own genome, increase their numbers by division, and synthesize some of their structural (constituent) proteins. Mitochondrial DNA is a closed circular molecule that encodes 13 enzymes involved in the oxidative phosphorylation pathway, two rRNAs, and 22 transfer RNAs (tRNAs) used in the translation of the mitochondrial mRNA.


Histology_Ross

Histology_Ross. Mitochondria possess a complete system for protein synthesis, including the synthesis of their own ribosomes. The remainder of the mitochondrial proteins is encoded by nuclear DNA; new polypeptides are synthesized by free ribosomes in the cytoplasm and then imported into mitochondria with the help of two protein complexes. These include translocase of the outer mitochondrial membrane (TOM complexes) and translocase of the inner mitochondrial membrane (TIM complexes). Translocation of proteins through mitochondrial membranes requires energy and assistance from several specialized chaperone proteins. Mitochondria are present in all cells except red blood cells and terminal keratinocytes.


Histology_Ross

Histology_Ross. Mitochondria are present in all cells except red blood cells and terminal keratinocytes. The number, shape, and internal structure of mitochondria are often characteristic for specific cell types. When present in large numbers, mitochondria contribute to the acidophilia of the cytoplasm because of the large amount of membrane they contain. Mitochondria may be stained specifically by histochemical procedures that demonstrate some of their constituent enzymes, such as those involved in ATP synthesis and electron transport. Mitochondria possess two membranes that delineate distinct compartments.


Histology_Ross

Histology_Ross. Mitochondria possess two membranes that delineate distinct compartments. Mitochondria display a variety of shapes, including spheres, rods, elongated filaments, and even coiled structures. All mitochondria, unlike other organelles described above, possess two membranes (Fig. 2.37). The inner mitochondrial membrane surrounds a space called the matrix. The outer mitochondrial membrane is in close contact with the cytoplasm. The space between the two membranes is called the intermembrane space. The following structural components of mitochondria possess specific characteristics related to their functions.


Histology_Ross

Histology_Ross.  Outer mitochondrial membrane. This 6to 7-nm-thick smooth membrane contains many voltage-dependent anion channels (also called mitochondrial porins). These large channels (approximately 3 nm in diameter) are permeable to uncharged molecules as large as 5,000 daltons. Thus, small molecules, ions, and metabolites can enter the intermembrane space but cannot penetrate the inner membrane. The environment of the intermembrane space is therefore similar to that of cytoplasm with respect to ions and small molecules. The outer membrane possesses receptors for proteins and polypeptides that translocate into the intermembrane space. It also contains several enzymes, including phospholipase A2, monoamine oxidase, and acetyl coenzyme A (CoA) synthase.


Histology_Ross

Histology_Ross.  Inner mitochondrial membrane. The TEM reveals that this membrane is thinner than the outer mitochondrial membrane. It is arranged into numerous cristae (folds) that significantly increase the inner membrane surface area (see Fig. 2.37). These folds project into the matrix that constitutes the inner compartment of the organelle. In some cells involved in steroid metabolism, the inner membrane may form tubular or vesicular projections into the matrix. The inner membrane is rich in the phospholipid cardiolipin, FIGURE 2.37 • Structure of the mitochondrion. a. This electron micrograph shows a mitochondrion in a pancreatic acinar cell. Note that the inner mitochondrial membrane forms the cristae (C) through a series of infoldings, as is evident in the region of the arrow. The outer mitochondrial membrane is a smooth continuous envelope that is separate and distinct from the inner membrane. 200,000.


Histology_Ross

Histology_Ross. b. Schematic diagram showing the components of a mitochondrion. Note the location of the elementary particles (inset), the shape of which refects the three-dimensional structure of ATP synthase. which makes the membrane impermeable to ions. The membrane forming the cristae contains proteins that have three major functions: (1) performing the oxidation reactions of the respiratory electron-transport chain, (2) synthesizing ATP, and (3) regulating transport of metabolites into and out of the matrix. The enzymes of the respiratory chain are attached to the inner membrane and project their heads into the matrix (Fig. 2.37, rectangle). With the TEM, these enzymes appear as tennis racquet–shaped structures called elementary particles. Their heads measure about 10 nm in diameter and contain enzymes that carry out oxidative phosphorylation, which generates ATP.


Histology_Ross

Histology_Ross.  Intermembrane space. This space is located between the inner and outer membranes and contains specific enzymes that use the ATP generated in the inner membrane. These enzymes include creatine kinase, adenylate kinase, and cytochrome c. The latter is an important factor in initiating apoptosis (see page 94).  Matrix. The mitochondrial matrix is surrounded by the inner mitochondrial membrane and contains the soluble enzymes of the citric acid cycle (Krebs cycle) and the enzymes involved in fatty-acid -oxidation. The major products of the matrix are CO2 and reduced NADH, which is the source of electrons for the electron-transport chain.


Histology_Ross

Histology_Ross. Mitochondria contain dense matrix granules that store Ca2 and other divalent and trivalent cations. These granules increase in number and size when the concentration of divalent (and trivalent) cations increases in the cytoplasm. Mitochondria can accumulate cations against a concentration gradient. Thus, in addition to ATP production, mitochondria also regulate the concentration of certain ions of the cytoplasmic matrix, a role they share with the sER. The matrix also contains mitochondrial DNA, ribosomes, and tRNAs. Mitochondria contain the enzyme system that generates ATP by means of the citric acid cycle and oxidative phosphorylation.


Histology_Ross

Histology_Ross. Mitochondria contain the enzyme system that generates ATP by means of the citric acid cycle and oxidative phosphorylation. Mitochondria generate ATP in a variety of metabolic pathways, including oxidative phosphorylation, the citric acid cycle, and -oxidation of fatty acids. The energy generated from these reactions, which take place in the mitochondrial matrix, is represented by hydrogen ions (H ) derived from reduced NADH. These ions drive a series of proton pumps located within the inner mitochondrial membrane that transfer H from the matrix to the intermembrane space (Fig. 2.38). These pumps constitute the electron-transport chain of respiratory enzymes (see Fig. 2.37). The transfer of H across the inner mitochondrial membrane establishes an


Histology_Ross

Histology_Ross. FIGURE 2.38 • Schematic diagram illustrating how mitochondria generate energy. The diagram indicates the ATP synthase complex and the electron-transport chain of proteins located in the inner mitochondrial membrane. The electron-transport chain generates a proton gradient between the matrix and intermembrane space that is used to produce ATP. Numbers represent sequential proteins involved in the electron-transport chain and ATP production: 1, NADH dehydrogenase complex; 2, ubiquinone; 3, cytochrome b-c1 complex; 4, cytochrome c; 5, cytochrome oxidase complex; and 6, ATP synthase complex.


Histology_Ross

Histology_Ross. electrochemical proton gradient. This gradient creates a large proton motive force that causes the movement of H to occur down its electrochemical gradient through a large, membrane-bound enzyme called ATP synthase. ATP synthase provides a pathway across the inner mitochondrial membrane in which H ions are used to drive the energetically unfavorable reactions leading to synthesis of ATP. This movement of protons back to the mitochondrial matrix is referred to as chemiosmotic coupling. The newly produced ATP is transported from the matrix to the intermembrane space by the voltage gradient–driven ATP/ADP exchange protein located in the inner mitochondrial membrane. From here, ATP leaves the mitochondria via voltage-dependent anion channels in the outer membrane to enter the cytoplasm. At the same time, ADP produced in the cytoplasm rapidly enters the mitochondria for recharging.


Histology_Ross

Histology_Ross. Several mitochondrial defects are related to defects in enzymes that produce ATP. Metabolically active tissues that use large amounts of ATP such as muscle cells and neurons are most affected. For example, myoclonic epilepsy with ragged red fbers (MERRF) is characterized by muscle weakness, ataxia, seizures, and cardiac and respiratory failure. Microscopic examination of muscle tissue from affected patients shows aggregates of abnormal mitochondria, providing a ragged appearance of red muscle fbers. MERRF is caused by mutation of the mitochondrial DNA gene encoding tRNA for lysine. This defect produces two abnormal complexes in the electron-transport chain of respiratory enzymes affecting ATP production. Mitochondria undergo morphologic changes related to their functional state.


