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Ovid: Koss' Diagnostic Cytology and Its Histopathologic Bases

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Editors: Koss, Leopold G.; Melamed, Myron R. Title: Koss' Diagnostic Cytology and Its Histopathologic Bases, 5th Edition Copyright 2006 Lippincott Williams & Wilkins
> Table of Contents > I - General Cytology > 2 - The Basic Structure of the Mammalian Cell

2 The Basic Structure of the Mammalian Cell

A cell is a self-contained fundamental unit of life. All cells are tridimensional, space-occupying structures, although when spread on a glass slide and viewed through the light microscope, they appear to be flat. Each mammalian cell has three essential components: cell membrane, cytoplasm, and nucleus (Fig. 2-1 and see Frontispiece and Fig. 3-1). The cell membrane encloses the transparent cytoplasm. Within the cytoplasm, enclosed in its own membrane or envelope, there is a smaller, approximately spherical dense structurethe nucleus. The nucleus is the principal repository of deoxyribonucleic acid (DNA), the molecule governing the genetic and functional aspects of cell activity (see Chap. 3). Although some mammalian cells, such as erythrocytes or squamous cells, may lose their nucleus in the final stages of their life cycle, even these final events are programmed by their DNA. All nucleated cells are classified as eukaryotic cells (from Greek, karion = kernel, nucleus) in contrast with primitive cells, such as bacteria, wherein the DNA is present in the cytoplasm but is not enclosed by a membrane as a distinct nuclear structure (prokaryotic cells). Many of the fundamental discoveries pertaining to the molecular biology of cells were made in prokaryotic cells, documenting that all basic biochemical manifestations of life have a common origin. Families of cells differ from each other by their structural features (morphology) and by their activities, all programmed by DNA. The recognition of these cell types and their alterations in health and disease is the principal task of diagnostic cytology. All cells share the fundamental structural components that will be described in these pages.


Cells can be examined by a variety of techniques, ranging from the commonly used light and electron microscopy to newer techniques of confocal and digital microscopy. Additional information on cell structure, derivation, and function can be obtained by immunocytochemistry and by in situ hybridization of cell components. The techniques required for special procedures will be described in the appropriate chapters. This brief summary will serve as an introduction to the description of the fundamental structure of the cell.

Light Microscopy
Bright-Field Light Microscopy
Bright-field light microscopes are optical instruments that allow the examination of cells at magnifications varying from 1 to 2,000, using an appropriate combination of lenses. The highest resolution of the commonly used light microscopes, that is, the ability of the instruments to visualize the smallest objects, is limited by the wavelength of the visible spectrum of light, which is about 0.5 m. The principles of bright-field light microscopy have been described in numerous books and manuals and need not be repeated here. It is assumed that the readers have a working knowledge of these instruments. Suffice it to say that the quality of the optics used, skill in the adjustment of the illumination, and the depth of the microscope's focus are essential P.22 in evaluating the cellular preparations. In practice of clinical cytology, bright-field microscopy satisfies nearly all requirements for the diagnostic assessment of cells. The same technique is used in assessing the results of special stains and of immunocytochemistry.

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Figure 2-1 Benign human fibroblasts from a female patient in tissue culture. A. Low-power view shows the relationship of the cells, which do not overlap each other. B. High-power view shows delicate cytoplasm, generally oval or round nuclei with small multiple nucleoli. Sex chromatin indicated by arrow (A: 250; B: 1,000) (Alcohol fixation, Papanicolaou stain. Culture by Dr. Fritz Herz, Montefiore Hospital. From Koss LG. Morphology of cancer cells. In Handbuch der allgemeinen Pathologie, vol. 6, Tumors, part I. Berlin, Springer, 1974, pp 1-139.)

Preparation of Cells for Bright-Field Light Microscopic Examination

The cells are usually prepared for a light microscopic examination in the form of direct smears on commercially available glass slides of predetermined thickness and optical quality. Samples of cells suspended in fluid may be placed on glass slides by means of a special centrifuge, known as a cytocentrifuge, or a similar apparatus. A cell suspension may also be filtered across a porous membrane. The cells deposited on the surface of such membranes may either be examined directly or may be placed on glass slides by a process of reverse filtration. Cell samples may also be studied in histologic-type sections, after embedding of the sediment in paraffin (a technique known as the cell block). For details of these techniques, see Chapter 44.

Fixation of cell preparations is a common procedure having for its purpose the best possible preservation of cell components after removal from the tissue of origin. A variety of fixatives may serve this purpose, all described in Chapter 44. However, diagnostic techniques may also be based on air-dried cell preparations, either unfixed or postfixed in methanol, which introduce a number of useful artifacts. Such techniques are used in hematology and in aspiration biopsy samples.

Optimal results in bright-field microscopy are obtained on stained preparations that provide visible contrast and discrimination among the cell components. A variety of stains, described in Chapter 44, can be used to best demonstrate various cell components. Common stain combinations use hematoxylin and its variants as the nuclear stain and eosin or its many variants as the cytoplasmic stain. Examples of stains of this type include the hematoxylin-eosin stain and the Papanicolaou stain, which allow for a good visualization of the principal components of the cell, by contrasting the nucleus and the cytoplasm. Other stains in common use include methylene blue, toluidine blue, and Giemsa colorant that provide less contrast among cell components but have the

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advantage of rapidity of use. An example of cells fixed in alcohol and stained by the Papanicolaou method is shown in Figure 2-4.

Phase-Contrast Microscopy
Phase-contrast microscopy utilizes the difference in light diffraction among the various cell components and special optics that allow the visualization of components of unstained cells. The Nomarski technique is a variant of phase contrast microscopy that is particularly useful in the study of cell surfaces. Either technique may be applied to the study of living cells in suspension or culture and, when coupled with time-lapse cinematography or a television system, may P.23 provide a continuous record of cell movements and behavior. These techniques are particularly useful in experimental systems, as they may document the differences in cell behavior under various circumstances, for example, after treatment of cultured cells with a drug or during a genetic manipulation. The systems also allow the study of events, such as movement of chromosomes during cell division, or mitosis. An example of the application of the Nomarski technique to a cell culture is shown in Figure 2-2 .

Fluorescent Microscopy
Cells or cell components stained with fluorescent compounds or probes can be visualized with the help of microscopes provided with special lenses and a source of fluorescent light, such as a mercury bulb or a laser, tuned to an appropriate wavelength, exciting fluorescence of the probe. In highly specialized commercial systems, the amount of fluorescence can be measured in individual cells or families of cells, and may serve to quantify various cell components. A somewhat similar system is used in flow cytometry (see Chap. 47). Fluorescence microscopy is particularly valuable in the procedures known as in situ hybridization, with the purpose of documenting the presence of chromosomes, chromosomal aberrations, or individual genes (see Fig. 2-31 and Figs. 4-26, 4-27, and 429). Fluorescent microscopy is also useful in identifying certain components of cell cytoplasms or cell membranes, using specific antibodies. Application of fluorescent microscopy and other techniques to the study of living cells was summarized in a series of articles on biologic imaging in the journal, Science, 2003.

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Figure 2-2 Time-lapse cinematography, using Nomarski interference contrast optics, shows events in the merging of two colonies of cultured human cancer cells, line C41. (In this technique the cell nuclei are seen in the form of craters wherein are located the nucleoli shown as small elevations.) A. Beginning of sequence: two adjacent colonies. B. Sixteen minutes later: a cytoplasmic bridge between the two colonies has been established. C. Twenty-six minutes later: the area of merger has increased in size. D. Ninety-five minutes later: the merger has progressed to the point at which several cells in both colonies are fused. (Courtesy of Dr. Robert Wolley, Montefiore Hospital.)

Confocal Microscopy
Using a system of complex optics and a laser, the technique, combined with phase and fluorescent microscopy in complex and costly instruments, allows the visualization of cells and tissues in slices, separated from each other by approximately 1 m. The images of the slices can be combined on a computer to give a three-dimensional picture of the cell or tissue and their components. This technique is applicable to individual cells or cell clusters that can be examined layer-by-layer. P.24

Digital Microscopy
With the wide availability of sophisticated computers, it has become possible to transform cell images into digits, that is, numerical values. The images are recorded by television or digital cameras, transformed into numerical values and stored in the computers' memory, on videotape, or on a videodisc. The original images can be reconstituted when needed. Such images, often of outstanding quality, can be manipulated with the help of special software. Images from several different sources can be assembled into plates suitable for publications or special displays. The colors of the displays can be adjusted for optimal quality of images. Many new plates in this book have been prepared with this technique. Digital microscopy can also be applied to electron microscopic images (Shotton, 1995). Digital microscopy has been extensively applied in analytical and quantitative studies of cells and cell components. These techniques allow discrimination among families of cells of similar appearance but different biologic or clinical significance. They can also be applied to a variety of measurements of cell components, such as DNA, as discussed in Chapter 46. Variants of these techniques have been used in commercial instruments for automated or semiautomated analysis of cell populations. Digital microscopy is suitable for direct transmission of images via cable or satellites to remote locations (telepathology or telecytology) for teaching or diagnostic purposes, as discussed in Chapters 1 and 46. Demonstration projects of this technology have documented that such images are of good quality when examined at the receiving stations. The images can be studied under variable magnification factors, thus allowing for diagnostic opinions. Transmissions of images by Internet have been extensively used for teaching. It is conceivable that, in the future, central telepathology consultation centers will be established to advise pathologists on difficult cases. At present, the systems are limited by cost, the speed of transmission, and by the availability of knowledgeable consultants to perform such services.

