a-textbook-of-histology-fawcett-don-w-don-wayne- 6.pdf

THE CELL The living substance of plants and animals tions can be localized in histological sections, is described by the general term protoplasm, or within intact cells, with greater precision and the smallest un...

THE CELL The living substance of plants and animals tions can be localized in histological sections, is described by the general term protoplasm, or within intact cells, with greater precision and the smallest unit of protoplasm capable of than is possible with the disruptive techniques independent existence is the cell. The simplest necessary for biochemical analysis. plants and animals consist of a single cell. Higher animals can be thought of as a com¬ plex society of interdependent cells of many THE CELL kinds that are specialized to carry out the func¬ tions essential for survival and reproduction There are hundreds of microscopically dis¬ of the animal as a whole. Cells serving the tinguishable cell types in the body, but all have same general function are bound together by certain structural features in common. This varying amounts of extracellular matrix to chapter describes the structural components form tissues (bone, muscle, etc.). Two or more of cells, in general, leaving an account of the tissues are combined to form larger functional distinctive identifying features of particular units called organs (skin, kidney, lung, etc.). cell types to later chapters. Several organs having interrelated functions The cell is partitioned into two major com¬ constitute an organ system, for example, the partments, the nucleus and the surrounding respiratory system (comprised of the nose, lar¬ cytoplasm, which are easily distinguished by ynx, trachea, and lungs) or the urinary system their form and staining characteristics. The (made up of the kidneys, ureters, urinary formed components within these compart¬ bladder, and urethra). ments are assigned to one of two categories, Although the term histology suggests that it organelles or inclusions, based on certain as¬ is a branch of morphological science primarily sumptions as to their function. Organelles are concerned with the tissues, its province is components found in all cells and are consid¬ much broader, encompassing the study of all ered to be metabolically active internal organs of the many cell types and extracellular com¬ carrying out specific essential functions (Fig. ponents of the body and the varying patterns 1—1). Inclusions, on the other hand, are meta¬ in which the cells are associated to form the bolically inert accumulations of cell products, functional units of the organs. Histology can such as pigment deposits, or stored metabo¬ be considered synonymous with microscopic lites, such as lipid and carbohydrate. Organ¬ anatomy. Its limits were formerly defined by elles are regarded as essential, whereas inclu¬ the resolving power of the light microscope, sions are dispensable and often temporary but the introduction of the electron micro¬ constituents of cells. This distinction between scope greatly extended the boundaries of the organelles and inclusions is still useful, but held. It now embraces the study of biological assignment of cell components to these cate¬ structure at all levels from the lower limit of gories was made at a time when too little was direct visual inspection down to the structure known about their ultrastructure and func¬ of large molecules. The development of inge¬ tion to make valid judgments as to whether nious histochemical and immunocytochemical they were metabolically active or inert, essen¬ methods has made it possible to identify the tial or dispensable. As our knowledge of cell intracellular sites of specific enzymatic activi¬ biology has progressed, the list of cell organ¬ ties and even to localize small peptides, and elles has lengthened and the traditional classi¬ the antigenic portions of larger molecules, by fication of some structures as inclusions has their binding of specific antibodies that have become debatable. For example, melano- been labeled to make them visible with the somes, pigment granules formerly considered microscope. By such methods, certain func¬ to be inert, have now been found to have a 1 2 THE CELL GOLGI COMPLEX SECRETION GRANULES Vocuoles Concentroting Secretory Product Agrooulor Membrones Vesicles CENTRIOLES ENDOPLASMIC RETICULUM Gronules Cristoe NUCLEAR ENVELOPE MITOCHODRION , Perinucleor Cisterno PLASMA NUCLEOLUS Glycogen MEMBRANE %mfl v - F, laments ( ~ 50 A) Nucleolonemo LIPID PARTICULATES DROPLETS OF THE MATRIX Figure 