Histology_Ross

Histology_Ross. Mitochondria undergo morphologic changes related to their functional state. TEM studies show mitochondria in two distinct configurations. In the orthodox confguration, the cristae are prominent, and the matrix compartment occupies a large part of the total mitochondrial volume. This configuration corresponds to a low level of oxidative phosphorylation. In the condensed confguration, cristae are not easily recognized, the matrix is concentrated and reduced in volume, and the intermembrane space increases to as much as 50% of the total volume. This configuration corresponds to a high level of oxidative phosphorylation. Mitochondria decide whether the cell lives or dies.


Histology_Ross

Histology_Ross. Mitochondria decide whether the cell lives or dies. Experimental studies indicate that mitochondria sense cellular stress and are capable of deciding whether the cell lives or dies by initiating apoptosis (programmed cell death). The major cell death event generated by the mitochondria is the release of cytochrome c from the mitochondrial intermembranous space into the cell cytoplasm. This event, regulated by the Bcl-2 protein family (see page 94), initiates the cascade of proteolytic enzymatic reactions that leads to apoptosis. Peroxisomes are single membrane-bounded organelles containing oxidative enzymes.


Histology_Ross

Histology_Ross. Peroxisomes are single membrane-bounded organelles containing oxidative enzymes. Peroxisomes (microbodies) are small (0.5 m in diameter), membrane-limited spherical organelles that contain oxidative enzymes, particularly catalase and other peroxidases. Virtually all oxidative enzymes produce hydrogen peroxide (H2O2) as a product of the oxidation reaction. Hydrogen peroxide is a toxic substance. The catalase universally present in peroxisomes carefully regulates the cellular hydrogen peroxide content by breaking down hydrogen peroxide, thus protecting the cell. In addition, peroxisomes contain D-amino acid oxidases, -oxidation enzymes, and numerous other enzymes.


Histology_Ross

Histology_Ross. Oxidative enzymes are particularly important in liver cells (hepatocytes), where they perform a variety of detoxification processes. Peroxisomes in hepatocytes are responsible for detoxification of ingested alcohol by converting it to acetaldehyde. The -oxidation of fatty acids is also a major function of peroxisomes. In some cells, peroxisomal fatty-acid oxidation may equal that of mitochondria. The proteins contained in the peroxisome lumen and membrane are synthesized on cytoplasmic ribosomes and imported into the peroxisome. A protein destined for peroxisomes must have a peroxisomal targeting signal attached to its carboxy-terminus.


Histology_Ross

Histology_Ross. Although abundant in liver and kidney cells, peroxisomes are also found in most other cells. The number of peroxisomes present in a cell increases in response to diet, drugs, and hormonal stimulation. In most animals, but not humans, peroxisomes also contain urate oxidase (uricase), which often appears as a characteristic crystalloid inclusion (nucleoid).


Histology_Ross

Histology_Ross. Various human metabolic disorders are caused by the inability to import peroxisomal proteins into the organelle because of a faulty peroxisomal targeting signal or its receptor. Several severe disorders are associated with nonfunctional peroxisomes. In the most common inherited disease related to nonfunctional peroxisomes, Zellweger syndrome, which leads to early death, peroxisomes lose their ability to function because of a lack of necessary enzymes. The disorder is caused by a mutation in the gene encoding the receptor for the peroxisome targeting signal that does not recognize the signal Ser-Lys-Leu at the carboxyterminus of enzymes directed to peroxisomes. Therapies for peroxisomal disorders have been unsatisfactory to date.


Histology_Ross

Histology_Ross. Microtubules are nonbranching and rigid hollow tubes of protein that can rapidly disassemble in one location and reassemble in another. In general, they grow from the microtubule-organizing center located near the nucleus and extend toward the cell periphery. Microtubules create a system of connections within the cell, frequently compared with railroad tracks, along which vesicular movement occurs. Microtubules are elongated polymeric structures composed of equal parts of -tubulin and -tubulin. Microtubules measure 20 to 25 nm in diameter (Fig. 2.39). The wall of the microtubule is approximately 5 nm thick and consists of 13 circularly arrayed globular dimeric tubulin molecules. The tubulin dimer has a molecular weight of 110 kilodaltons (kDa) and is formed from an -tubulin and a -tubulin molecule, each with a molecular weight of 55 kDa (Fig. 2.40). The dimers polymerize in an end-to-end fashion, head to tail, with the molecule of one dimer bound to the


Histology_Ross

Histology_Ross. FIGURE 2.39 • Electron micrographs of microtubules. a. Micrograph showing microtubules (arrows) of the mitotic spindle in a dividing cell. On the right, the microtubules are attached to chromosomes. 30,000. b. Micrograph of microtubules (arrows) in the axon of a nerve cell. In both cells, the microtubules are seen in longitudinal profle. 30,000. molecule of the next dimer in a repeating pattern. Longitudinal contacts between dimers link them into a linear structure called a protoflament. Axial periodicity seen along the 5-nm-diameter dimers corresponds to the length of the protein molecules. A small, 1-m segment of microtubule contains approximately 16,000 tubulin dimers. The arrangement of -tubulin and -tubulin molecules within the microtubule is visible in Figure 2.41. Microtubules grow from -tubulin rings within the MTOC that serve as nucleation sites for each microtubule.


Histology_Ross

Histology_Ross. Microtubule formation can be traced to hundreds of tubulin rings that form an integral part of the MTOC (Fig. 2.42). The and -tubulin dimers are added to a -tubulin ring in an end-to-end fashion (see Fig. 2.40). Polymerization of tubulin dimers requires the presence of guanosine triphosphate (GTP) and Mg2. Each tubulin molecule binds GTP before it is incorporated into the forming microtubule. The GTP–tubulin complex is then polymerized, and at some point GTP is hydrolyzed to guanosine diphosphate (GDP). As a result of this polymerization pattern, microtubules are polar because all of the dimers have the same orientation. Each microtubule possesses a minus (nongrowing) end that corresponds to -tubulin; in the cell, it is usually embedded in the MTOC. The plus (growing) end of microtubules corresponds to -tubulin and extends the cell periphery. Tubulin dimers dissociate from microtubules in the steady state, which adds a pool of free tubulin dimers to the cytoplasm. This pool is in


Histology_Ross

Histology_Ross. corresponds to -tubulin and extends the cell periphery. Tubulin dimers dissociate from microtubules in the steady state, which adds a pool of free tubulin dimers to the cytoplasm. This pool is in equilibrium with the polymerized tubulin in the micro-tubules; therefore, polymerization and depolymerization are in equilibrium. The equilibrium can be shifted in the direction of depolymerization by exposing the cell or isolated microtubules to low temperatures or high pressure. Repeated exposure to alternating low and high temperature is the basis of the purification technique for tubulin and microtubules. The speed of polymerization or depolymerization can also be modified by interaction with specific microtubule-associated proteins (MAPs). These proteins, such as MAP-1, 2, 3, and 4, MAP-, and TOG , regulate microtubule assembly and anchor the microtubules to specific organelles. MAPs are also responsible for the existence of stable populations of nondepolymerizing microtubules in the


Histology_Ross

Histology_Ross. and TOG , regulate microtubule assembly and anchor the microtubules to specific organelles. MAPs are also responsible for the existence of stable populations of nondepolymerizing microtubules in the cell, such as those found in cilia and ﬂagella.


Histology_Ross

Histology_Ross. The length of microtubules changes dynamically as tubulin dimers are added or removed in a process of dynamic instability.