Electron Microscopy
Transmission Electron Microscopy
Transmission electron microscopic technique utilizes certain optical properties of a fixed beam of electrons to illuminate the object. The images are captured on photographic plates. Extremely thin sections of tissues or cells (50 to 100 nm) and staining with heavy metals are required. Special fixation and embedding techniques must be used. The method allows a unique insight into the fine structure of the cell. Most of the images in this chapter were obtained by this technique.

Scanning Electron Microscopy

In the commonly used mode, the scanning electron microscopy technique utilizes a rapidly moving beam of electrons to scan the surface of cells or other objects. The cells are dehydrated, fixed, and coated with a thin

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metallic layer, usually of gold and palladium. The metal forms an exact replica of the cell surface. The beam of electrons glides over the metallic surface, and the reflected electrons form an image that may be registered on a photographic plate (Fig. 2-3) or on a fluorescent screen. Scanning electron microscopy is also applicable to the freeze-fracture technique, described below.

Figure 2-3 Scanning electron microscope view of cells in pleural effusion. The small doughnut-like cells are erythrocytes, the large chestnut-like cells are cancer cells. Intermediate-sized cells are macrophages, mesothelial cells, and leukocytes. The surfaces of the large cancer cells are covered by microvilli. ( 300.) (Courtesy of Dr. W. Domagala, Montefiore Hospital.)

Other Techniques
Several other special techniques, such as interference microscopy and x-ray diffraction microscopy, have been used for a variety of investigative purposes. Scanning-tunneling microscopy is a new tool for visualization of surfaces of molecules such as DNA. This technique has no applications to diagnostic cytology. Magnetic resonance, a technique widely used in imaging of the human body (MRI), is applicable to the study of tissues in vitro and to histologic sections as magnetic resonance microscopy (Huesgen et al, 1993; Sbarbati and Osculati, 1996; Johnson et al, 1997). The technique is based on magnetic gradients that produce a shift in hydrogen ions' alignment in water content of the living tissues, creating images that can be captured by computer and recorded on a photographic plate. Because of its low resolution, the practical value of this technique remains to be determined.


The components of the cell will be described under three main headings: the cell membrane, the cytoplasm, and

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the nucleus (see Frontispiece). Whenever possible, the description will comprise light and electron microscopic P.25 observations. The purely morphologic description has limited bearing on the intimate biochemical interrelationship of the cell components. The reader is referred to Chapter 3 and the appended references for further information.

The Cell Membrane

The cell membrane is the outer boundary of the cell, facilitating and limiting the exchange of substances between the cell and its environment. In light microscopy, the membrane of well-fixed mammalian cells cannot be seen. The cell's periphery appears as a thin condensation (Fig. 2-4). In transmission electron microscopy, the cell membrane appears as a well-defined line measuring approximately 75 in width (Fig. 2-5). The membrane is composed of three layers, each about 25 thick (see Frontispiece and Fig. 2-18). The inner and the outer dense (electronopaque) layers are separated by a somewhat wider lucent central layer. Similarly constructed membrane systems are observed in a variety of intracytoplasmic components within the cell, such as the mitochondria and the endoplasmic reticulum (see below). The term unit membrane is often used in reference to cell membranes in general. Davson and Danielli (1952) proposed that the plasma membrane is composed of a double lipid layer coated by polypeptide chains of protein molecules. This concept was acceptable so long as it readily explained certain physicochemical characteristics (semipermeability) of cell membranes. However, it has become evident that the cell membrane, far from being a passive envelope of cell contents, plays a critical role in virtually every aspect of cell function. Thus, the cell membrane regulates the internal environment of the cell, participates actively in recognition of the external environment and in transport of substances to and from the cell, determines the immunologic makeup of the cells, and accounts for the interrelationship of cells.

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Figure 2-4 Human bronchial cells, oil immersion. A. The focus was on the region of the cell membrane (M) and the nucleus. Within the latter there is a single nucleolus (NL) and several chromocenters. A sex chromatin body (S) adherent to the nuclear membrane may be observed. In this photograph the cilia appear to be anchored in a thick portion of the cytoplasm or a terminal plate. B. The focus was on cilia and their points of attachment within the cell. These are dense granules or basal corpuscles. The basal corpuscles form the socalled terminal plate.

The initial insight into the makeup and function of the cell membrane was based on the study of erythrocytes. Their membrane is made up of a double layer (bilayer) of lipids, formed by molecules provided with chains of fatty acids. The lipid molecules have one water-soluble (or hydrophilic) end and a water-insoluble (or hydrophobic) end. In the cell membrane, the electrically charged hydrophilic ends of the lipid molecules form the inner and the outer surfaces of the cell membrane, whereas the uncharged, hydrophobic chains of fatty acids are directed toward the center of the cell membrane, away from the two surfaces. Cholesterol molecules add structural rigidity to the cell membrane. Protein molecules of various sizes, functions, and configurations are located within the lipid bilayer (integral proteins) but also extend beyond the cell membrane, either to the outside or to the inside of the cell or both. Such transmembrane proteins provide communication between the cell environment and cell interior. The number, makeup, position, and mobility of the protein molecules account for specific, individual properties of cells and tissues by forming specific receptor molecules. Cell membranes are further characterized by molecules of carbohydrates that attach either to the lipids (glycolipids) or to the proteins (glycoproteins) and which are the repository of the immunologic characteristics of the cell. On the inner (cytoplasmic) aspect of the cell membrane, other protein molecules have been identified (peripheral proteins). Their function appears to be structural in maintaining the integrity of the cell membrane and in providing communication between the cell membrane and the interior of the cell (Fig. 2-6). This complex asymmetric structure of the cell membrane cannot be demonstrated by conventional electron microscopy. P.26 Therefore, to study the problem, special techniques have been applied, such as freeze-fracture. The freezefracture technique consists of three steps: very rapid freezing of cells and tissues, fracturing the tissue with an instrument, and preparation of a metal replica of the fractured surface that can be examined in the scanning electron microscope. It has been determined that the fracture lines are not distributed in a haphazard fashion but, rather, run along certain predetermined planes.

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Figure 2-5 Electron micrograph of a segment of an arteriole. L = lumen, E = endothelial cells, M = smooth muscle cell, N = nucleus. Caveolae (CAV) and microvilli (MV) are evident in the endothelial cell. C = cell membrane; CF = collagen fibers with characteristic periodicity. Basement laminae (membranes) (BL) separate the endothelial cells from the muscle cells and the muscle cells from the connective tissue. ( 16,000.)

Freeze-fracture of cell membranes disclosed two surfaces that, by agreement, have been named the P face and E face (Fig. 2-7). The P face represents the inner aspect of the cell membrane and contains numerous protruding protein particles. The E face represents the outer part of the cell membrane, which is relatively smooth, except for pits corresponding to the protein particles attached to the P face. A few protein particles usually remain attached to the E face. The density and distribution of the protein particles varies from cell type to cell type and may be substantially modified by immunologic and chemical methods, indicating that the position of these particles within the cell membrane is not fixed. Thus, the cell membrane is now thought to be a fluid-mosaic membrane, as first proposed by Singer and Nicholson (1972). It may be conceived as a viscous structure that can adapt itself to changing needs and conditions by being permissive to movements of large molecules, such as protein particles. Fixation of cells solidifies the membrane. The freeze-fracture images represent only snapshots of the position of the protein particles at the time of fixation. The freeze-fracture technique may also be used to study the structure of cell junctions (see Fig. 2-16) and the interior of other cell membranes, such as the nuclear envelope (see Fig. 2-27). The basic structure of intracellular membranes, such as those composing the endoplasmic reticulum or mitochondria, appears to be essentially similar to that of the cell membrane, but differs in lipid/protein ratios and associated proteins and enzymes, reflecting the diversity of functions.

Cytoplasmic Interactions
Extensive work has been performed in recent years to establish links between the cell membrane and the cytoplasm. It is quite evident that this must be a very intimate association, as cell function depends on signals and nutrients received through the cell membrane. Also, the export of substances manufactured by the cell (or products of cell metabolism) must be regulated by interaction between the cytoplasm and the cell membrane. Molecular biologic investigations of recent years have identified numerous protein molecules that contribute to the

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function of the cell membrane as a flexible link between the environment and the interior of the cell. Each one of these molecules interacts with other molecules and theseinteractions are growing increasingly complex. So far, only P.27 small fragments of this knowledge have emerged. At thetime of this writing (2004), no clear, cohesive picture has been formulated to explain how the cell membrane functions. Suffice it to say that there is good evidence that the cell membrane plays an important role in virtually every aspect of cell function in health and disease. Luna and Hitt (1992) discussed the interaction between the cell skeleton and cell membrane as one example of these interactions. Among the components of the cell skeleton that interact with the cell membrane are the intermediate filaments and tubules, described further on in this chapter.

Figure 2-6 Schematic representation of the current concepts of cell membrane. The membrane is made up of two layers of lipids (pins), with points directed toward the center (uncharged hydrophobic ends) and pinheads (electrically charged hydrophilic ends) toward the two surfaces. The black pinheads indicate molecules of cholesterol, which add rigidity to the cell membrane. Integral protein molecules, represented by geometric figures of various shapes, are located within the bilipid layer, but also protrude from both surfaces. Symbolic representation of an emitting and receiving (dish) antennae show the cell's communications with its environment. On the inner aspect of the cell membrane, peripheral proteins (spectrin, actin) have been identified. These probably lend structural support to the membrane and provide communication between the cell membrane and the cytoplasm.

The cell membrane is also the site of molecules that define the immunologic features of the cell. For example, the clusters of differentiation (CD) and blood group antigens discussed elsewhere in this book, are located on the cell membranes.

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Figure 2-7 Principle of freeze-fracture. The sharp wedge (arrow) separates the frozen membrane into two faces (P and E; see text) without disturbing the position of intermembrane protein particles or structures (see Figs. 2-16 and 2-27).