1-1. In the center, a depiction of the cell and its organelles as they appear with the light microscope. Around the periphery are drawings of the same components as these appear in electron micrographs. The substructure of the plasma membrane, encircled, is not directly visualized, but represents the bimolecular layer of phospholipids inferred from indirect methods of analysis. highly organized internal structure and to or lamellar structures, but the electron micro¬ have enzymatic activities that require their re¬ scope has shown that these, and newly de¬ classification as an organelle. Similarly, secre¬ scribed organelles, have a complex structure tory granules, formerly considered to be sim¬ and all are bounded by membranes composed ply stores of cell product, are now found to of lipid and protein. Most of the physiologi¬ be bounded by an enzymatically active mem¬ cally important processes takeJ place at sur¬ brane and, therefore, qualify as organelles. faces and interfaces, and the demonstration The electron microscope has revealed a of the extensive compartmentation of the cy¬ number of fibrillar elements in the cytoplasm toplasm by membranes has been an important that escaped detection with the light micro¬ contribution of modern ultrastructural stud¬ scope. These are neither organelles nor inclu¬ ies of the cell. The partitioning of the cyto¬ sions, but are grouped together in a third cate¬ plasm achieved by membrane-bounded or¬ gory of cell components, the cytoskeleton. ganelles enhances the efficiency of countless The organelles visible with the light micro¬ chemical reactions by amplifying the area of scope (mitochondria, Golgi complex, ergas- the physiologically active interfaces within the toplasm) were interpreted as solid granular cell. It also facilitates control of cell metabo-  THE CELL 3 lism by enabling the cell to maintain a separa¬ the diversity of its functions. It contains local tion of enzymes and their substrates at some specializations for cell attachment and cell-to- times, and, at other times, permitting their cell communication. Its permeability proper¬ controlled interaction by varying the perme¬ ties permit diffusion of ions and gases in solu¬ ability of a particular membrane or the rate tion into and out of the cell, but prevent passive of active transport across it. If there were un¬ entry of most larger molecules. limited diffusion and interaction within the In micrographs of high magnification, the cell, it would be impossible to maintain the membrane appears as two electron-dense high degree of chemical heterogeneity charac¬ lines (2.5-3.0 nm) separated by an electron- teristic of the cytoplasm. Enzymes would at¬ lucent intermediate zone (3.5-4.0 nm) (Fig. tack their substrates and all of the potential 1-2). Except for minor differences in thick¬ interactions of the countless chemical constit¬ ness, all membranes of the cell have this same uents of the cytoplasm would race out of con¬ appearance. They consist of a bimolecular trol. This does not occur. The cell is able to layer of mixed phospholipids with their hy¬ regulate its metabolic processes and to hold drophilic portions at the outer and inner sur¬ in reserve a large repertoire of unexpressed faces of the membrane and their hydrophobic biochemical reactions. It can activate each of chains projecting toward the middle of the these at the appropriate time and control its bilayer. The two dense lines, seen in electron rate to conform to the varying needs of the micrographs of osmium-fixed tissue, are due whole organism. That this is possible is due to deposition of the heavy metal in the hydro¬ in large measure to the segregation of bio¬ philic ends of the phospholipid molecules, chemical processes in the membrane- whereas the intervening pale zone represents bounded organelles of the cytoplasm. their unstained hydrocarbon chains. Choles¬ terol and varying amounts of proteins, glyco¬ proteins, and glycolipids are intercalated in CELL MEMBRANE the phospholipid bilayer (Fig. 1-3). The posi¬ tion of the integral proteins within the mem¬ All cells are bounded by a cell membrane brane depends on the location of hydrophilic (plasma membrane, plasmalemma). This is not and hydrophobic regions along the length of resolved in thin sections viewed with the light the molecule. The majority are transmem¬ microscope, but in electron micrographs, it ap¬ brane proteins that have nonpolar regions pears as a thin dense line, 8.5 to 10 nm in thick¬ traversing the hydrophobic interior of the ness around the periphery of the cell. Its ap¬ membrane and polar regions, at either end of pearance at low magnification gives no hint of the molecule, exposed on its outer and inner the complexity of its molecular organization or surfaces. The lipid bilayer behaves as a two- Figure 1-2. Electron micrograph of the plasma membrane of two adjacent cells, showing its trilaminar appearance, with two dense layers and a light intermediate layer. The intercellular space is occupied by material rich in carbohydrate, consisting, in part, of the glycocalyx of the apposed membranes. 