Histology_Ross

Histology_Ross. Microtubules observed in cultured cells with real-time video microscopy appear to be constantly growing toward the cell periphery (by addition of tubulin dimers) and then suddenly shrink in the direction of the MTOC (by removal of tubulin dimers). This constant remodeling process, known as dynamic instability, is linked to a pattern of GTP hydrolysis during the microtubule assembly and disassembly process. The MTOC can be compared to a feeding chameleon, which fires its long, projectile tongue to make contact with potential food. The chameleon then retracts its tongue back into its mouth and repeats this process until it is successful in obtaining food. The same strategy of “firing” microtubules from the MTOC toward the cell periphery and subsequently retracting them enables the cell to establish an organized system of mi- ( end) ( end) tubulin dimer bound to GTP tubulin dimer bound to GDP 24 nm Pi Pi -tubulin -tubulin -tubulin capping proteins


Histology_Ross

Histology_Ross. FIGURE 2.40 • Polymerization of microtubules. On the left, the diagram depicts the process of polymerization and depolymerization of tubulin dimers during the process of microtubule assembly. Each tubulin dimer consists of an -tubulin and a -tubulin subunit. On the right is a diagram showing that each microtubule contains 13 tubulin dimers within its cross section. The minus ( ) end of the microtubule contains a ring of -tubulin, which is necessary for microtubule nucleation. This end is usually embedded within the MTOC and possesses numerous capping proteins. The plus ( ) end of the microtubule is the growing end to which tubulin dimers bound to guanosine triphosphate (GTP) molecules are incorporated. Incorporated tubulin dimers hydrolyze GTP, which releases the phosphate groups to form polymers with guanosine diphosphate (GDP)–tubulin molecules.


Histology_Ross

Histology_Ross. crotubules linking peripheral structures and organelles with the MTOC. As mentioned earlier, the association of a micro-tubule with MAPs, such as occurs within the axoneme of a cilium or ﬂagellum, effectively blocks this dynamic instability and stabilizes the microtubules. FIGURE 2.41 • Three-dimensional reconstruction of an intact microtubule. This image was obtained using cryo-electron microscopy. Tomographic (sectional) images of a frozen hydrated microtubule were collected and digitally reconstructed at a resolution of 8 angstroms (Å). The helical structure of the -tubulin molecules is recognizable at this magnifcation. 3,250,000. (Courtesy of Dr. Kenneth Downing.) The structure and function of microtubules in mitosis and in cilia and ﬂagella are discussed later in this chapter and in Chapter 5. Microtubules can be visualized in the light microscope and are involved in intracellular transport and cell motility.


Histology_Ross

Histology_Ross. Microtubules can be visualized in the light microscope and are involved in intracellular transport and cell motility. Microtubules may be seen in the light microscope by using special stains, polarization, or phase-contrast optics. Because of the limited resolution of the light microscope, in the past microtubules were erroneously called fibers, such as the “fibers” of the mitotic spindle. Microtubules may now be distinguished from filamentous and fibrillar cytoplasmic components even at the light microscopic level by using antibodies to tubulin, the primary protein component of microtubules, conjugated with ﬂuorescent dyes (Fig. 2.42). In general, microtubules are found in the cytoplasm, where they originate from the MTOC; in cilia and ﬂagella, where they form the axoneme and its anchoring basal body; in centrioles and the mitotic spindle; and in elongating processes of the cell, such as those in growing axons.


Histology_Ross

Histology_Ross. Microtubules are involved in numerous essential cellular functions:  Intracellular vesicular transport (e.g., movement of secre tory vesicles, endosomes, and lysosomes),  Movement of cilia and ﬂagella,  Attachment of chromosomes to the mitotic spindle and their movement during mitosis and meiosis,  Cell elongation and movement (migration), and  Maintenance of cell shape, particularly its asymmetry. FIGURE 2.42 • Staining of microtubules with fuorescent dye.


Histology_Ross

Histology_Ross. FIGURE 2.42 • Staining of microtubules with fuorescent dye. This confocal immunofuorescent image shows the organization of the microtubules within an epithelial cell in tissue culture. In this example, the specimen was immunostained with three primary antibodies against tubulin (green), centrin (red), and kinetochores (light blue) and then incubated in a mixture of three different fuorescently tagged secondary antibodies that recognized the primary antibodies. Nuclei were stained (dark blue) with a fuorescent molecule that intercalates into the DNA double helix. Note that the microtubules are focused at the MTOC or centrosome (red) located adjacent to the nucleus. The cell is in the S phase of the cell cycle, as indicated by the presence of both large unduplicated kinetochores and smaller pairs of duplicated kinetochores. 3,000. (Courtesy of Dr. Wilma L. Lingle and Ms. Vivian A. Negron.)


Histology_Ross

Histology_Ross. Movement of intracellular organelles is generated by molecular motor proteins associated with microtubules. In cellular activities that involve movement of organelles and other cytoplasmic structures—such as transport vesicles, mitochondria, and lysosomes—microtubules serve as guides to the appropriate destinations. Molecular motor proteins attach to these organelles or structures and ratchet along the microtubule track (Fig. 2.43). The energy required for the ratcheting movement is derived from ATP hydrolysis. Two families of molecular motor proteins have been identified that allow for unidirectional movement:  Dyneins constitute one family of molecular motors. They move along the microtubules toward the minus end of the tubule. Therefore, cytoplasmic dyneins are capable of transporting organelles from the cell periphery toward the


Histology_Ross

Histology_Ross. MTOC. One member of the dynein family, axonemal dynein, is present in cilia and ﬂagella. It is responsible for the sliding of one microtubule against an adjacent micro- tubule of the axoneme that effects their movement.  Kinesins, members of the other family, move along the microtubules toward the plus end; therefore, they are ca FIGURE 2.43 • The molecular motor proteins associated with microtubules. Microtubules serve as guides for molecular motor proteins. These ATP-driven microtubule-associated motor proteins are attached to moving structures (such as organelles) that ratchet them along a tubular track. Two types of molecular motors have been identifed: dyneins that move along microtubules toward their minus ( ) end (i.e., toward the center of the cell) and kinesins that move toward their plus ( ) end (i.e., toward the cell periphery). pable of moving organelles from the cell center toward the cell periphery.


Histology_Ross

Histology_Ross. pable of moving organelles from the cell center toward the cell periphery. Both dyneins and kinesins are involved in mitosis and meiosis. In these activities, dyneins move the chromosomes along the microtubules of the mitotic spindle. Kinesins are simultaneously involved in movement of polar microtubules. These microtubules extend from one spindle pole past the metaphase plate and overlap with microtubules extending from the opposite spindle pole. Kinesins located between these microtubules generate a sliding movement that reduces the overlap, thereby pushing the two spindle poles apart toward each daughter cell (Fig. 2.44). Actin filaments are present in virtually all cell types.


Histology_Ross

Histology_Ross. Actin molecules (42 kDa) are abundant and may constitute as much as 20% of the total protein of some nonmuscle cells (Fig. 2.45). Similar to the tubulin in microtubules, actin molecules also assemble spontaneously by polymerization into a linear helical array to form filaments 6 to 8 nm in diameter. They are thinner, shorter, and more ﬂexible than microtubules. Free actin molecules in the cytoplasm are referred to as G-actin (globular actin), in contrast to the polymerized actin of the filament, which is called F-actin (flamentous actin). An actin filament is a polarized structure; its fast-growing end is referred to as the plus (barbed) end, and its slow-growing end is referred to as the minus (pointed) end. The dynamic process of actin polymerization requires the presence of K , Mg2 , and ATP, which is hydrolyzed to ADP after each G-actin molecule is incorporated into the filament (Fig. 2.46). The control and regulation of the polymerization process depends on the local


Histology_Ross

Histology_Ross. K , Mg2 , and ATP, which is hydrolyzed to ADP after each G-actin molecule is incorporated into the filament (Fig. 2.46). The control and regulation of the polymerization process depends on the local concentration of G-actin and the interaction of actinbinding proteins (ABPs), which can prevent or enhance polymerization.


Histology_Ross

Histology_Ross. In addition to controlling the rate of polymerization of actin filaments, ABPs are responsible for the filaments’ organization. For example, a number of proteins can modify or act on actin filaments to give them various specific characteristics:


Histology_Ross

Histology_Ross. FIGURE 2.44 • Distribution of kinesinlike motor protein within the mitotic spindle. This confocal immunofuorescent image shows a mammary gland epithelial cell in anaphase of mitosis. Each mitotic spindle pole contains two centrioles (green). A mitosis-specifc kinesinlike molecule called Eg5 (red ) is associated with the subset of the mitotic spindle microtubules that connect the kinetochores (white) to the spindle poles. The motor action of Eg5 is required to separate the sister chromatids (blue) into the daughter cells. This cell was frst immunostained with three primary antibodies against Eg5 (red), centrin (green), and kinetochores (white) and then incubated in three different fuorescently tagged secondary antibodies that recognize the primary antibodies. Chromosomes were stained with a fuorescent molecule that intercalates into the DNA double helix. 3,500. (Courtesy of Dr. Wilma L. Lingle and Ms. Vivian A. Negron.)  Actin-bundling proteins cross-link actin filaments into parallel


Histology_Ross

Histology_Ross. fuorescent molecule that intercalates into the DNA double helix. 3,500. (Courtesy of Dr. Wilma L. Lingle and Ms. Vivian A. Negron.)  Actin-bundling proteins cross-link actin filaments into parallel arrays, creating actin filament bundles. An example of this modification occurs inside the microvillus, where actin filaments are cross-linked by the actin-bundling proteins fascin and fmbrin. This cross-linkage provides support and imparts rigidity to the microvilli.