Coated Pits, Vesicles, and Caveolae: Mechanisms of Import and Export

Import, export, and transport of a variety of molecules within the cytoplasm takes place through pits and vesicles formed by invagination of cellular membranes. The largest of such vesicles observed on cell surfaces are known as pinocytotic vesicles. The pits and vesicles are coated by molecules of a complex protein, clathrin, which appears to be present in all cells. Clathrin is composed of three heavy and three light protein chains that form the scaffolds of the coats. Clathrin requires the cooperation of other proteins known as adaptors to fulfill its many functions, which include capturing, sorting, and transporting molecules. The molecular mechanisms of endocytosis have been extensively studied (Gillooly and Stemark, 2001). It may be assumed that each pit or vesicle is provided with specific receptors to a molecule or molecules of importance to the cell, and that it will recognize and selectively capture this molecule or molecules from thousands of molecules circulating within the fluid bathing the cell. Once the selected substance is captured, the vesicle closes and sinks into the cytoplasm to deliver its cargo to its appropriate destination. However, nature is extremely economical, and there is excellent evidence that the fragment of cell membrane that is used to form a vesicle is recirculated and returned to the surface in a different location to serve again. A similar mechanism is observed in removal or phagocytosis of hostile substances (or organisms, such as bacteria) that are recognized by the receptors on the cell surface. Removal of accumulated extracellular debris is another phagocytic function usually performed by specialized cells (macrophages) in a similar manner (see Fig. 513). A number of genetic disorders are now thought to be associated with defective mechanisms of intracellular membrane transport (Olkkonen and Ikonen, 2000). A reverse mechanism occurs in export of molecules, which are packaged into vesicles formed within the cell (mainly in the Golgi apparatus) (see below) and travel to the surface. The vesicles attach to the inner aspect of the cell membrane by means of specific receptors. After the merger, the cell membrane splits open, and the content of the vesicles is discharged into the circulating fluid bathing the cell. Besides clathrin-coated pits, the cell membrane also forms specific small invaginations (50 to 100 nm in diameter) that are known as caveolae. In cross-section, the caveolae appear as small, spherical vesicles in the adjacent cytoplasm (see Fig. 2-5). They are particularly prevalent in endothelial cells, smooth muscle cells, and type I pneumocytes (Schlegel et al, 1998; Couvet et al, 1997). The caveolae are composed of caveolins, a family of integrated membrane proteins, which interact with a number of signaling molecules and thus regulate the cell's responses to its environment (Okamoto et al, 1998). Thus, caveolins have been implicated in cells' response to injury and may play a role in human breast cancer (Engelman, 1998). P.28

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Specialized Structures of Cell Surfaces

Transmission electron microscopy has been helpful in elucidating some of the structural details of specialized structures of cell surfaces and the manner in which cells are attached to each other.

The Glycocalix
Specialized techniques of electron microscopy serve to demonstrate an ill-defined, fuzzy layer of material on the free surfaces of cells. This layer is referred to as glycocalix and appears to be composed primarily of glycoproteins containing residues of sialic acid. Although the thickness and, presumably, chemical makeup of glycocalix vary from one type of cell to another, its occurrence is a rather generalized phenomenon, the exact function of which is not well understood.

Cilia and Flagella: Motile Cell Processes

The cilia and flagella may be readily identified by light microscopy. Both are mobile extensions of the cell membrane and are capable of rapid movements. A flagellum is usually a single, elongated mobile part of the cell, as observed in spermatozoa. Cilia are shorter and multiple, usually functioning (batting) in a synchronous manner, for example, in cells lining the bronchial epithelium (see Fig. 2-4), or other epithelia, such as that of the fallopian tube and the endocervix. Cells bearing cilia are usually polarized; that is, they have a specific spatial orientation in keeping with their function: the cilia are usually oriented toward the lumen of an organ or tissue. The cilia are anchored in a thick, flat portion of the cell cytoplasm immediately adjacent to the surface, referred to as a terminal plate (see Fig. 2-4A). Careful observation reveals that the terminal plate is composed of a series of dense granules, or basal corpuscles, each belonging to a single cilium (see Figs. 2-4B and 2-8). Cilia are rare in cancer cells.

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Figure 2-8 Diagrammatic representation of the structure of the ciliary apparatus (A) of a mollusk (Elliptio), (B) an amphibian (Rana), and (C) a mammal (mouse). Note the differences in attachment to the cytoplasm. (Fawcett DW. Laryngoscope 64:557-567, 1954.)

There is a remarkable uniformity of ultrastructure of the motile cell processes throughout the animal and the plant kingdoms. Each cilium or flagellum contains 11 microtubules, of which two are single and located within the center, and nine are double (doublets) and located at the periphery (Figs. 2-9 and 2-10). The structure of the cilia and flagella is very similar to that of the centrioles (see below). Species differences do exist in the manner in which the cilia and the flagella are anchored within the cytoplasm (see Fig. 2-8). Within recent years, considerable insight has been gained into the function of the cilia and flagella. These cell processes are composed of an intricate system of protein fibrils that glide against each other in executing the movements, which require a substantial input of energy, provided by mitochondria. For details of the current concepts of movements, see Satir (1965) and Sale and Satir (1977).

Microvilli and Brush Border

Microvilli are short, slender, regular projections on free surfaces of cells that can be visualized in electron microscopy

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P.29 or light microscopy. The term brush border or striated border is applied to specialized cell surfaces provided with microvilli. The brush border is observed on the free surface of the intestinal mucosa (Fig. 2-11A and see Fig. 2-15). The regular, finger-like intestinal microvilli, delimited by the plasma membrane, measure approximately 1 m in length and serve the function of increasing the useful surface of the cell. A similarly organized brush border is observed in the proximal segment of the renal tubules. Microvilli may be observed by light microscopy on the surface of various normal human cells, as short, delicate, hair-like striations, best observed in air-dried and stained cells, spread on glass slides. Scanning electron microscopy shows microvilli, as finger-like, slender structures, projecting from the surface of the cell. Long and irregular microvilli that occur on the surfaces of cancer cells are much easier to see in light microscopy and are occasionally of diagnostic help. These observations are discussed in detail in Chapter 7 and are illustrated in Figures 7-7, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13 and 7-14.

Figure 2-9 Diagrammatic representation of a cilium (A) and of the principal piece of mammalian sperm flagellum (B). Note the similarity of the basic structure, with two single microtubules in the center and nine double microtubules at the periphery. This structure of cilia is encountered throughout the plant and the animal kingdoms. (Fawcett DW. Laryngoscope 64:557-567, 1954.)

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Figure 2-10 Electron micrograph of cross- and longitudinal sections of cilia from human endocervical cells. The nine peripheral double microtubules and the two central single microtubules are well shown. ( 80,000.) (Courtesy of Dr. H. Dembitzer, Montefiore Hospital.)

Cell Contacts
The relationship of cells to one another within the same tissue or within adjoining tissues is of paramount importance for the structural integrity and function of all organs (see Fig. 2-11). These relationships are regulated by cell membranes, which form a variety of cell contacts and cell attachments. It is not known as yet whether the cell attachments are formed on predetermined specialized areas of cell surfaces, or incidental to haphazard cell contacts. From the morphologic point of view, a number of structural cell contacts have been identified. These are the desmosomes, the junctional complexes, and the gap junctions (Fig. 2-12).

The Desmosomes and Hemidesmosomes

The structure of cell attachments, especially within the epithelia, has been of interest to biologists and pathologists alike for over a century. Early on, it has been noted in light microscopy that, within the squamous stratified epithelia, the cells are attached to each other by means of cytoplasmic extensions, named intercellular bridges. In phase microscopy, fine fibrils, named tonofibrils, may be seen converging on the areas connecting the unfixed, unstained cells. For many years, it has been known that, in the centers of the intercellular bridges, there existed small dense structures, variously referred to as granules (Ravier) or nodes (Bizzozero) and currently referred to as desmosomes. Electron microscopic studies have demonstrated that the desmosomes represent points of adhesion of two adjacent cells (see Figs. 2-11, 2-12 and 2-13). The cytoplasm of adjacent cells remains firmly attached at the points of desmosomal adherence but, owing to artifacts of P.30 fixation, it shrinks elsewhere. The elongated desmosomebound portions of the cytoplasm constitute the intercellular bridges seen in light microscopy. Recent studies show that molecules of C-cadherin are an essential component of

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desmosomes (He et al, 2003).

Figure 2-11 Diagrammatic representation of several types of specialization found on the surfaces of contact between adjacent cells. A. On the interface between columnar epithelial cells of the intestine, desmosomes (arrow) are frequently seen near the free surface showing striated border. B. On the contact surfaces of liver cells, desmosomes occur (arrows) on either side of the bile capillary. Near these are stud-like processes that project into concavities on the surface of the adjacent cell. C. In the stratified squamous epithelium of the rodent vagina, the cell surfaces are adherent at the desmosomes and retracted between, giving rise to the so-called intercellular bridges of light microscopy. A continuous system of intercellular spaces exists between bridges. Projecting into these spaces are a few short microvilli. D. In the stratum spinosum of the tongue, adjoining cells have closely fitting corrugated surfaces. Numerous desmosomes are distributed over the irregular surface. E. The partially cornified cells of the superficial layers of stratified squamous epithelium apparently lack desmosomes, but the ridges and grooves of the cell surfaces persist. F. An extraordinarily elaborate intercrescence of cell surfaces is found in the distal convoluted segment of the frog nephron. (Fawcett DW. Structural specializations of the cell surface. In Palsy SL (ed). Frontiers in Cytology. New Haven, Yale University Press, 1958.)