4 THE CELL Figure 1-3. Schematic drawing of the fluid mosaic model of the cell membrane. Globular protein molecules are positioned in the lipid bilayer at different depths depending on the distribution of their hydrophilic and hydrophobic regions. Some extend through the bilayer (transmembrane proteins). The lipid bilayer is fluid and the integral proteins are free to diffuse laterally within the plane of the membrane if not resisted by binding to peripheral proteins in the underlying cytoplasm. Terminal oligosaccharides of the glycoproteins extend outward contributing to the surface coat or glycocalyx. dimensional viscous solution, within which the plane of fracture follows the path of least resis¬ proteins can move about if they are not bound tance through the hydrophobic region of the to filaments in the underlying cytoplasm. The lipid bilayers and, thus, cleaves the membranes lipid constituents of the membrane are largely in half (Fig. 1-4). A replica of the exposed sur¬ responsible for its form and its permeability face is then made by evaporation of a heavy properties, whereas receptors, ion pumps, metal, such as platinum, from a source at an and enzymatic activities reside in its proteins. acute angle to the fracture surface. Carbon is The integral proteins of the plasma mem¬ then deposited uniformly over this surface by brane are not visible in electron micrographs of evaporation from a separate electrode directly tissue sections, but they can be studied in tissues over the specimen, forming a stable replica of prepared by the freeze—fracture method. In all irregularities on the fracture face. The tis¬ this procedure, tissue is rapidly frozen in liquid sue is then digested away and the replica is re¬ Freon and then fractured under vacuum, by covered for examination with the electron mi¬ impact of a blade cooled to — 196°C. The croscope. Figure 1-4. In the freeze-fracture method of study, the fracture plane follows the hydrophobic region of the membrane, exposing two fracture faces—the E-face, the inwardly facing outer half, and the P-face, the outwardly facing inner half! which usually contains the majority of the integral protein particles.  THE CELL 5 Figure 1-5. A freeze-fracture preparation of the plasma membrane of two adjacent cells. The fracture plane has broken across from one to the other, exposing the E-face of one and the P-face of the other. In such preparations, the cleaved cell mem¬ basal membranes of epithelial cells differ in brane presents two distinct appearances. The their enzymatic activities. Although the pro¬ outwardly facing inner half-membrane, called teins are able to move laterally within the lipid the P-face, shows numerous randomly distrib¬ bilayer, translocation of proteins from the api¬ uted 6-9-nm convexities that are replicas of cal to the basolateral domain of epithelial cells the membrane proteins (Fig. 1-5). The in¬ is prevented by a circumferential tight-junc- wardly directed outer half-membrane, called tion between adjoining cells that constitutes a the E-face, is relatively smooth but may show barrier to diffusion between these two do¬ shallow depressions, corresponding, in their mains, and therefore serves to maintain their distribution, to the convexities on the oppos¬ distinctive properties. It is possible that move¬ ing P-face that represent the protein mole¬ ment of some transmembrane proteins is also cules of the membrane. In the various intra¬ prevented by their binding to cytoskeletal ele¬ cellular membranes, the concentration of ments in the underlying cytoplasm. particles observed by this method correlates The lipid bilayer is permeable to water, oxy¬ well with the protein content of the same gen, nitrogen, and certain small uncharged membranes as determined by biochemical polar molecules. It has very low permeability analysis. Because many enzymes are mem¬ to larger uncharged molecules and to all brane proteins, it is not surprising that the charged molecules. Entry of these substances concentration of intramembrane particles is into cells depends on active