Histology_Ross

Histology_Ross. FIGURE 2.45 • Distribution of actin flaments in pulmonary artery endothelial cells in culture. Cells were fxed and stained with NDB phallacidin stain conjugated with fuorescein dye. Phallacidin binds and stabilizes actin flaments, preventing their depolymerization. Note the accumulation of actin flaments at the periphery of the cell just beneath the plasma membrane. These cells were also stained with two additional dyes: a mitochondria-selective dye (i.e., MitoTracker Red) that allows the visualization of mitochondria (red ) in the middle of the cell and DAPI stain that reacts with nuclear DNA and exhibits blue fuorescence over the nucleus. 3,000. (Courtesy of Molecular Probes, Inc., Eugene, OR.) proteins they contain. There are two types of filaments (myoflaments) present in muscle cells: 6to 8-nm actin filaments (called thin flaments; Fig. 2.47) and 15-nm filaments (called thick flaments) of myosin II, which is the predominant protein in muscle cells. Myosin II is a double-headed


Histology_Ross

Histology_Ross. 6to 8-nm actin filaments (called thin flaments; Fig. 2.47) and 15-nm filaments (called thick flaments) of myosin II, which is the predominant protein in muscle cells. Myosin II is a double-headed molecule with an elongated rodlike tail. The specific structural and functional relationships among actin, myosin, and other ABPs in muscle contraction are


Histology_Ross

Histology_Ross. FIGURE 2.46 • Polymerization of actin flaments. Actin flaments are polarized structures. Their fast-growing end is referred to as the plus ( ) or barbed end; the slow-growing end is referred to as the minus ( ) or pointed end. The dynamic process of actin polymerization requires energy in the form of an ATP molecule that is hydrolyzed to ADP after a G-actin molecule is incorporated into the flament.  Actin flament–severing proteins cut long actin filaments into short fragments. An example of such a protein is gelsolin, a 90-kDa ABP that normally initiates actin polymerization but at high Ca2 concentrations causes severing of the actin filaments, converting an actin gel into a ﬂuid state.


Histology_Ross

Histology_Ross.  Actin-capping proteins block further addition of actin molecules by binding to the free end of an actin filament. An example is tropomodulin, which can be isolated from skeletal and cardiac muscle cells. Tropomodulin binds to the free end of actin myofilaments, regulating the length of the filaments in a sarcomere.  Actin cross-linking proteins are responsible for cross-linking actin filaments with each other. An example of such proteins can be found in the cytoskeleton of erythrocytes. Several proteins—such as spectrin, adductin, protein 4.1,and protein 4.9—are involved in cross-linking actin filaments.  Actin motor proteins belong to the myosin family, which hydrolyzes ATP to provide the energy for movement along the actin filament from the minus end to the plus end. Some cells, such as muscle cells, are characterized by the size, amount, and nature of the filaments and actin-motor discussed in Chapter 11 (Muscle Tissue).


Histology_Ross

Histology_Ross. In addition to myosin II, nonmuscle cells contain myosin I, a protein with a single globular domain and short tail that attaches to other molecules or organelles. Extensive studies have revealed the presence of a variety of other nonmuscle myosin isoforms that are responsible for motor functions in many specialized cells, such as melanocytes, kidney and intestinal absorptive cells, nerve growth cones, and inner ear hair cells. Actin filaments participate in a variety of cell functions. Actin filaments are often grouped in bundles close to the plasma membrane. Functions of these membrane-associated actin filaments include the following.  Anchorage and movement of membrane protein. Actin filaments are distributed in three-dimensional networks throughout the cell and are used as anchors within specialized cell junctions such as focal adhesions.


Histology_Ross

Histology_Ross.  Formation of the structural core of microvilli on absorptive epithelial cells. Actin filaments may also help maintain the shape of the apical cell surface (e.g., the apical terminal web of actin filaments serves as a set of tension cables under the cell surface).  Locomotion of cells. Locomotion is achieved by the force exerted by actin filaments by polymerization at their growing ends. This mechanism is used in many migrating cells—in particular, on transformed cells of invasive tumors. As a result of actin polymerization at their leading edge, cells extend processes from their surface by pushing the plasma membrane ahead of the growing actin filaments. The leading-edge extensions of a crawling cell are called lamellipodia; they contain elongating organized


Histology_Ross

Histology_Ross. FIGURE 2.47 • Thin flament organization and structure in cardiac cells. a. Immunofuorescence micrograph of a chick cardiac myocyte stained for actin (green) to show the thin flaments and for tropomodulin (red ) to show the location of the slow-growing ( ) ends of the thin flaments. Tropomodulin appears as regular striations because of the uniform lengths and alignment of the thin flaments in sarcomeres. 320. (Courtesy of Drs. Velia F. Fowler and Ryan Littlefeld.) b. Diagram of a thin flament. The polarity of the thin flament is indicated by the fast-growing ( ) end and the slow-growing ( ) end. Only a portion of the entire thin flament is shown for clarity. Tropomodulin is bound to actin and tropomyosin at the slow-growing ( ) end. The troponin complex binds to each tropomyosin molecule every seven actin monomers along the length of the thin flament. (Courtesy of Drs. Velia F. Fowler and Ryan Littlefeld.) bundles of actin filaments with their plus ends directed to ward the plasma


Histology_Ross

Histology_Ross. every seven actin monomers along the length of the thin flament. (Courtesy of Drs. Velia F. Fowler and Ryan Littlefeld.) bundles of actin filaments with their plus ends directed to ward the plasma membrane. Extension of cell processes. These processes can be ob called flopodia, located around their surface. As in lamel lipodia, these protrusions contain loose aggregations of 10 to 20 actin filaments organized in the same direction, again with their plus ends directed toward the plasma mem brane. Actin filaments are also essential in cytoplasmic streaming (i.e., the streamlike movement of cytoplasm that can be observed in cultured cells).


Histology_Ross

Histology_Ross. Intermediate flaments play a supporting or general structural role. These ropelike filaments are called intermediate because their diameter of 8 to 10 nm is between those of actin filaments and microtubules. Nearly all intermediate filaments consist of subunits with a molecular weight of about 50 kDa. Some evidence suggests that many of the stable structural proteins in intermediate filaments evolved from highly conserved enzymes, with only minor genetic modification. Intermediate filaments are formed from nonpolar and highly variable intermediate filament subunits.


Histology_Ross

Histology_Ross. Intermediate filaments are formed from nonpolar and highly variable intermediate filament subunits. Unlike those of microfilaments and microtubules, the protein subunits of intermediate filaments show considerable diversity and tissue specificity. In addition, they do not posses enzymatic activity and form nonpolar filaments. Intermediate filaments also do not typically disappear and re-form in the continuous manner characteristic of most microtubules and actin filaments. For these reasons, intermediate filaments are believed to play a primarily structural role within the cell and to compose the cytoplasmic link of a tissuewide continuum of cytoplasmic, nuclear, and extracellular filaments (Fig. 2.48).


Histology_Ross

Histology_Ross. Intermediate flament proteins are characterized by a highly variable central rod-shaped domain with strictly conserved globular domains at either end (Fig. 2.49). Although the various classes of intermediate filaments differ in the amino acid sequence of the rod-shaped domain and show some variation in molecular weight, they all share a homologous region that is important in filament self-assembly. Intermediate filaments are assembled from a pair of helical monomers that twist around each other to form coiled-coil dimers. Then, two coiled-coil dimers twist around each other in antiparallel fashion (parallel but pointing in opposite directions) to generate a staggered tetramer of two coiled-coil dimers, thus forming the nonpolarized unit of the intermediate filaments (see Fig. 2.49). Each tetramer, acting as an individual unit, is aligned along the axis of the filament. The ends of the tetramers are bound together to form the free ends of the filament. This assembly process provides a


Histology_Ross

Histology_Ross. tetramer, acting as an individual unit, is aligned along the axis of the filament. The ends of the tetramers are bound together to form the free ends of the filament. This assembly process provides a stable, staggered, helical array in which filaments are packed together and additionally stabilized by lateral binding interactions between adjacent tetramers.