The fine structure of a desmosome, or macule adherens (from Latin = adhesive area; plural, maculae adherentes), is fairly uniform in most tissues examined to date: within each cell, at the region of localized contact of two cells, there is a dense plaque adjacent to the cell membrane, made up of converging cytoplasmic actin microfilaments (tonofibrils). The two cell membranes do not appear modified. Within the intercellular substance, there is a dense central lamina. Very slender filaments run between the central lamina and the adjacent cell membranes (see Fig. 213).

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Figure 2-12 Diagrammatic representation of the three principal types of cell junctions. The tight junction (TJ) is formed by fusion of the two outer layers of adjacent cells. It is impermeable to most molecules. The gap junction (GJ) serves the purposes of cell-to-cell communication. The desmosomes (D) are button-like, extremely tough cell junctions that are particularly well developed in protective epithelia, such as the squamous epithelium.

The desmosomal apparatus is operational in all epithelia and many other tissues, but the details of the structure may vary from one tissue type to another. For instance, the squamous epithelium of the genital tract may be structurally somewhat different from the squamous epithelium of other P.31 organs. Burgos and Wislocki (1956) demonstrated the existence of intercellular canaliculi in the rodent vagina during estrus. Such canaliculi conceivably serve as channels for metabolites, etc. and, perhaps, are instrumental in bringing about the marked cyclic changes in the vaginal epithelium in these animals (see Fig. 2-11).

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Figure 2-13 Desmosomes and actinfilaments (tonofibrils). Epidermis of human vulva. Electron micrograph of a portion of two adjoining epithelial cells showing actin filaments attached to two desmosomes (D). The filaments do not transverse cell boundaries. Note within the intercellular space a central dense lamina (arrow), a part of the desmosome structure. Bundles of filaments (T) may be observed within the cytoplasm. ( 54,400.)

Recent investigations of cytoskeleton (see below) disclosed that desmosomes are biochemically complex structures containing many different filamentous proteins, some of which are desmosome specific. Among the latter, specific adhesion proteins (adherins) have been identified in cytoplasmic plaques. Other protein components of desmosomes are desmoplakins and desmogleins. The desmosomes also contain intermediate filaments of various molecular weights. It has been documented that the makeup of desmosomes varies in different cell and tissue types (Franke et al, 1982, 1994). With the development of specific monoclonal antibodies to these proteins, the presence of desmosomal proteins may now be used as a means of tissue identification and diagnosis of diseases (Franke et al, 1989, 1994; Schmidt et al, 1994). Hemidesmosomes (half-desmosomes) are observed at the attachment points of epithelial basal cells to the basement lamina. The half-desmosome is morphologically somewhat similar to the desmosome: there is a thickening of a limited area of the cytoplasm of a basal cell adjacent to the cell membrane, upon which converge cytoplasmic fibrils. However, the apposed basement membrane shows merely a slight thickening, which contains slender filaments. An intermediate thickening, or membrane, is usually present within the fibrils of the hemidesmosome (Fig. 2-14). Jones et al (1994) documented that the hemidesmosomes serve as connectors between the extracellular matrix and the intermediate filaments in the cytoplasm of the cell. The mechanisms of cell adhesion molecules to the extracellular matrix were reviewed by Hutter et al (2000).

The Junctional Complexes

Farquhar and Palade (1963) described a particular type of attachment of epithelial cells, known as the junctional complex, located along the lateral surfaces of the cells adjacent to the lumen (Fig. 2-15). The junctional complex is composed of three parts. The tight junction (zonula occludens), closest to the lumen, represents an area of fusion

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of the outer leaflets of the plasma membranes of two adjacent cells. The molecular mechanisms of formation of this junction were discussed by Knox and Brown (2002). This cell junction contains the adhesion molecule, E-cadherin (Franke et al, 1994). The intermediate junction (zonula adherens) is characterized by the presence of an intercellular space, separating areas of cytoplasmic density occurring in each of the participating cells. The third part of the junctional complex is a desmosome (macula adherens). On the surface of certain epithelia, for example, in the small intestine, the tight junctions form an occlusive network that is essentially not permeable to molecules, even of a very small size, and presumably, synchronizes the function of these epithelia. Thus, nutrients cannot penetrate the seal between the cells, but are absorbed by the cell surfaces facing the lumen. A similar arrangement is encountered on the surfaces of many other epithelia in contact with a fluid medium, such as the renal tubules, bile canaliculi, and ependymal cells. Freeze-fracture of tight junctions shows a continuous network of ridges and grooves at the site of membrane fusion (Fig. 2-16A). P.32

Figure 2-14 Half-desmosomes. Electron micrograph of the basal portion of the epithelial cell (E) of rat bladder and the basement lamina (BL). The half-desmosomes (D) are fan-shaped areas of increased density owing to numerous converging fine fibrils. An intermediate membrane (IM) is present between the cell membrane (CM) and the basement lamina. Dense material, possibly fibrillar, located between the cell membrane and the basement lamina completes the half-desmosome. ( 54,600.)

The Gap Junctions (Nexus Junctions)

First observed in the cardiac muscle and, subsequently in a variety of other tissues, the gap or nexus junctions were identified as specialized areas of cell contact. In transmission electron microscopy, gap junctions appear as welldemarcated areas of merger between two adjacent cells, somewhat less than 200 in thickness. The junction is composed of seven layers, three of which are electrontranslucent and are sandwiched in between electron-dense layers (see Fig. 2-12). The central electron-lucent zone (or gap) is composed of small hexagonal subunits, forming

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the channels of communication between adjacent cells (Revel and Karnovsky, 1967). Freeze cleaving confirmed that the gap junction is a highly specialized area of cell contact, displaying membrane-associated particles in a hexagonal array (see Fig. 2-16B). There are at least two different types of gap junctions, with a somewhat different arrangement of particles. The gap junction channels are composed of a diverse family of proteins, named connexins (Donaldson et al, 1997). The gap junctions have multiple functions: they provide cell-to-cell communications of essential metabolites and ions and may serve as electrical synapses (Leitch, 1992). It has been shown that defects in connexins may be associated with human diseases (Paul, 1995; Spray, 1996). Thus, the gap junctions and the associated proteins are essential to function and integrity of tissues.

The Cytoplasm and Organelles

The cytoplasm is the component of the cell, located between the nucleus and the cell membrane. Depending on the type and origin of the cell, the cytoplasm may present a variegated light microscopic appearance. Its shape, size, and staining properties vary greatly and will be described in detail for the various tissues and organs. In living cells, there is an intense movement of particles within the cytoplasm. In conventional light microscopy, various products of cell metabolism may be seen in the cytoplasm, often appearing as granules or vacuoles. The latter are round or oval structures, generally with an unstained or a faintly stained center. Their contents may be identified by special techniques. Electron microscopic investigation of cells, coupled with sophisticated biochemical methods, has shed considerable light on the basic structure of the cytoplasm and of the major organized cytoplasmic components or organelles. P.33

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Figure 2-15 Junctional complex. Electron micrograph of intestinal-type epithelium observed in a rare nasal tumor of man. The component of the junctional complex may be observed: tight junction (TJ), intermediate junction (IJ), and the desmosome (D). Other desmosomes (D, D) may be observed below. Note also the microvilli (MV), seen in longitudinal and cross section, and mitochondria (M), some with intramitochondrial dense granules. Also note dense bodies (DB), which may represent secretory granules ( 22,800.) (Courtesy of Dr. Robert Erlandson, Sloan-Kettering Institute for Cancer Research, New York.)

Ultrastructure of the Cytoplasm

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The cytoplasm is composed of organized cell components, or organelles, the cytoskeleton, and a cytoplasmic matrix. The organized components of the cytoplasm comprise the membranous systems, ribosomes, mitochondria, lysosomes, centrioles, microbodies, and miscellaneous structures.

The Membranous System

The membranous system is composed of the endoplasmic reticulum and the Golgi complex.

The Endoplasmic Reticulum

The endoplasmic reticulum is a closed system of unit membranes forming tubular canals and flattened sacs or cisternae that subdivide the cytoplasm into a series of compartments. The membranes of the endoplasmic reticulum may be rough, that is, covered with numerous attached granules composed of ribonucleic acid (RNA) and proteins (RNP granules or ribosomes; see below), or smooth, free of any particles. The amount and structural forms of endoplasmic reticulum vary from one cell type to another. In general, rough endoplasmic reticulum is abundant in cells with marked synthesis of proteins for exportfor instance, in the pancreas or the salivary glands, see Figure 2-17. In light microscopy, the RNA-rich cytoplasmic areas (once named ergastoplasm) stain bluish with hematoxylin. This feature is commonly observed in metabolically active cells. Smooth cytoplasmic reticulum is abundant in cells that synthesize various steroid hormones. P.34

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Figure 2-16 Electron micrographs of freeze-fracture preparations showing a tight junction (A) and a gap junction (B). A. The tight junction (zonula occludens) appears in freeze-fracture images as a continuous meshwork of ridges and grooves representing the sites of membrane fusion (arrows). Epidermis of the transparent catfish (Kryptoterus). B. The appearance of gap junctions is quite different from the tight junction in that they are made up of plaques (GJ) of closely packaged particles. The particles measure about 9 nm in diameter and are believed to be the sites at which hydrophilic channels bring about electrical coupling between cells. Myocardium of a tunicate (Ciona). (Unpublished data of RB Hanna and GD Pappas, Albert Einstein College of Medicine, New York. Courtesy of Dr. Pappas.)

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Figure 2-17 Rough endoplasmic reticulum. Electron micrograph of an epithelial cell of a human submaxillary gland. Note the ribosomes (RNP particles) attached to the membranes of the endoplasmic reticulum. Free ribosomes are also present in the space between the membranes. (43,000.) (Courtesy of Dr. Bernard Tandler, Sloan-Kettering Institute for Cancer Research, New York.)