transport by inte¬ greater in organelles that have a high degree gral proteins of the membrane. Some of the of metabolic activity. transmembrane proteins transport glucose The seemingly random distribution of par¬ and amino acids; others form channels per¬ ticles within the plasma membrane gives a mis¬ mitting passive diffusion of certain ions; and leading impression of uniformity of mem¬ still others function as pumps, moving so¬ brane function over the entire cell. It is dium, potassium, hydrogen, and calcium into known, however, that the luminal, lateral, and and out of cells against a concentration gradi- 6 THE CELL Figure 1-6. (A) A high-magnification micrograph of the plasma membrane and the unusually thick glycocalyx on an insect cell. (B) A low-magnification micrograph of the brush border of an intestinal epithelial cell, showing the glycocalyx on the tips of the microvilli. ent. Such pumps maintain, in the cytoplasm, subunits. A lipophilic segment of the core pro¬ stable concentrations of ions that are essential tein spans the lipid bilayer, whereas the por¬ for normal cell function. tion of the long molecule bearing the carbohy¬ Other transmembrane proteins are recep¬ drate side-chains projects from the outer tors that enable the cell to recognize and bind surface of the membrane. The carbohydrate- specific molecules. There are receptors for rich chains of many such molecules form a neurotransmitters, hormones, and certain es¬ surface coat on the cell, called the glycocalyx sential nutrients. Receptors for the neuro¬ (Fig. 1-6). Such a coat is present on all cells, transmitter, acetylcholine, contain an ion but it is especially conspicuous on the coherent channel that opens upon ligand binding, and layer of cells that form the epithelium lining the resulting ion flux through the channel in¬ the gastrointestinal tract. In electron micro¬ duces a response in the cell. Receptors for graphs, the glycocalyx appears as a mat of hormones form a complex that induces an delicate branching polysaccharide filaments. associated membrane protein to generate a The chemical properties of this coat endow it messenger molecule that diffuses into the cy¬ with a high degree of selectivity with respect to toplasm to trigger the cells response. After the substances that can bind to the cell surface. ligand binding, other receptors for specific Ionized carboxyl and sulfate groups on the macromolecules needed by the cell aggregate polysaccharides have a strong negative charge into a small area of membrane that invaginates and avidly bind cationic ferritin and the dyes and pinches off, carrying the ligand into the Alcian-blue and ruthenium-red which are cytoplasm in a small vesicle. This process, commonly used to stain the glycocalyx. called receptor-mediated, endocytosis, will be dis¬ Organogenesis during embryonic life re¬ cussed more fully later in this chapter. quires recognition and adhesion of like cells, The membrane proteins include proteogly¬ and in postnatal life, immunological defenses cans, molecules consisting of a core protein require recognition between unlike cells. The bearing multiple glucosaminoglycan side- specificity of these cell interactions may be chains that are linear polymers of disaccharide provided by a stereochemical fit between com-  THE CELL 7 plimentary molecules on membranes of the form distinct plaques or circumferential two cells. Carbohydrates offer a greater struc¬ bands that are visible in micrographs of tural diversity for recognition than proteins. freeze-fracture preparations. These so-called As few as four different monosaccharides can junctional complexes will be discussed more fully form very great numbers of distinct tetrasac- in the following chapter on epithelia. charides. Thus, the sequence of glycosidic subunits of the large polysaccharides proj¬ ecting from the cell membranes can present NUCLEUS an infinite variety of molecular configurations as a basis for cell recognition. Recent studies The nucleus, the largest organelle of the suggest that cell—cell recognition during histo¬ cell, is centrally situated and usually round or genesis involves modulation of a relatively ellipsoidal, but, in some cell types, it may be small number of integral membrane proteins deeply infolded or lobulated. In stained tissue now called cell-adhesion molecules (CAMs). An sections, irregular clumps of chromatin of vary¬ increasing number of these are being identi¬ ing size may be scattered throughout the nu¬ fied. Among them are, one for brain cells and cleoplasm, but they tend to adhere to the inner