Histology_Ross

Histology_Ross. Intermediate filaments are a heterogeneous group of cytoskeletal elements found in various cell types. FIGURE 2.48 • Electron micrograph of the apical part of an epithelial cell demonstrating intermediate flaments. This electron micrograph, obtained using the quick-freeze deep-etch technique, shows the terminal web (TW ) of an epithelial cell and underlying intermediate flaments (IF ). The long, straight actin flament cores or rootlets (R) extending from the microvilli are cross-linked by a dense network of actin flaments containing numerous actin-binding proteins. The network of intermediate flaments can be seen beneath the terminal web anchoring the actin flaments of the microvilli. 47,000. (Reprinted with permission from Hirokawa N, Keller TC 3rd, Chasan R, Mooseker MS. Mechanism of brush border contractility studied by the quick-freeze, deep-etch method. J Cell Biol 1983;96:1325–1336.)


Histology_Ross

Histology_Ross. Intermediate filaments are organized into six major classes on the basis of gene structure, protein composition, and cellular distribution (Table 2.3).  Classes 1 and 2. These are the most diverse groups of intermediate filaments and are called keratins (cytokeratins). These classes contain more than 50 different isoforms and account for most of the intermediate filaments (about 54 genes out of the total of 70 human intermediate filament genes are linked to keratin molecules). Keratins only assemble as heteropolymers; an acid cytokeratin (class 1) and a basic cytokeratin (class 2) molecule form a heterodimer. Each keratin pair is characteristic of a particular type of epithelium; however, some epithelial cells may express more than one pair. Keratin filaments are found in different cells of epithelial origin. According to new nomenclature, keratins are divided into three expression groups: keratins of simple epithelia,


Histology_Ross

Histology_Ross. FIGURE 2.49 • Polymerization and structure of intermediate flaments. Intermediate flaments are self-assembled from a pair of monomers that twist around each other in parallel fashion to form a stable dimer. Two coiled-coil dimers then twist around each other in antiparallel fashion to generate a staggered tetramer of two coiled-coil dimers. This tetramer forms the nonpolarized unit of the intermediate flaments. Each tetramer, acting as an individual unit, aligns along the axis of the flament and binds to the free end of the elongating structure. This staggered helical array is additionally stabilized by lateral binding interactions between adjacent tetramers.


Histology_Ross

Histology_Ross. keratins of stratifed epithelia, and structural keratins, also called hard keratins. The latest are found in skin appendages such as hair and nails. Keratin filaments span the cytoplasm of epithelial cells and, via desmosomes, connect with keratin filaments in neighboring cells. Keratin subunits do not co-assemble with other classes of intermediate filaments; therefore, they form a distinct cell-specific and tissuespecific recognition system.


Histology_Ross

Histology_Ross.  Class 3. This group contains four proteins: vimentin, the most widely distributed intermediate filament protein in the body, and vimentin-like proteins such as desmin, glial fbrillary acidic protein (GFAP), and peripherin. They represent a diverse family of cytoplasmic filaments found in many cell types. In contrast to keratins, class 3 proteins (with the exception of desmin) preferentially form homopolymeric filaments containing only one type of intermediate protein. Vimentin is the most abundant intermediate filament found in all mesoderm-derived cells, including fibroblasts (Fig. 2.50); desmin is characteristic of muscle cells; GFAP is found in glial cells (highly specific for astrocytes), and peripherin is found in many peripheral nerve cells.


Histology_Ross

Histology_Ross.  Class 4. Historically this group has been called neuroflaments; they contain intermediate filament proteins that are expressed mostly in axons of nerve cells. The three types of neurofilament proteins are of different molecular weights: NF-L (a low-weight protein), NF-M (a medium-weight protein), and NF-H (a high-weight protein). They co-assemble to form a heterodimer that contains one NF-L molecule and one of the others. All three proteins form neurofilaments that extend from the cell body into the ends of axons and dendrites, providing structural support. However, genes for class 4 proteins also encode several other intermediate filament proteins. These include nestin and -internexin in nerve cells as well as synemin, syncoilin, and paranemin in muscle cells. Members of this group preferentially co-assemble in tissues as heteropolymers.


Histology_Ross

Histology_Ross.  Class 5. Lamins, specifically nuclear lamins, form a network-like structure that is associated with the nuclear envelope. Lamins are represented by two types of proteins, lamin A and lamin B. In contrast to other types of intermediate filaments found in the cytoplasm, lamins are located within the nucleoplasm of almost all differentiated cells in the body. A description of their structure and function can be found on page 82.  Class 6. This is a lens-specific group of intermediate filament, or “beaded flaments” containing two proteins, phakinin and flensin. The periodic beadlike surface appearance of these filaments is attributed to the globular structure of the carboxy-terminus of the filensin molecule, which projects out from the assembled filament core. Intermediate filament–associated proteins are essential for the integrity of cell-to-cell and cell-to-extracellular matrix junctions.


Histology_Ross

Histology_Ross. Intermediate filament–associated proteins are essential for the integrity of cell-to-cell and cell-to-extracellular matrix junctions. A variety of intermediate flament–associated proteins function within the cytoskeleton as integral parts of the molecular architecture of cells. Some proteins, such as those of the plectin family, possess binding sites for actin filaments,


Histology_Ross

Histology_Ross. TABLE Classes of Intermediate Filaments Their Location and Associated Diseases 2.3 Molecular Weight Examples of Type of Protein (kDa) Where Found Associated Diseases Class 1 and 2: Keratins Acid cytokeratins 40–64 All epithelial cells Epidermolysis bullosa simplex Basic cytokeratins 52–68 All epithelial cells Keratoderma disorders caused by keratin mutations Meesman corneal dystrophy Class 3: Vimentin and Vimentin-like Vimentin 55 Cells of mesenchymal origin Desmin-related myopathy (including endothelial cells, (DRM) myofbroblasts, some smooth Dilated cardiomyopathy muscle cells) and some cells of Alexander disease neuroectodermal origin Amyotrophic lateral sclerosis Desmin 53 Muscle cells; co-assembles with (ALS) nestin, synemin and paranemin Glial fbrillary acidic protein 50–52 Neuroglial cells (mainly astrocytes; (GFAP) to lesser degree, ependymal cells), Schwann cells, enteric glial cells, satellite cells of sensory ganglia, and pituicytes Peripherin 54 Peripheral neurons Class 4:


Histology_Ross

Histology_Ross. cells (mainly astrocytes; (GFAP) to lesser degree, ependymal cells), Schwann cells, enteric glial cells, satellite cells of sensory ganglia, and pituicytes Peripherin 54 Peripheral neurons Class 4: Neurofilaments Neuroflament L (NF-L) 68 Neurons Charcot-Marie-tooth disease Co-assembles with NF-M or NF-H Parkinson disease Neuroflament M (NF-M) 110 Neurons Co-assembles with NF-L Neuroflament H (NF-H) 130 Neurons Co-assembles with NF-L Nestin 240 Neural stem cells, some cells of neuroectodermal origin, muscle cells Co-assembles with desmin Α-Internexin 68 Neurons Synemin Α/ΒA 182 Muscle cells Co-assembles with desmin Syncoilin 64 Muscle cells Paranemin 178 Muscle cells Co-assembles with desmin Class 5: Lamins Lamin A/CB 62–72 Nucleus of all nucleated cells Emery-Dreyfuss muscular dystrophy Lamin B 65–68 Nucleus of all nucleated cells Limb girdle muscular dystrophy Class 6: Beaded Filaments Phakinin (CP49)C 49 Eye lens fber cells Juvenile-onset cataracts Co-assembles with flesin


Histology_Ross

Histology_Ross. Lamin B 65–68 Nucleus of all nucleated cells Limb girdle muscular dystrophy Class 6: Beaded Filaments Phakinin (CP49)C 49 Eye lens fber cells Juvenile-onset cataracts Co-assembles with flesin Congenital cataracts Filesin (CP115) 115 Eye lens fber cells Co-assembles with phakinin A Synemin and synemin represent two alternative transcripts of the DMN gene. BLamin C is a splice product of lamin A. C The molecular weight of filensin/phakinin heterodimer is 131 kilodaltons.