The Golgi Complex

First described by Golgi in 1898, this organelle consists of a series of parallel, doughnut-shaped flat spaces or

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cisternae and spherical or egg-shaped vesicles demarcated by smooth membranes (Fig. 2-18). In epithelial cells with secretory function, the Golgi complex is usually located between the nucleus and the luminal surface of the cells. Present evidence suggests that the Golgi complex synthesizes and packages cell products for the cells' own use and for export (Fig. 2-19). For example, the Golgi complex synthesizes structural proteins, such as the components of the asymmetric unit membrane observed in the urothelium (Hicks, 1966; Koss, 1969; see Chapter 22). The synthesis of the protein products occurs within the cisternae of the Golgi complex. The products for export are packaged in the form of vesicles lined by a single smooth membrane derived from pinched off ends of the cisternae and is released into the cytoplasm (Fig. 2-20). A review of the mechanisms of protein sorting by the Golgi apparatus was provided by Allan and Balch (1999).

The Ribosomes
The ribosomes are submicroscopic particles measuring between 150 and 300 in diameter, depending on the technique used, and are composed of RNA and proteins in approximately equal proportions. They are ubiquitous and have been identified in practically all cells of animal and plant origin. In the cytoplasm, the ribosomes may be either floating free or they may be attached to the outer surface of the endoplasmic reticulum (see Fig. 2-17). It appears likely that the two types of ribosomes exercise different functions: the free ribosomes are primarily engaged in the production of proteins for the cell's own use, whereas attached ribosomes are responsible for protein production for export. A marked concentration of ribosomes (and hence proteins) confers upon the cytoplasm a basophilic staining (see above). Each ribosome is composed of two, approximately round subunits of unequal size and has been compared to a Russian doll. Ribosomes may be joined together by strands of messenger RNA (mRNA) to form aggregates or polyribosomes that thus resemble a string of beads. The string may be either P.36 open or closed. Ribosomes are attached to the membranes of the endoplasmic reticulum by the larger subunit.

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Figure 2-18 Inactive Golgi complex. Electron micrograph of human labial salivary gland. In this type of cell, the Golgi complex (GC) is composed mainly of a series of parallel membranes made up of smooth reticulum (SR). Note the absence of ribosomes (see Fig. 2-17). C = cell (plasma) membrane; its three-layer structure, with a translucent middle layer is well seen in this photograph. ( 17,300.) (Courtesy of Dr. Bernard Tandler, Sloan-Kettering Institute for Cancer Research, New York.)

The ribosomal RNA (rRNA) is manufactured in the nucleolus and transferred into the cytoplasm where it becomes associated with the protein component. At the conclusion of the process of protein synthesis, the ribosomal subunits are separated and return to the cytoplasmic pool. The details of the mechanism of protein synthesis are discussed

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in Chapter 3. Ribosome-like structures may also be observed within the nucleus, presumably representing various types of RNA.

The Mitochondria
Although the mitochondria were first observed in light microscopy in the latter part of the 19th century, their structure and function have become better known only within the last 50 years. These organelles are present in all eukaryotic cells. Mitochondria are small, usually elongated structures, usually less than 0.5 m in width and less than 7 m in length. Even within the same cell, the mitochondria may vary substantially in size and configuration, assuming spherical, cigar-, club-, or tennis racquet-like shapes. However, the basic structure of a mitochondrion, initially described by Palade in 1953, is uniform. Each mitochondrion is composed oftwo membranes, located one within the other. The outer shell of the mitochondrion is a continuous, closed-unit membrane. Running parallel to the outer membrane is a morphologically similar inner membrane that forms numerous crests or invaginations (cristae mitochondriales), subdividing the interior of the organelle into a series of communicating compartments (Fig. 2-21 and see Frontispiece and Fig. 2-15). Frequently, the cristae are approximately at a right angle to the long axis of the mitochondrion, but they may also be oblique or, for that matter, longitudinal. There is no P.37 known relationship between the orientation of the cristae and the function of the organelle. A homogeneous material or mitochondrial matrix, containing a mixture of molecules and enzymes, fills the interior of the organelle.

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Figure 2-19 Active Golgi complex. Electron micrograph of a human labial salivary gland. Note the enormous accumulation of mucous granules (MG) within the Golgi complex (GC) and above it, toward the lumen (L) of the acinus. The basic structure of the Golgi complex is maintained. C = cell (plasma) membrane (see Fig. 218). (8,700.) (Courtesy of Dr. Bernard Tandler, Sloan-Kettering Institute for Cancer Research, New York.)

The size and configuration of the mitochondria may vary according to the nutritional status of an organ. For instance, the mitochondria of the liver may become very large in some deficiency states, only to return to normal with resumption of a normal diet. Mitochondrial enlargement may also be caused by poor fixation of material. The latter is the probable background of a cell change known as cloudy swelling to light microscopists.

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Accumulation of fat, hemosiderin, and proteins may be observed in the immediate vicinity of the mitochondria. This probably occurs because of the role of the mitochondria in energy-producing oxidative processes. Indeed, the key role of the mitochondria within the cell is that of carriers of energy-producing complex enzyme systems. Several oxidative systems have been identified within the mitochondria: Krebs cycle enzymes, fatty acid cycle enzymes, and the enzymes of the respiratory chain, including the cytochromes. Most importantly, the formation of energyproducing adenosine triphosphate (ATP) from phosphorus and adenosine diphosphate (ADP) takes place within the mitochondria. The ATP is exported into the cytoplasm where it serves as an essential source of energy for the cell. It has been documented that the mitochondria possess their own DNA that is independent of nuclear DNA and is responsible for independent protein synthesis and for the mitochondrial division cycle. This supports the concept that the mitochondria are quasi-independent organelles, living in symbiosis with the host cell, which they supply with energy. It is a matter for an interesting P.38 speculation that mitochondria may represent primitive bacteria that, at the onset of biologic events, became incorporated into the primordial cell, and this association became permanent for mutual benefit. Thus, two genetic systems exist within a cell, one vested in the mitochondria and the other in the nucleus. The two systems are interdependent, although the exact mechanisms of this association are not understood.

Figure 2-20 Ultrastructural features of a calcitonin-producing medullary carcinoma of the thyroid. Numerous electron-opaque secretory granules bound by a single membrane may be noted (arrowheads). The peripheral cisternae of the Golgi complex (G) show accumulation of electronopaque substance; hence, the assembly of the secretory granules is probably a function of the Golgi apparatus. ( 54,400.) (Koss LG. Morphology of cancer cells. In Handbuch der allgemeinen Pathologie, vol. 6, Tumors, part I. Berlin, Springer, 1974, pp 1-139.)

The mitochondrial DNA has been extensively studied, and its structure has been determined. It is a small molecule of double-stranded DNA containing only 37 genes (13 structural genes encoding proteins, 22 transfer RNA genes, and 2 genes encoding ribosomal RNAs). All mitochondria of the zygote are contributed by the ovum; hence, all of mitochondrial DNA is of maternal origin. Because muscle function depends heavily on energy systems vested in mitochondria, it is not surprising that various muscular disorders have been observed in association with abnormalities of mitochondrial DNA (Moraes et al, 1989; Fadic and Johns, 1996; and DiMauro and Schon, 2003). Such disorders are transmitted exclusively by females to their offspring. There is also recent evidence that mitochondria participate in the phenomenon of programmed cell death or apoptosis. The issue is discussed at

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length in Chapter 6.

Figure 2-21 Schematic representation of a mitochondrion shown in longitudinal section (left) and crosssection (right). For details, see text.

In cells characterized by an abundance of mitochondria (oncocytes, sometimes named Hrthle cells, and tumors composed of oncocytes oncocytomas), which may occur in the salivary glands, thyroid, kidney, breast, and sometimes in other organs, the mitochondrial DNA may be modified (Welter et al, 1989). For description of oncocytes and oncocytomas, see appropriate chapters.

The Lysosomes (Lytic Bodies) and the Autophagic Vacuoles

The lysosomes, or cell disposal units, are the organelles participating in the removal of phagocytized foreign material. Occasionally, the lysosomes also digest obsolete fragments P.39 of cytoplasm and organelles, such as mitochondria, for which the cell has no further use. The term autophagic vacuoles or residual bodies has been suggested for such structures. In electron microscopic preparations, the lysosomes may be identified as spherical or oval structures of heterogeneous density and variable diameter (Fig. 222). The lysosomes contain several hydrolytic enzymes, acid phosphatase being the first one identified, that serve to digest the phagocytized material. It is of interest to note that granules commonly observed in neutrophilic leukocytes belong to the family of lysosomes inasmuch as they contain packaged digestive enzymes that assist in the dissolution of phagocytized bacteria.

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Figure 2-22 Electron micrograph of epithelial cell, rat urinary bladder. Large oval body containing droplets of dense lipid-like material and clear vesicles. The body is probably a disposal unit and, as such, related to autophagic vacuoles and lysosomes. ( 38,000.)

The origin of at least some lysosomes has been traced to certain regions of smooth endoplasmic reticulum (Novikoff et al, 1973) that is intimately associated with the inner (active) face of the Golgi complex. It appears that, in some cells at least, the outer membrane of the lysosome may merge with the cell membrane. This is followed by extrusion of the contents of the lysosome into the extracellular space. This process is the reverse of pinocytosis, or phagocytosis (see above). The lysosomes appear to play an important role in certain storage diseases, for example, in Tay Sachs disease. This is one of several known inborn or hereditary defects of metabolism wherein the deficiency of an enzyme (hexosaminidase A) results in accumulation of a fatty substance, ganglioside, in lysosome-like vesicles in cells of the central nervous system. In several other uncommon diseases (such as metachromatic leukodystrophy) and certain granulomatous disorders (malakoplakia, see Chap. 22), abnormalities of lysosomes play a major role.