muscle (N-CAM), one for liver cells (L-CAM), aspect of the nuclear membrane. Chromatin and one responsible for adhesion of glial cells consists of nucleic acids and associated histone to neurons (NG-CAM). These consist of a proteins that strongly bind hematoxylin and polypeptide chain linked to a carbohydrate other basic dyes. The principal nucleic acid such as sialic acid. of chromatin deoxyribonucleic acid (DNA) can In addition to the widely distributed adhe¬ be specifically stained with the Feulgen reac¬ sion molecules that are of submicroscopic di¬ tion. A portion of the DNA that is inactive mensions, there are local specializations of the is condensed, and therefore stainable, whereas membranes of epithelial cells for cell attach¬ other portions that are being transcribed are ment or cell-to-cell communication. In these, in an extended state and are not visible with membrane particles are closely aggregated to the microscope (Fig. 1-7). The nucleus also Nuclear pores Figure 1-7. Schematic interpretation of the state of the chromatin in the interphase nucleus. The condensed portions of the chromosomes (heterochromatin) are relatively inactive. The extended or uncoiled segments (euchromatin) are the sites of active transcription. Also shown is the nuclear envelope consisting of a perinuclear cisterna which is often continuous with the endoplasmic reticulum in the cytoplasm. Figure 1-8. Micrograph of a typical nucleus showing a prominent nucleolus and large aggregations of heterochromatin against the nuclear membrane, which is traversed by pores (at arrows). Inset upper left: two nuclear pores and their pore diaphragms. Inset lower right: the fibrous lamina present on the inner aspect of the nuclear envelope. 8  THE CELL 9 contains one or two nucleoli that consist mainly croscope reveals that it consists of two parallel of ribonucleoprotein and these are unstained membranes separated by a 10-30-nm space, by the Feulgen reaction. the perinuclear cisterna. The outer membrane The nucleus is the archive of the cell, the may have small granules (ribosomes) adhering repository of its genetic material. Encoded in to its outer surface and it is often continuous the sequence of nucleotides in its long DNA with membrane-bounded tubular elements molecules is the information necessary for extending throughout the cytoplasm (the en¬ synthesis of all of the integral proteins and the doplasmic reticulum). At many sites around secretory products of the cell. The synthetic the circumference of the nucleus, the inner activities of the cell are directed by informa¬ and outer membranes of the nuclear envelope tional macromolecules formed on the tem¬ are continuous with one another, around cir¬ plate of DNA in the nucleus and transported cular nuclear pores that serve as avenues of to the cytoplasm to direct protein synthesis. communication between the nucleoplasm and the cytoplasm (Fig. 1—8). The pores are not always uniformly distributed (Fig. 1-9), and NUCLEAR ENVELOPE their number varies from a few dozen to sev¬ eral thousand in cell types that are metaboli- The nucleus is bounded by a nuclear envelope cally very active. that participates in the organization of the In thin sections, the pores appear to be chromatin and controls the movement of mac¬ closed by a \hinpore diaphragm (Fig. 1—8, inset). romolecules between the nucleoplasm and the Negatively stained preparations of the iso¬ surrounding cytoplasm. Its structure was not lated nuclear envelope reveal several non- resolved with the light microscope but it was membranous structures associated with the assumed to be a membrane. The electron mi¬ pores. Attached to the membrane at the inner 'm?-- Figure 1-9. A freeze-fracture preparation of a portion of the nuclear membrane showing numerous nuclear pores. Their number and distribution changes as the cell differentiates or changes its activity. (D.W. Fawcett and H.E. Chemes. 1979. Tissue and Cell 11:147). Inset: nuclear pores as they appear with a different method of preparation (courtesy of E. Bearer and L. Orci). 