Histology_Ross

Histology_Ross. microtubules, and intermediate filaments and are thus important in the proper assembly of the cytoskeleton. Lamins, the intermediate filaments in the nucleus, are associated with numerous proteins in the inner nuclear membrane, including emerin, lamin B receptor (LBR), nurim, and several lamina-associated polypeptides. Some of these proteins have multiple binding sites to intermediate filaments, actin, chromatin, and signaling proteins; thus, they function in chromatin organization, gene expression, nuclear architecture, and cell signaling and provide an essential link between the nucleoskeleton and cytoskeleton of the cell. Another important family of intermediate filament–associated proteins consists of desmoplakins, desmoplakinlike proteins, and plakoglobins. These proteins form the attachment plaques for intermediate filaments, an essential part of desmosomes and hemidesmosomes. The interaction of intermediate filaments with cell-to-cell and cell-toextracellular matrix junctions


Histology_Ross

Histology_Ross. plaques for intermediate filaments, an essential part of desmosomes and hemidesmosomes. The interaction of intermediate filaments with cell-to-cell and cell-toextracellular matrix junctions provides mechanical stren gth and resistance to extracellular forces. Table 2.4 summarizes the characteristics of the three types of cytoskeletal filaments.


Histology_Ross

Histology_Ross. Centrioles represent the focal point around which the MTOC assembles. Centrioles, visible in the light microscope, are paired, short, rodlike cytoplasmic cylinders built from nine microtubule triplets. In resting cells, centrioles have an orthogonal


Histology_Ross

Histology_Ross. FIGURE 2.50 • Distribution of intermediate flaments in human fetal lung fbroblasts. Distribution of vimentin (red) and actin flaments (green) is shown in cultured fbroblasts from human fetal lung. Vimentin is an intermediate flament protein expressed in all cells of mesenchymal origin. In cultured fbroblasts, vimentin flaments are visible centrally within the cell cytoplasm, whereas the actin flaments are aggregated primary near the cell surface. This immunofuorescent image was obtained using the indirect immunofuorescence techniques in which vimentin flaments were treated with mouse anti-vimentin primary antibodies followed by goat anti-mouse secondary antibodies conjugated with Texas red fuorescent dye. Actin flaments were counter-stained with phalloidin conjugated with a green fuorescent dye. Nuclei were stained blue with Hoechst fuorescent stain. 3,500. (Reprinted with permission from Michael W. Davidson, Florida State University.) orientation: One centriole in the pair is arrayed


Histology_Ross

Histology_Ross. dye. Nuclei were stained blue with Hoechst fuorescent stain. 3,500. (Reprinted with permission from Michael W. Davidson, Florida State University.) orientation: One centriole in the pair is arrayed at a right angle to the other. Centrioles are usually found close to the nucleus, often partially surrounded by the Golgi apparatus, and associated with a zone of amorphous, dense pericentriolar material. The region of the cell containing the centrioles and pericentriolar material is called the microtubule-organizing center or centrosome (Fig. 2.51). The MTOC is the region where most microtubules are formed and from which they are then directed to specific destinations within the cell. Therefore, the MTOC controls the number, polarity, direction, orientation, and organization of microtubules formed during the interphase of the cell cycle. During mitosis, duplicated MTOCs serve as mitotic spindle poles. Development of the MTOC itself depends solely on the presence of centrioles. When


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Histology_Ross. formed during the interphase of the cell cycle. During mitosis, duplicated MTOCs serve as mitotic spindle poles. Development of the MTOC itself depends solely on the presence of centrioles. When centrioles are missing, the MTOCs disappear, and formation of microtubules is severely impaired.


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Histology_Ross. The pericentriolar matrix of MTOC contains nume rous ring-shaped structures that initiate microtubule formation. The MTOC contains centrioles and an amorphous pericentriolar matrix of more than 200 proteins, including tubulin that is organized in ring-shaped structures. Each -tubulin ring serves as the starting point (nucleation site) for the growth of one microtubule that is assembled from tubulin dimers; and -tubulin dimers are added with specific orientation to the -tubulin ring. The minus end of the micro-tubule remains attached to the MTOC, and the plus end


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Histology_Ross. TABLE Summary Characteristics of Three Types of Cytoskeletal Elements 2.4 Shape Double-stranded linear Ropelike fibers Nonbranching long, hollow helical array cylinders Diameter 6–8 8–10 20–25 (nm) Basic Monomer of G-actin Various intermediate filament Dimers of and -tubulin protein (MW 42 kDa) proteins (MW ~50 kDa) (MW 54 kDa) -tubulin subunit found in MTOC is necessary for nucleation of microtubules -, -, -, -tubulins are associated with MTOC and basal bodies Enzymatic ATP hydrolytic activity None GTP hydrolytic activity activity Polarity Yes Nonpolar structures Yes Minus ( ) or pointed Minus ( ) end is nongrowing end is slow-growing end end embedded in MTOC Plus ( ) or barbed end Plus ( ) end is the growing end is faster-growing end Assembly Monomers of G-actin are Two pairs of monomers At the nucleation site, and process added to growing flament form two coiled-coil -tubulin dimers are added Polymerization requires dimers; then two coiled-to -tubulin ring in an presence of K , Mg2


Histology_Ross

Histology_Ross. At the nucleation site, and process added to growing flament form two coiled-coil -tubulin dimers are added Polymerization requires dimers; then two coiled-to -tubulin ring in an presence of K , Mg2 , and coil dimers twist around end-to-end fashion ATP, which is hydrolyzed to each other to generate Each tubulin dimer ADP after each G-actin a staggered tetramer, molecule binds GTP before it molecule is incorporated which aligns along the becomes incorporated into into the flament axis of the flament and the microtubule binds to the free end of Polymerization also requires the elongating structure presence of Mg2 GTP–tubulin complex is polymerized; after incorporation, GTP is hydrolyzed to GDP Source of ATP N/A GTP energy required for assembly Characteristics Thin, fexible flaments Strong, stable structures Exhibit dynamic instability Associated Variety of ABPs with different Intermediate flament– Microtubule-associated proteins functions: fascin bundling; associated proteins: plectins


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Histology_Ross. stable structures Exhibit dynamic instability Associated Variety of ABPs with different Intermediate flament– Microtubule-associated proteins functions: fascin bundling; associated proteins: plectins proteins: MAP-1, 2, 3, gelsolin flament severing; bind micro tubules, actin, and 4, MAP-, and TOG CP protein capping; and intermediate flaments; regulate assembly, stabilize, spectrin cross-linking; desmoplakins and plakoglobins and anchor microtubules to myosin I and II motor attach intermediate flaments specifc organelles; motor functions to desmosomes and proteins—dyneins and kinesins— hemidesmosomes required for organelle movement Actin Filaments (Microfilaments) Intermediate Filaments Microtubules chapter 2 Location Core of microvilli Extend across cytoplasm Core of cilia in cell Terminal web connecting desmosomes Emerge from Concentrated beneath and hemidesmosomes MTOC and spread plasma membrane In nucleus just beneath into periphery of Contractile elements inner nuclear membrane


Histology_Ross

Histology_Ross. web connecting desmosomes Emerge from Concentrated beneath and hemidesmosomes MTOC and spread plasma membrane In nucleus just beneath into periphery of Contractile elements inner nuclear membrane cell of muscles Mitotic spindle, Contractile ring Centrosome in dividing cells Major Provide essential components Provide mechanical strength Provide network functions to contractile elements and resistance to shearing (“railroad tracks”) for of muscle cells (sarcomeres) forces movement of organelles within cell Provide movement for cilia and chromosomes during cell division TABLE Summary Characteristics of Three Types of Cytoskeletal Elements, Cont.2.4 Actin Filaments (Microfilaments) Intermediate Filaments Microtubules


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Histology_Ross. FIGURE 2.51 • Structure of the MTOC. This diagram shows the represents the growing end directed toward the plasma membrane (see Fig. 2.51). Centrioles provide basal bodies for cilia and flagella and align the mitotic spindle during cell division. Although centrioles were discovered more than a century ago, their precise functions, replication, and assembly are still under intense investigation. The known functions of centrioles can be organized into two categories:  Basal body formation. One of the important functions of the centriole is to provide basal bodies, which are necessary for the assembly of cilia and flagella (Fig. 2.52).