The Peroxisomes or Microbodies

The peroxisomal family of organelles is characterized by storage of enzymes involved in metabolism of hydrogen peroxide. The most commonly encountered enzyme is catalase. Morphologically, peroxisomes are vesicular structures that, in nonhuman cells, are often provided with a dense central core or nucleoid (Fig. 2-23). Occasionally, the core has a crystalloid structure. Microbodies were extensively studied in liver cells and cells of the renal proximal convoluted tubules of rats. It has been shown that, under certain circumstances, peroxisomes are capable of becoming very large and, apparently, of dividing (Lavin and Koss, 1973). Whether these organelles have an independent DNA system, such as that of the mitochondria, is not known.

The Centrioles
The centrioles are cytoplasmic organelles that play a key role during cell division. Each interphase animal cell contains a pair of centrioles, short tubular structures, usually located in the vicinity of the concave face of the Golgi complex. As the cell is about to enter mitosis, another pair of centrioles appears, and each pair travels to the

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opposite poles of the cell and becomes the anchoring point of the mitotic spindle. The formation of the mitotic spindle from microtubules is described below. The origin of the second pair of centrioles has not been fully clarified; apparently it is synthesized de novo from precursor P.40 molecules in the cytoplasm (Johnson and Rosenbaum, 1992). This event is induced and directed in an unknown fashion by the original pair of centrioles. Each pair of centrioles is surrounded by a clear zone, the centrosome, which, in turn, is surrounded by a slightly denser area or the astrosphere. Within each pair, the centrioles are placed at right angles to each other. Thus, in a fortuitous electron micrograph, one centriole will appear in a longitudinal section and the other in cross section. In the cross section, each centriole appears as a cylindrical structure with a clear center and nine triplets or groups of three microtubules (Fig. 2-24). Thus, the basic structure of the centriole, first described by de Harven and Bernhard in 1956, closely approximates that of cilia and flagella (see Figs. 2-9 and 2-10). It has been suggested that the centrioles are at the origin of cilia. If this were the case, it would indicate that the centrioles might multiply manyfold. It has been observed that formation of the sperm flagellum takes place from one of the centrioles, while the other remains inactive.

Figure 2-23 Peroxisomes (P) or microbodies in proximal tubules of rat kidney. Note the central dense core or nucleoid. Ly = lysosomes; MV = microvilli. ( 19,800.) (Lavin P, Koss LG. Effect of a single dose of cyclophosphamide on various organs in the rat. IV. The kidney. Am J Pathol 62:169, 1971.)

The Cytoskeleton
The skeleton of the cells and, hence, the structures maintaining their physical shape, facilitating their motion, and providing structural support to all cell functions, is provided by a family of fibrillar proteins. Several techniques were developed that allow the isolation of these proteins and the production of specific monoclonal or polyclonal antibodies that can be used to identify these proteins and to localize them within cells. By techniques of molecular biology, the precise composition of such proteins has been determined and the genes responsible for their formation identified and sequenced (see Chap. 3). This work is not only of theoretical value but has also led to strides in immunocytochemistry, particularly relative to intermediate filaments (see below and Chap. 45).

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The cytoskeleton is fundamentally composed of three types of fibrillar proteins, initially classified by their diameter in electron microscopic photographs: the actin filaments (microfilaments, tonofilaments), intermediate filaments, and microtubules. They will be described in sequence.

Actin Filaments (Microfilaments, Tonofilaments)

The ubiquitous actin filaments, measuring 5 to 7 nm in diameter, are observed in all cells of all vertebrate species. In electron microscopy, they can be recognized as bundles of longitudinal cytoplasmic filaments crisscrossing the cytoplasm and often converging on specific targets such as desmosomes (see Fig. 2-13). The actin filaments are found within virtually all structural cell components and interact with many other proteins that regulate their length. The fundamental structure of these elongated fibrillar proteins is helical, with two different ends: this latter feature allows the filaments to attach to two different molecules and function as an intermediary polarized link. The actin filaments are easily polymerized (i.e., they form structures composed of several actin units). This is probably the mechanism that allows actin filaments to form tight meshworks in conjunction with other proteins. Among the latter, it is important to mention P.41 the links of actin filaments to a contractile protein, myosin, accounting for motion and contractility of cells and of cell appendages such as cilia and flagella. Other linkages occur with transmembrane proteins, such as spectrin, ensuring the communications between the cell membrane and cell interior. Thus, actin microfilaments perform several essential functions within cells as linkage filaments coordinating the activity of divergent cell components.

Figure 2-24 Centrioles. Electron micrograph of thymus of DBA mouse. Two centrioles are seen in this electron micrograph: one (C) in cross section, showing nice triplets of tubules, and the other (C) in oblique section and apparently at a right angle to (C). Centriole satellite (S) is attached to C. This may represent the point of anchorage of the tubules of the mitotic spindle. N = nucleus; NM = nuclear membrane. (94,000.) (Courtesy of Dr. Etienne de Harven, Sloan-Kettering Institute for Caner Research, New York.)

Intermediate Filaments
The group of cytoplasmic filaments was initially identified in electron microscopy because of their diameter (7 to 11 nm); hence, intermediate filaments (IFs) are larger than actin microfilaments and smaller than microtubules (see

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the following section). This group of filaments assumed an important role in immunocytochemistry and histochemistry as markers of cell derivation and differentiation by means of specific antibodies that serve to identify the presence and the distribution of IFs in cells and tissues (see Chap. 45). The genes governing the synthesis of IFs have been identified by molecular biology techniques and applied to studies of cell differentiation across species, documenting that these genes belong to the fundamental cellular genes in primitive multicellular organisms, such as worms, mollusks, and perhaps even plants (Nagle, 1988 and 1994). It is of interest, though, that the precise function of the IF proteins is obscure, as they do not appear to participate in any life cycle events. Several subspecies of IF proteins have been identified, differing from each other by relative molecular mass (Mr) and anatomic distribution (Table 2-1). Their significance in immunocytochemistry is discussed in Chapter 45. Perhaps the best known of the IFs are the keratins, which have been extensively studied in the epidermis of the skin (Sun et al, 1984; Franke et al, 1989). As shown in Figure 2-25 , there are several subfamilies of keratin filaments (proteins) forming pairs, each composed of one basic and one acidic protein (see Fig. 2-25A). Each type of squamous epithelium (skin, cornea, other epithelia) may be represented by a special pair of proteins of high relative molecular mass. With the change of epithelial type from a single layer to multilayer epithelium, different keratin genes, producing proteins of increasing molecular mass are activated (see Fig. 2-25B). This mechanism may be important in understanding the change known as squamous metaplasia (see Chap. 6). Of note is the identification of lamins, structural proteins of the nucleus, and its components. These proteins contribute to the formation of the nuclear membrane and the nuclear pore complexes. They may play a role in the organization of interphase chromosomes (see below).

Microtubules, measuring between 22 and 25 nm in diameter, have long been recognized and identified by light microscopy as the constituents of the mitotic spindle. The determination of their existence in the interphase cells required P.42 electron microscopy. The understanding of their chemical makeup, function, and molecular biology is an ongoing process. Microtubules are hollow, tube-like structures, which appear to be universally present in all cells, and are synthesized from precursor molecules of tubulin. As described earlier (see Figs. 2-9 and 2-10), microtubules are an integral component of cilia, flagella, and centrioles (see Fig. 2-24). Microtubules, like actin filaments (see above), are polarized, that is, they have one minus and one plus end; hence, they can be attached to two different molecules and form a bridge between them.



Mr (daltons)

Tissue Distribution


Form: acid types 9-19


Epithelia (specific types associated with specific epithelial types and their maturation)

Pairs: neutral basic types 1-8



Muscle fibers of all types



Cells of mesenchymal origin and some epithelial cells, such as mesothelium, thyroid, endometrium

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Glial fibrillary proteins (GPF)


Glial cells, Schwann cells


68,000; 160,000; 200,000

Dendrites and axons; body of neuronal cells



Form nuclear skeleton and various nuclear structures; similar to cytoplasmic IF

For further discussion of intermediate filaments, see Chapter 45.

Modified with permission from Nagle RB. Intermediate filaments: A review of the basic biology. Am J Surg Pathol, 12 (Suppl. 1): 4-16, 1988.

Figure 2-25 A. A unifying model of keratin expression. Keratins of subfamilies A (acidic) and B (basic) are arranged vertically, according to their relative molecular mass (molecular weights). The drawing indicates that keratin proteins of A and B type form pairs, with proteins of increasing relative molecular mass (Mr) making their appearance as epithelia mature from simple to stratified. K = kilodaltons; s.e. = stratified epithelia. B. A schematic drawing showing the embryonic development as well as the postulated evolutionary history of human epidermis. The bottom part of the drawing shows a simplified diagram of electrophoretic analysis of keratins of increasing Mr, expressed in kilodaltons (numbers on right) corresponding to the evolution of epithelia from simple to stratified to keratinized. K = kilodaltons; s.e. = stratified epithelium. (Sun TT, et al. Classification, expression, and possible mechanisms of evolution of mammalian epithelial keratins: A unifying model. In Levin AJ, et al (eds). Cancer Cells, vol. 1. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory, 1984, pp 169-176.)