10 THE CELL synthesized proteins that enter the nucleus, and ribonucleoproteins that leave it, are of a size that would require an opening 20 nm or more in diameter. Transport of such mole¬ cules appears to depend on their possession of a signal sequence of amino acids that targets them to the nuclear envelope. It is speculated that binding of the signal sequence to the nu¬ clear pores may trigger opening of the chan¬ nel from 10 nm to more than 20 nm to permit mediated transport of such large molecules. Much remains to be learned about how this gating of the pores works. If a monoclonal antibody to the nuclear pore complex is in¬ jected into the cytoplasm, it completely inhib¬ its nucleocytoplasmic transport of ribosomal and transport RNAs, but does not interfere with diffusion of small molecules. Three gly¬ coproteins, designated nucleoporins, have been identified as components of the pore complex, but their exact location and function in facili¬ tating transport remain unclear. A lectin that binds to one of these nucleoporins completely inhibits transport into the nucleus. A continuous meshwork of fine filaments is Figure 1-10. Negatively stained preparation of the nuclear pore complexes isolated from an amphibian oocyte (P.N.T. interposed between the inner nuclear mem- Unwin. 1982. J. of Cell Biol. 93:63. and outer rims of the pore are particles ar¬ ranged in two distinct coaxial rings, about 120 nm in diameter. Each ring is composed of eight subunits 15-20 nm in diameter (Figs. 1- 10 and 1—11). Projecting inward from these subunits are eight radially arranged spokes that converge at what appears to be a central granule or plug. The eight subunits of the rings, their radial spokes, and connecting links form a structural framework that imposed on the pore an octagonal symmetry around an axis perpen¬ dicular to the plane of the nuclear envelope. The term nuclear pore complex is now used to in¬ clude both the membranous and nonmembra- nous constituents of this structure. The spokes and central granule are evidently responsible for the specious appearance of a pore dia¬ phragm in micrographs of low magnification. The central granule and the radial spokes are obviously the principal barrier to movement through the pores, but the complex seems to have a central channel that acts as a molecular sieve with an effective diameter of 10 nm. In the two-way traffic between nucleoplasm ring and cytoplasm, molecules less than 10 nm in Figure 1-11. Diagrammatic representation of the nuclear diameter pass through the pores by passive pore complex (A) viewed from the cytoplasmic surface and diffusion, but larger molecules require an en¬ (B) in thin sections. The nuclear lamina on the inner surface ergy-dependent transport mechanism. Newly of the envelope is not depicted.  THE CELL 11 brane and the peripheral heterochromatin terphase chromosomes constitute the hetero¬ (Fig. 1-8, inset). This nuclear lamina varies in chromatin of the nucleus, whereas the thickness (30-100 nm) in different cell types, dispersed portions that are not microscopi¬ and in the same cell type in different species’ cally identifiable are the euchromatin. Only a but it seems to be a ubiquitous adjunct to the fraction of the total complement of units of nuclear envelope. Its filaments are polymers heredity (genes) is actively involved, at any of polypeptides called lamins that range in given time, in directing protein synthesis. This mass from 60 to 75 kilodaltons (kD). A-type portion is in the euchromatin, whereas the and B-type lamins are distinguished on the ba¬ genes that are not being expressed are in the sis of their location and chemical properties. heterochromatin. The numerous cell types in Those of the A-type are located mainly on the the body differ greatly in their synthetic activi¬ inner aspect of the nuclear lamina. The B- ties and, accordingly, in the proportion of type predominate near its outer surface and their DNA that is in the active extended form. are responsible for its binding to integral The pattern of heterochromatin in the nu¬ membrane proteins that are specific to the cleus, therefore, differs from cell type to cell inner nuclear membrane. type and provides one of the criteria for cell- When the nuclear envelope disintegrates type identification. during cell division, the lamins are disassem¬ I he appearance of chromatin in histologi¬ bled into monomeric form, permitting nu¬ cal sections gives no hint of the high degree clear membrane dissolution. At telephase of of order that would be expected from genetic mitosis, reconstruction of the nuclear mem¬ considerations, and progress in understand¬ brane takes place by fusion of membrane-lim¬ ing its organization has been relatively recent. ited vesicles that are adherent to the telophase In electron micrographs of thin sections, het¬ chromosomes. The concurrent self-assembly erochromatin seems to be made up of closely of the lamins into a continuous nuclear lamina packed 20-30-nm subunits, but whether these plays an important role in this process. If anti¬ are small granules or cross sections of highly