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Histology_Ross. Basal bodies are formed either by de novo formation without contact with the pre-existing centrioles (the acentriolar pathway) or by duplication of existing centrioles (the centriolar pathway). About 95% of the centrioles are generated through the acentriolar pathway. Both pathways give rise to multiple immediate precursors of centrioles, known as procentrioles, which mature as they migrate to the appropriate site near the apical cell membrane, where they become basal bodies (Fig. 2.53). The basal body acts as the organizing center for a cilium. Microtubules grow upward from the basal body, pushing the cell membrane outward, and elongate to form the mature cilium. The process of centriole duplication is described later on page 70.


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Histology_Ross.  Mitotic spindle formation. During mitosis, the position location of the MTOC in relation to the nucleus and the Golgi apparatus. In some species, the MTOC is tethered to the nuclear envelope by a contractile protein, the nucleus–basal body connector (NBBC). The MTOC contains the centrioles and an amorphous protein matrix with an abundance of -tubulin rings. Each -tubulin ring serves as the nucleation site for the growth of a single microtubule. Note that the minus (–) end of the microtubule remains attached to the MTOC, and the plus ( ) end represents the growing end directed toward the plasma membrane.


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Histology_Ross. of centrioles determines the location of mitotic spindle poles. Centrioles are also necessary for the formation of a fully functional MTOC, which nucleates mitotic spindle– associated microtubules. For instance, astral microtubules are formed around each individual centriole in a starlike fashion. They are crucial in establishing the axis of the developing mitotic spindle. In some animal cells, the mitotic spindle itself (mainly kinetochore microtubules) is  FOLDER 2.2 Clinical Correlation: Abnormalities in Microtubules and Filaments Abnormalities related to the organization and structure of microtubules, actin, and intermediate flaments underlie a variety of pathologic disorders. These abnormalities lead to defects in the cytoskeleton and can produce a variety of defects related to intracellular vesicular transport, intracellular accumulations of pathologic proteins, and impairment of cell mobility.


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Histology_Ross. Defects in the organization of microtubules and microtubule-associated proteins can immobilize the cilia of respiratory epithelium, interfering with the ability of the respiratory system to clear accumulated secretions. This condition, known as Kartagener’s syndrome (see page 120), also causes dysfunction of microtubules, which affects sperm motility and leads to male sterility. It may also cause infertility in women because of impaired ciliary transport of the ovum through the oviduct.


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Histology_Ross. Microtubules are essential for vesicular transport (endocytosis and exocytosis) as well as cell motility. Certain drugs, such as colchicine, bind to tubulin molecules and prevent their polymerization; this drug is useful in the treatment of acute attacks of gout, to prevent neutrophil migration, and to lower their ability to respond to urate crystal deposits in the tissues. Vinblastine and vincristine (Oncovin) represent another family of drugs that bind to microtubules and inhibit the formation of the mitotic spindle essential for cell division. FIGURE F.2.2.1 • Photomicrograph of Mallory bodies. Ac-These drugs are used as antimitotic and antiproliferative cumulation of keratin intermediate flaments forming intercelluagents in cancer therapy. Another drug, paclitaxel (Taxol), lar inclusions is frequently associated with specifc cell injuries. is used in chemotherapy for breast cancer. It stabilizes mi-In alcoholic liver cirrhosis, hepatocytes exhibit such inclusions crotubules,


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Histology_Ross. is frequently associated with specifc cell injuries. is used in chemotherapy for breast cancer. It stabilizes mi-In alcoholic liver cirrhosis, hepatocytes exhibit such inclusions crotubules, preventing them from depolymerizing (an action (arrows), which are known as Mallory bodies. Lymphocytes and opposite to that of colchicine), and thus arrests cancer cells macrophages responsible for an intense infammatory reaction in various stages of cell division. surround cells containing Mallory bodies. 900.


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Histology_Ross. Actin flaments are essential for various stages of leukocyte in neuroflaments within brain tissue are characteristic of migration as well as for the phagocytotic functions of vari Alzheimer’s disease, which produces neurofbrillary ous cells. Some chemical substances isolated from fungi, tangles containing neuroflaments and other microtubule such as cytochalasin B and cytochalasin D, prevent associated proteins.


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Histology_Ross. actin polymerization by binding to the plus end of the actin Another disorder of the central nervous system,flament inhibiting lymphocyte migration, phagocytosis, and Alexander disease is associated with mutations in the cell division (cytokinesis). Several toxins of poisonous coding region of the GFAP gene. The pathologic feature of mushrooms, such as phalloidin, also bind to actin flathis disease is the presence of cytoplasmic inclusionsments, stabilizing them and preventing their depolymeriza tion. Conjugated with fuorescein dyes, derivatives of the tion of intermediate flament protein GFAP. Altered GFAP phallotoxin family (i.e., NDB-phallacidin) are frequently prevents the assembly not only of intermediate flamentsused in the laboratory to stain actin flaments (see Figs.


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Histology_Ross. but also of other proteins that contribute to the structural2.45 and 2.50). Prolonged exposure of the cell to these integrity and function of astrocytes. Infants with Alexander substances can disrupt the dynamic equilibrium between disease develop leukoencephalopathy (infection of theF-actin and G-actin, causing cell death.


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Histology_Ross. brain) with macrocephaly (abnormally large head), seizures, and psychomotor retardation, leading to death As noted, the molecular structure of intermediate flaments usually within the frst decade of life. is tissue specifc and consists of many different types of A prominent feature of alcoholic liver cirrhosis is the proteins. Several diseases are caused by defects in the presence of eosinophilic intracytoplasmic inclusions com-proper assembly of intermediate flaments. These defects posed predominantly of keratin intermediate flaments. have also been induced experimentally by mutations in in-These inclusions, called Mallory bodies, are visible in light termediate flament genes in laboratory animals. Changes microscopy within the hepatocyte cytoplasm (Fig. F.2.2.1).


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Histology_Ross. FIGURE 2.52 • Basal bodies and cilia. This electron micro-graph shows the basal bodies and cilia in cross-sectional profle as seen in an oblique section through the apical part of a ciliated cell in the respiratory tract. Note the 9 2 microtubule arrangement of the cilia in which nine microtubules at the periphery of the cilia surround two central microtubules. The basal bodies lack the central tubule pair. On several cross sections, the basal foot is visible as it projects laterally from the basal body (asterisks).


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Histology_Ross. 28,000. (Courtesy of Patrice C. Abell-Aleff.) formed by MTOC-independent mechanisms and consists of microtubules that originate from the chromosomes. Recent experimental data indicate that in the absence of centrioles, astral microtubules fail to develop, causing errors in mitotic spindle orientation (Fig. 2.54). Thus, the primary role of centrioles in mitosis is to position the mitotic spindle properly by recruiting the MTOC from which astral microtubules can grow and establish the axis for the developing spindle. The dominant feature of centrioles is the cylindrical array of triplet microtubules with associated proteins. The TEM reveals that each rod-shaped centriole is about 0.2 m long and consists of nine triplets of microtubules that are oriented parallel to the long axis of the organelle and run in slightly twisted bundles (Fig. 2.55). The three micro-


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Histology_Ross. FIGURE 2.53 • Two pathways of basal body formation. In the centriolar pathway, a pair of existing centriole serves as an organizing center for the duplication of new centrioles. Utilizing this pathway, ciliated cells have the ability to assemble large number of centrioles in the vicinity of an old mature centriole. In the acentriolar pathway, which plays a major role in the formation of basal bodies in ciliated cells, new centrioles are formed de novo from fbrous granules located in close proximity of nonmicrotubular structures called deuteresomes. Both pathways give rise to procentrioles, which mature as they migrate to the appropriate site near the apical cell membrane, where they become basal bodies. Fibrous granules contribute to the formation of the striated rootlet. (Based on Hagiwara H, Ohwada N, Takata K. Cell biology of normal and abnormal ciliogenesis in the ciliated epithelium. Int Rev Cytol 2004;234:101–139.) tubules of the triplet are fused, with adjacent microtubules


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Histology_Ross. H, Ohwada N, Takata K. Cell biology of normal and abnormal ciliogenesis in the ciliated epithelium. Int Rev Cytol 2004;234:101–139.) tubules of the triplet are fused, with adjacent microtubules sharing a common wall. The innermost or A microtubule is a complete ring of 13 protofilaments containing and tubulin dimers; the middle and outer B and C micro-tubules, respectively, appear C-shaped because they share tubulin dimers with each other and with the A microtubule. The microtubules of the triplets are not equal in length. The C microtubule of the triplet is usually shorter than A and B.