The principal role for microtubules and associated proteins

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P.43 is their participation in cellular events requiring motion. Cilia and flagella are a good example of this function in which microtubules perform a sliding movement in association with a protein, dynein, and an energy-producing system, adenosine triphosphate (ATP). The mitotic spindle is synthesized by the cells undergoing mitosis from molecules of tubulin. The spindle formation may be inhibited by some drugs, such as colchicine and vinblastine, or enhanced by Taxol, a potent anti-cancer drug, derived from the bark of a tree, the western yew (Taxus brevifolia). These drugs are commonly used in experimental work involving cell division. During cell division, the centrioles serve as an organizing center for the mitotic spindle (see above). From the centrioles, located at the opposite poles of the cell, the microtubules attach to the condensed double chromosomes arranged at the metaphase plate (see Chap. 4) and participate in the migration of the single chromosomes into the two daughter cells. Once the mitosis is completed, the spindle microtubules are depolarized and redistributed in the cytoplasm. Undoubtedly, microtubules perform yet other functions within the cell: they may be associated with movements of coated pits and pinocytotic vesicles to and from cell membranes and are associated with cell motion.

Storage of Products of Cell Metabolism Within the Cytoplasm

The identification of the many varied materials produced and stored within the cells was successfully accomplished before the era of electron microscopy. The identification of lipids, glycogen, mucin, and pigments, such as bile, hemosiderin, melanin, and lipofuscin, goes back to the 19th century. Electron microscopy has shed considerable light on their ultrastructure, the mechanisms of accumulation, and their relationship to various cytoplasmic organelles. Thus, lipids often accumulate in close rapport with mitochondria (see above). The role of the Golgi complex in the production of mucus and other cell products, and in formation of storage vesicles, was discussed above. The production of various polypeptide hormones in the pancreatic islet cells and other cells with endocrine function, accumulating in the form of endocrine cytoplasmic vesicles, has been documented (see Fig. 2-20). The histochemical or immunocytochemical identification of the nature of various cell products stored in the cytoplasm may play a crucial role in diagnosis of some cell and tissue disorders. As an example, the presence of mucin may be of value in the differential diagnosis of an adenocarcinoma, whereas the presence of melanin may establish the diagnosis of a malignant melanoma. The identification of specific hormones by immunocytochemistry is often of assistance in classifying tumors with endocrine function (see Chap. 45).

The Cytoplasmic Matrix

The space within the cytoplasm, not occupied by the membranous system, the cell skeleton, or by the organelles, is referred to as the cytoplasmic matrix. The matrix is composed of proteins and free ribosomes. There is still little knowledge about the makeup of the proteins constituting the bulk of the cytoplasmic matrix. It is quite certain that the matrix contains all of the amino acids necessary for protein synthesis, various forms of RNA, and enzymes (see Chap. 3). Under the impact of various chemicals or heat, the matrix may be irreversibly coagulated; this is the principle of cell fixation. In electron micrographs, the matrix appears as a homogeneous substance, occasionally containing fine granules, fibrils, or filaments.

The Nucleus and Its Membrane

The Nuclear Membrane
The nucleus is enclosed within the nuclear membrane, or nuclear envelope, composed of two electron-dense membranes, each measuring approximately 75 in thickness and separated from each other by a clear zone measuring from 200 to 400 in width. On the inner (nuclear) side of the nuclear membrane, there is a layer of filaments (fibrous lamina), about 300 in thickness, which presumably enhances the resilience of the membrane and may play a role in the anchorage of chromosomes. The outer membrane of the nuclear membrane resembles rough endoplasmic reticulum because numerous ribosomes are attached to it; thus, it may be considered as a part of the cell's inner membrane system. The nuclear membrane is characterized by the presence of nuclear pores (Fig. 226). A pore is an area where there is a fusion of the two dense layers of the nuclear envelope. A complex array of protein molecules with a central channel, about 9 nm in diameter (nuclear pore complex), constitutes the nuclear pore. The nuclear pores serve as exchange channels between the nucleus and the cytoplasm. Freeze-fracture of the nuclear membrane discloses that the distribution of the nuclear pores is random and does not follow any geometric pattern (Fig. 2-27). Still, the nuclear pores form a close relationship with individual chromosomes and their number may be chromosome dependent. For example, it has been shown that the number of nuclear pores is increased in aneuploid cancer cells with elevated DNA content and, hence, elevated number of chromosomes

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(Czerniak et al, 1984). This is in keeping with the new data on the organization of the normal interphase nucleus (see below). The nuclear membrane disappears during the late prophase of the mitosis and is reformed during the late telophase (for stages of mitosis, see Chap. 4). The probable mechanism of formation of the nuclear membrane is discussed below. The intact nuclear envelope shows a remarkable resistance to trauma or corrosive chemicals such as acids or alkali. When a cell is exposed to such agents, the cytoplasm usually disintegrates fairly rapidly, but the nuclear envelope usually remains intact, protecting the contents of the nucleus. This remarkable property of the nuclear envelope is utilized in many techniques of nuclear isolation, for example, in measuring DNA content by flow cytometry (see Chap. 47).

The Nucleus
The nucleus is the principal repository site of DNA and, therefore, is the center of events governing metabolic and P.44 reproductive processes of the cell. The basic concepts pertaining to the mechanism of DNA structure and function are described in Chapter 3. The events in cell division (cell cycle and mitosis) are described in Chapter 4.

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Figure 2-26 Area of nucleus. Electron micrograph of an epithelial cell, rat bladder; N = nucleus. Note the nuclear envelope, consisting of two membranes, the inner (IL) and the outer (OL), separated by a translucent space. The inner (nuclear) aspect of the nuclear membrane appears thick because of the presence of a fibrous lamina. Nuclear pores (NP) are well in evidence. Nuclear contents appear granular; CY = cytoplasm. (64,000.)

Resting or Interphase Nucleus

In light microscopy of appropriately stained preparations, the resting or interphase nuclei of normal cells are seen as a large, usually spherical structure located within the cytoplasm. In stained preparations, the nucleus is surrounded by a distinct, thin peripheral ring, representing the nuclear membrane. The location of the nucleus depends on cell shape: in cells of approximately spherical, oval, or spindly configuration, the nucleus usually occupies a central position; in cells of columnar shape, which are usually polarized, the nucleus is frequently located in the vicinity of the distant cell pole, away from the lumen of the organ. The shape of the normal nucleus may vary: it is usually spherical but may be oval, elongated, or even indented, and, hence, kidney-shaped, depending on cell type. In polymorphonuclear leukocytes and megakaryocytes, the nuclei form two or more lobes. Located within the nucleus is an important organelle, the nucleolus, which may be single or multiple (see below). The dominant chemical component of the interphase nucleus is a mixture of DNA and associated histones and nonhistone proteins (known in the aggregate as nuclear chromatin) that readily reacts with dyes such as hematoxylin, that confer upon the nucleus a bluish stain of variable intensity (see Frontispiece and Fig. 2-1). The double-stranded DNA within the nucleus can also be stained with a highly specific stain, the Feulgen stain (Fig. 2-28), which is extensively used in quantitative analysis of DNA. The total DNA can also be visualized and quantitated with the use of specific fluorescent reagents (probes), such as propidium iodide or DAPI, extensively P.45 used in molecular biology and quantitative and analytical cytology (see Chap. 47).

Figure 2-27 Freeze-fracture replica of the nuclear membrane of a urothelial cell, showing random

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distribution of the nuclear pores (arrows) on face E and face P. Note the fine granules of intermembrane proteins in the background. (Approx. 50,000.) (Courtesy of Dr. Bogdan Czerniak.)

Figure 2-28 Feulgen-stained cultured malignant cells from an experimental carcinoma of the bladder (line BC 7, probably fibroblastic). The stain is specific for double-stranded DNA; hence, only the nuclei are stained. Note the increase in the intensity of staining of the condensed chromosomal DNA in the mitotic figures. ( 1,000.) (Culture by Dr. Fritz Herz, Montefiore Hospital. Koss LG. Morphology of cancer cells. In Handbuch allgemeinen Pathologie, vol. 6, Tumors, part I. Berlin, Springer, 1974, pp 1-139.)

The size of the nucleus depends substantially, but not absolutely, on its DNA content. During the cell cycle, described in Chapter 4, the DNA content of the nucleus doubles during the synthesis phase (S-phase) and remains double until the cell divides. The diameter of nuclei with a double amount of DNA is about 40% larger than that of nuclei in the resting phase of the cell cycle. Thus, the assessment of the nuclear size, an important feature in recognition of cancer cells, must always be compared with a population of normal cells. For further discussion of this issue, see Chapter 7. In well-fixed and stained cells, within the homogeneous background of the nucleus (sometimes referred to as nuclear sap), one can observe a fine network of thin, thread-like linear condensations, known as the linin network.

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Located at various points in the network are small, dark granules of odd shapes, the chromocenters. The chromocenters are composed of an inactive form of DNA, composed of sequences that do not participate in the biologic activities; therefore, they are designated as constitutive heterochromatin. Constitutive heterochromatin may also be identified in chromosomal preparations around the centromeres (see Chap. 4). This form of chromatin should be distinguished from another form of condensed chromatin that may occur in only some cells and that is called facultative P.46 heterochromatin. An example of the latter is the sex chromatin body (also known as the Barr's body after the person who described it), which is a condensed portion of one of the two X chromosomes and, therefore, is seen only in females or male individuals with genetic abnormalities, such as excess of X chromosome (Klinefelter's syndrome) (see Chaps. 4 and 9 for further discussion of this condition). The sex chromatin body is seen as a triangular dark structure, attached by its base to the inner side of the nuclear membrane, with the tip of the triangle pointed toward the center of the nucleus. The identification of the sex chromatin body is of value in the recognition of some genetic disorders and occasionally cancer cells (see Chaps. 7, 26, and 29).