bodies to lamins are injected into cells during convoluted fibrils is not apparent. However, if mitosis, the daughter cells are able to complete isolated nuclei are disrupted and their content cytokinesis but are not able to form normal spread on the surface of water, the dispersed nuclei. The chromatin remains condensed chromatin can be picked up on specimen and inactive and nucleoli are not reconstitu¬ grids, dried, and examined directly with the ted. Thus, it appears that the lamins are in¬ electron microscope. In such preparations, volved in the functional organization of the the chromatin appears as a tangle of 30-nm interphase nucleus. fibrils. With higher shearing forces, these can be further extended, and then they appear as a beaded strand of regularly spaced discoid CHROMATIN subunits, called nucleosomes, connected by a thin filament 4 nm in diameter (Fig. 1—12). Deoxyribonucleic acid (DNA), the genetic More detailed analysis is beyond the reach material of the nucleus, resides in the chromo¬ of microscopy and has depended on X-ray somes. These are not identifiable, as such, in crystallographic and biochemical studies. the nondividing interphase nucleus, but in cells These have identified the thin filament con¬ preparing to divide, they become visible as necting the nucleosomes as a double-stranded basophilic rod-like or thread-like structures DNA molecule and have shown that the core 3—6 jiim in length and 0.5—0.8 fxm in diameter. of the nucleosome is an octomer of two mole¬ During reconstitution of the nucleus after cell cules each, of four histones (H4, H3, H2A, and division, some segments of the chromosomes H2B). A segment of the DNA molecule is remain condensed and are visible as clumps of coiled around each nucleosome with straight chromatin at the periphery (Fig. 1-8). Other spacer segments of DNA, of varying length, segments of the chromosomes become un¬ between successive nucleosomes (Fig. 1-13). coiled and dispersed in the nucleoplasm. In An additional histone H1; is associated with this loosely packed state, the DNA and associ¬ the connecting segments of the DNA mole¬ ated protein have little affinity for basic dyes cule. In condensed chromatin of the intact and in electron micrographs are indistin¬ cell nucleus, the beaded filaments are believed guishable from other granular or flocculent to be helically coiled to form the 30-nm fibrils. components of the nucleoplasm. The stain- The granular appearance of heterochromatin able, electron-dense portions of the in¬ in electron micrographs is probably attribut- 12 THE CELL Figure 1-12. (A) Chromatin from a salamander erythrocyte, spread on water, fixed in formalin, critical-point dried, and shadowed with carbon-platinum. It appears as a tangled mass of 20-30-nm fibrils (Micrograph courtesy of H.Ris). (B) If chromatin fibrils are extended sufficiently by shear forces, the fibrils uncoil and appear as delicate beaded strands of nucleosomes connected by segments of double-stranded DNA. (Micrograph courtesy of A. Olins.) able to closely packed transverse and oblique scaffold. Examined at high magnification, this sections of these fibrils. The configuration of halo appears as an elaborate labyrinthine pat¬ the euchromatin is largely conjectural, but it tern traced by a continuous, highly tortuous is assumed that in these portions of the chro¬ 4-nm filament of DNA, devoid of associated mosomes, the 30-nm fibrils have uncoiled and nucleosomes (Fig. 1—15). extended, exposing the DNA to facilitate ex¬ The DNA molecule consists of two poly¬ pression of its genes. nucleotide chains intertwined in an antiparal¬ In dividing cells, all of the chromatin con¬ lel double helix. Each strand is a linear poly¬ denses and is organized into a number of mer of nucleotide subunits, each of which chromosomes that is characteristic of each ani¬ consists of a phosphate group, a pentose sugar mal species. Electron micrographs of thin sec¬ (deoxyribose), and an organic base. The bases tions provide little insight into their substruc¬ are of four kinds: adenine, cytosine, guanine, ture, but when isolated from cells in division and thymidine. These project toward, and are and studied in whole mounts, chromosomes linked to, complementary bases on the other are found to be made up of a 30-nm fibril strand. The genetic information in the DNA forming closely spaced loops arranged radi¬ molecule is encoded in the sequence of bases ally around an axial scaffold of structural pro¬ along the polynucleotide chains. The unit of tein (Fig. 1-14). The fibril is thought to be heredity, the gene, is