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Histology_Ross. The microtubule triplets of the centriole surround an internal lumen. The distal part of the lumen (away from the nucleus) contains a 20-kDa Ca2 -binding protein, centrin (Fig. 2.56). The proximal part of the lumen (close to the nucleus) is lined by -tubulin, which provides the template for the arrangement of the triplet microtubules. In addition, a family of newly discovered -, -, -, and -tubulin molecules as well as pericentrin protein complexes have also been localized with the centrioles. Other proteins, such a protein p210, form a ring of molecules that appears to link the distal end of the centriole to the plasma membrane. Filamentous connections between the centriole pair have been identified in human lymphocytes. In other organisms, two protein bridges, the proximal and distal connecting fbers, connect each centriole in a pair (see Fig. 2.56). In dividing cells, these connections participate in segregating the centrioles to each daughter cell. In some organisms, the proximal


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Histology_Ross. fbers, connect each centriole in a pair (see Fig. 2.56). In dividing cells, these connections participate in segregating the centrioles to each daughter cell. In some organisms, the proximal end of


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Histology_Ross. FIGURE 2.54 • Mitotic spindle during normal cell division and in cells lacking centrioles. a. This schematic drawing shows the orientation of the mitotic spindle in a normal cell undergoing mitosis. Note the positions of the centrioles and the distribution of the spindle microtubules. b. In a cell that lacks centrioles, mitosis occurs and a mitotic spindle containing only kinetochore micro-tubules is formed. However, both poles of the mitotic spindle lack astral microtubules, which position the spindle in proper plane during the mitosis. Such a misoriented spindle is referred to as an anastral bipolar spindle. (Based on Marshall WF, Rosenbaum JL. How centrioles work: lessons from green yeast. Curr Opin Cell Biol 2000;12:119–125.) each centriole is attached to the nuclear envelope by contractile proteins called nucleus–basal body connectors (NBBCs). Their function is to link the centriole to the mitotic spindle poles during mitosis. In human cells, the centrosome–nucleus connection


Histology_Ross

Histology_Ross. proteins called nucleus–basal body connectors (NBBCs). Their function is to link the centriole to the mitotic spindle poles during mitosis. In human cells, the centrosome–nucleus connection appears to be maintained by filamentous structures of cytoskeleton. A distinctive feature of mammalian centrioles is the difference between individual centrioles in the pair. One centriole (termed the mature centriole) contains stalklike satellite processes and sheetlike appendages whose function is not known (see Fig. 2.56). The other centriole (termed the immature centriole) does not possess satellites or appendages.


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Histology_Ross. Centrosome duplication is synchronized with the cell-cycle events and linked to the process of ciliogenesis. Centrosome dynamics such as duplication or formation of basal bodies for ciliogenesis are synchronized with the cell- FIGURE 2.55 • Electron micrograph showing parent and daughter centrioles in a fbroblast. Note that the transverse-sectioned centriole in each of the pairs reveals the triplet confguration of microtubules. The lower right centriole represents a mid-longitudinal section, whereas the upper left centriole has also been longitudinally sectioned, but along the plane of its wall.


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Histology_Ross. 90,000. (Courtesy of Drs. Manley McGill, D. P. Highfeld, T. M. Monahan, and Bill R. Brinkley.) cycle progresion. Cilia are assembled during the G1 phase; they are most abundant in GO, and are disassembled before cell enters M phase of the cell cycle. These events are depicted in Figure 2.57, which shows an association between centrosome duplication, primary cilium formation, and progression through the cell cycle. Since each daughter cell receives only one pair of centrioles after cell division, the daughter cells must duplicate existing centrioles prior to cell division. In most somatic cells, duplication of centrioles begins near the transition between the G1 and S phases of the cell cycle. This event is closely associated with the activation of the cyclin E-Cdk2 complex during the S phase of the cell cycle (see Fig. 3.11). This


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Histology_Ross. FIGURE 2.56  Schematic structure of centrioles. In nondividing cells, centrioles are arranged in pairs in which one centriole is aligned at a right angle to the other. One centriole is also more mature (generated at least two cell cycles earlier) than the other centriole, which was generated in the previous cell cycle. The mature centriole is characterized by the presence of satellites and appendages. Centrioles are located in close proximity to the nucleus. The basic components of each centriole are microtubule triplets that form the cylindrical structure surrounding an internal lumen. The proximal part of the lumen is lined by -tubulin, which provides the template for nucleation and arrangement of the microtubule triplets. The distal part of each lumen contains the protein centrin. In some species, two protein bridges, the proximal and distal connecting fbers, connect each centriole in a pair. In some species, but not in humans, the proximal end of each centriole is attached to the


Histology_Ross

Histology_Ross. species, two protein bridges, the proximal and distal connecting fbers, connect each centriole in a pair. In some species, but not in humans, the proximal end of each centriole is attached to the nuclear envelope by a contractile protein known as the nucleus–basal body connector.


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Histology_Ross. complex directly phosphorylates the nucleus-chaperoning protein nucleophosmin/B23, which is responsible for initiating the duplication of centrioles.


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Histology_Ross. In most cells, duplication begins with the splitting of a centriole pair, followed by the appearance of a small mass of fibrillar and granular material at the proximal lateral end of each original centrioles. Because the existing pair of centrioles serves as a core for new organelle formation, this process of centriole duplication is referred as the centriolar pathway (see Fig. 2.53). The fbrous granules coalesce into dense spherical structures called deuterosomes, and they give rise to the procentriole (or bud), which gradually enlarges to form a right-angle appendage to the parent (see Fig. 2.53). Microtubules begin to develop in the mass of fibrous granules as it grows (usually during the S to late G2 phases of the cell cycle), appearing first as a ring of nine single tubules, then as doublets, and finally as triplets. As procentrioles mature during the S and G2 phases of the cell cycle, each parent–daughter pair migrates around the nucleus. Before the onset of mitosis,


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Histology_Ross. then as doublets, and finally as triplets. As procentrioles mature during the S and G2 phases of the cell cycle, each parent–daughter pair migrates around the nucleus. Before the onset of mitosis, centrioles with surrounding amorphous pericentriolar material position themselves on opposite sides of the nucleus and produce astral microtubules. In doing so, they define the poles between which the bipolar mitotic spindle develops.


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Histology_Ross. The important difference between duplication of centrioles during mitosis and during ciliogenesis is the fact that during mitosis only one daughter centriole buds from the lateral side of parent organelle, whereas during ciliogenesis as many as ten centrioles may develop around parent centriole. Development of cilia on the cell surface requires the presence of basal bodies, structures derived from centrioles.


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Histology_Ross. Each cilium requires a basal body. The generation of centrioles, which occurs during the process of ciliogenesis, is responsible for the production of basal bodies. The newly formed centrioles migrate to the apical surface of the cell and serve as organizing centers for the assembly of the microtubules of the cilium. The core structure (axoneme) of a motile cilium is composed of a complex set of microtubules consisting of two central microtubules surrounded by nine microtubule doublets (9 2 configuration). The organizing role of the basal body differs from that of the MTOC. The axonemal microtubule doublets are continuous with the A and B microtubules of the basal body from which they develop by addition of and -tubulin dimers at the growing plus end. A detail description of the structure of cilia, basal bodies, and the process of ciliogenesis can be found in Chapter 5, Epithelial Tissue.