Interphase Nucleus in Electron Microscopy

Except for the nuclear membrane, described above, the ultrastructure of the interphase nucleus does not cast much light on its organization. The area of the nucleus is filled with finely granular material, or nuclear sap (nucleoplasm), wherein one can observe scattered ribosomes. The filamentous proteins, lamins, may sometimes be observed as a network of fine filaments attached to the nuclear membrane. The chromatin may be seen as overlapping electron-dense or dark areas at the periphery of the nucleus, undoubtedly representing fragments of chromosomes attached to the nuclear membrane (see belowstructure of interphase nucleus). The correlation of the electron microscopic images with specific chromosomes has been poor, even with the use of immunoelectron microscopy, wherein specific genes or proteins can be identified by antibodies usually labeled with colloidal gold.

The Nucleus in Cycling Cells

In a cell population that is proliferating and, therefore, is characterized by mitotic activity, the appearance of the nonmitotic nucleus may change. Besides the enlargement, caused by the increase in DNA during the S-phase of the cell cycle (see above), the granularity of the nucleus may increase substantially during the prophase of the mitosis because of early condensation of parts of chromosomes in the form of chromatin granules. Although such events are more common in cancer cells (see Chap. 7), they may also occur in normal cells undergoing cell division.

The Nucleolus
In a normal interphase resting nuclei, the nucleoli are seen as round or oval structures of variable sizes, averaging about 1 m in diameter, occupying a small area within the nucleus. The location of the nucleoli is variable but, in light microscopy, they are usually located close to the approximate center of the nucleus, rarely at the periphery. The number of nucleoli per nucleus varies from one to four but usually only one nucleolus is observed. The reason for the variable number of nucleoli is their origin in the nucleolar organizer loci, located on each of the two homologues of chromosomes 13, 14, 15, 21, and 22. Thus, theoretically, 10 nucleoli per cell should be seen. However, the small nucleoli merge shortly after the birth of the cell, thus reducing the total number of these organelles. Thanks to the work of Caspersson and his colleagues in Sweden (1942, 1950), much is known about the natural sequence of events in the life of a nucleolus. The nucleoli are born within the nucleolar organizer loci in the designated portion of the chromosomes by accumulation of proteins and ribonucleic acid (RNA), which explodes the center of the chromosomal fragment (Figs. 2-29 and 2-30). The chromosomal DNA of the nucleolus organizing locus forms a rim surrounding the RNA-rich central space and is easily recognized as the nucleolus-associated chromatin. After merger of small nucleoli, the larger nucleolus, or nucleoli, occupies a central role in the life of a cell as the center of production of RNA (see Chap. 3). The nucleolus disappears at the onset of cell division, only to be reborn again in the daughter cells after mitosis. The size of the nucleoli in interphase cells varies according to the function of the cell. In metabolically active cells, such as cells processing or secreting various products, the nucleoli are larger than in quiescent cells with limited metabolic activities. For example, in mucus-secreting intestinal epithelial cells, the nucleoli are larger than in squamous cells, which perform an essentially passive protective function. Under some circumstances, such as an injury requiring rapid repair when the cells are forced to produce a large amount of protein, the accumulation of large amounts of RNA causes the nucleoli to become multiple and very large and measure up to 4 or 5 m in

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diameter. Large nucleoli of irregular configuration are common in cancer cells (see Chap. 7). An important feature of the nucleoli in light microscopy is their staining affinities. The center of the nucleolus accepts acidophilic dyes, such as eosin, and therefore stains red. The periphery, that is, the nucleolus-associated chromatin, retains the staining features of DNA and, therefore, stains blue with basophilic dyes. In Feulgen stains, the nucleolus-associated chromatin accepts the dye, but the center of the nucleolus remains unstained.

The Nucleolus in Electron Microscopy

The ultrastructure of the nucleolus has been extensively studied because of its role as the center of production of RNA (see Chap. 3). The nucleolus is composed of electron-dense and electron-lucent areas. Occasionally, at the periphery of the nucleolus, a distinct dense zone corresponding to the nucleolus-organizing region of a chromosome may be distinguished. The core of the nucleolus corresponds to the granular and fibrillar products of ribosomal RNA in various stages of synthesis.

Organization of the Interphase Nucleus

Although the light microscopic structure and ultrastructure of the nucleus have been well known for many years, as summarized above, until the 1980s, no tools were available to probe the organization of the interphase nucleus. It was commonly thought that during interphase, the nuclear chromatin represented uncoiled chromosomal DNA, forming a structure of incredible complexity. Although individual P.47 genes could be identified and localized on individual chromosomes by molecular biologic techniques (see Chap. 3), the overall organization of the interphase nucleus remained a mystery. On the other hand, considerable knowledge was accumulated in reference to the nucleus during mitosis, giving rise to the study of cytogenetics (see Chap. 4). Thus, it became known that the normal human cell contains 46 chromosomes, arranged in 22 pairs of nonsex chromosomes or autosomes and two sex chromosomes, either 2 X (in females) or XY (in males). Thus, each chromosome had its double and both are known as homologues.

Figure 2-29 Diagram of development of nucleolus from nucleolus-associated chromatin. (Caspersson TO. Cell Growth and Cell Function-A Cytochemical Study. New York, WW Norton, 1950.)

The introduction of fluorescent probes, first to specific segments of individual chromosomes and then to whole chromosomes, has now allowed us to study the position and configuration of chromosomes in interphase cells. The techniques are known as fluorescent in situ hybridization (FISH), and chromosomal painting techniques. A number of initial studies, conducted mainly on human cells in culture, suggested that, contrary to previous assumptions, individual chromosomes could be identified in interphase cells. However, only a recent study of terminally differentiated human bronchial cells (Koss, 1998) could document that all

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chromosomes retain their identity during the interphase (Fig. 2-31). Further, it was shown that the two homologues of the same chromosome were located in different portions of the nucleus and were in close apposition to the nuclear membrane. By measuring angles formed by two homologues, it could be documented that the position of individual chromosomes in interphase cells is constant and is probably maintained in normal cells throughout the entire cell cycle. It was also documented that, in the bronchial cells, the configuration of the two homologues was somewhat different, suggesting that they may participate differently in cell function, as has been previously documented for X chromosome (Lyon's hypothesis, see Chap. 4). These studies strongly suggest that the fundamental organization of the nuclear DNA is orderly throughout the life of the cell and explains the orderly transmission of the genetic material from one generation of cells to another. The peripheral position of the chromosomes on the nuclear membrane also strongly suggested that each homologue might be responsible for the formation of its own proprietary segment of the nuclear membrane during the telophase. It was also suggested that the nuclear pores, which are the portals of exit (or entry) of the nuclear products (such as RNA) into the cytoplasm, might be formed at the points of junction of adjacent segments of the nuclear P.48 membrane. The consequences of these observations may have a significant impact on our understanding of nuclear structure and function.

Figure 2-30 Actual photographs of development of nucleolus inside the nucleolus-associated chromatin in a neurocyte (Feulgen stain). (Caspersson TO. Cell Growth and Cell Function-A Cytochemical Study. New York, WW Norton, 1950.)

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Figure 2-31 The position and configuration of chromosomes in terminally differentiated bronchial cells (oval nuclei) or goblet cells (spherical nuclei) stained with FISH. The two homologues of each chromosome are clearly located in different territories of the nucleus. The location of the autosomes on or adjacent to the nuclear membrane is evident. Identification numbers of chromosomes and the sex of the donor (F or M) are indicated. Only one signal was generated for the X chromosome in a male (XM). The differences in configuration and size of territories of the two autosomes (one compact and one open) are best seen in chromosomes 1F, 1M, 5M, 5F, 7F, 8F, 9M, 10F, 12F, 15M, 20F, and XF. Similar differences were noted for other chromosomes but are not well shown.

The Basement Membrane

The basement membrane is a complex structure that occurs at the interface of epithelia and the underlying connective tissue. There are several component parts to the basement membrane. Best seen in the electron micrograph is a thin, condensed, usually uninterrupted electron-opaque layer, known as basal lamina (see Figs. 2-5 and 2-14). Basal lamina is separated from the epithelial cell membranes by a narrow, electron-lucent layer known as lamina lucida. Crossing the lamina lucida are the cell junctions, known as hemidesmosomes, described above, that anchor epithelial cells to the basal lamina (see above and Fig. 2-14). On the side of the connective tissue, the basal lamina is in close contact with collagen fibrils. Basal lamina is also observed in nonepithelial tissues, for example, surrounding smoothmuscle cells. Within recent years, the basement membranes have been the subject of intensive studies, for several reasons. The basement membranes are a product of interaction between the epithelial cells and the connective tissue; hence, they form a barrier that has been shown to be important in a variety of diseases. Cell surface receptor molecules, known as integrins, are an important factor in regulating the relationship of the cells to the extracellular matrix (Giancotti and Ruoslanti, 1999). Some examples of diseases affecting the basement membrane are disorders of the renal glomeruli, certain skin disorders, and invasive cancer. Cancer cells, even in invasive or metastatic cancers, are capable of reproducing the basal lamina, although it may be functionally deficient. The principal functions of the basement membrane appear to be the support and anchorage of cells, such as epithelial cells, and, most likely, a regulatory role in the activity of some other cells, such as the smooth muscle. Basal lamina also serves as a template in epithelial regeneration. Major chemical components of the basement membrane include several complex proteins, such as laminins, collagen types IV and V, fibronectin, proteoglycans, and other adhesion molecules. The interrelationship of these components with each other, and the cells that

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produce it, is complex and not fully understood at this time. The relationship of cancer suppressor genes with various adhesion molecules and, hence, the basement membrane, in the genesis of benign tumors and formation of metastases in malignant tumors, is discussed in Chapters 3 and 7. P.49

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