a sequence of bases in formed by supercoiling of the 10-nm coiled the DNA that contain the information neces¬ filament of DNA and nucleosomes observed sary for the synthesis of a nucleic acid or a in dissociated chromatin from interphase nu¬ protein. In directing protein synthesis, this clei. If isolated chromosomes are treated with information is first transcribed from DNA to a dextran sulfate and heparin to extract the his¬ messenger ribonucleic acid (mRNA), which is a tones, the 30-nm fibril straightens, uncoils, linear polymer of the nucleotides adenylate, and spreads in a broad halo around the axial cytodylate, guanylate, or urodylate. Along the  THE CELL 13 in length, making it possible to pack a very large amount of genetic information into a nucleus that is only 6 /u,m in diameter. How this orderly process of DNA compaction is controlled at each cell division, so as to main¬ tain a consistent chromosomal form and the same arrangement of genes along their length, still defies explanation. NUCLEOLUS The nucleolus is visible in the living cell as a rounded refractile body usually eccentrically placed in the nucleus. In histological sections, it is deeply stained by basic dyes due to its content of ribonucleoprotein. It is usually un¬ reactive with the Feulgen reaction for deoxyri- bonucleoprotein, but it is often surrounded by an intensely stained rim of nucleolus-associated chromatin. In electron micrographs, the nucle¬ olus appears as a three-dimensional network of anastomosing dense strands, bounding electron-lucid interstices that are occupied by material indistinguishable from the sur¬ rounding nucleoplasm. This network, called the nucleolonema or pars granulosa, is made up Figure 1-13. Diagram of a 30-nm chromatin fiber showing of 15-nm ribonucleoprotein particles in a ma¬ its postulated helical structure around a central channel. In the lower part of the figure, the fiber is drawn out to trix of fine filaments. Rounded areas with a maximal extension to illustrate the nucleosome core of lower density and a fibrillar texture are found histones with the RNA double helix wrapped around it. at two or more sites within the nucleolus (Fig. Successive nucleosomes are joined by spacer segments of DNA and their associated histones. (Redrawn after R. Bradbury. 1978. La Recherche 9:644; and A. Worcel and C. Benyaj. 1978. Cell 12:88 from Amer. Scientist 66:704). length of the mRNA molecule, specific sets of three successive nucleotides, called codons, designate each of the 20 amino acids that are the building blocks of proteins. Messenger RNA is transported to the cytoplasm for trans¬ lation, in which the sequence of its nucleotides determines the order in which amino acids are assembled in the synthesis of a specific protein. Two other ribonucleic acids, ribosomal ribonucleic acid (rRNA) and transfer ribonucleic acid (tRNA) are transcribed in the nucleus and transported to the cytoplasm to play essential roles in protein synthesis that will be discussed later. Each chromosome is believed to contain a single very long molecule of DNA. The total length of the DNA in the human diploid ge¬ Figure 1-14. Electron micrograph of unsectioned meta¬ nome is estimated to be 1.8 m, containing 10 phase chromosome from a cultured cell showing the chro¬ to 3 x 108 nucleotide base pairs. The several matids, primary constriction, and what appear to be closely orders of coiling and supercoiling described packed loops of filaments radially arranged around the long above achieve nearly a 10,000-fold reduction axis of the chromatids. (Micrograph courtesy of H. Ris.) Figure 1-15. (Inset) If isolated chromosomes are treated with dextran sulfate and heparin, the histones are extracted permitting the DNA to uncoil, forming a broad halo around the core structural proteins. If an area such as that in the rectangle is examined at higher magnification, the halo is revealed as a labyrinthine pattern of 4-nm filaments of DNA (Micrograph from J.R. Paulsen and U.K. Laemmli. 1977. Cell 12:817). 14  THE CELL 15 Figure 1-16. Examples of nucleoli from two different cell types, showing variations in the pattern of the nucleolonema and in the number and size of the nucleolar organizer regions. 1—16). These so-called fibrillar centers contain Most of the events described above are not the nucleolar-organizer regions of those chromo¬ accessible to morphological study, but it has somes possessing nucleolar genes. Immedi¬ been possible to isolate from amphibian oo¬ ately surrounding each of these paler areas is cytes DNA-containing transcriptionally active a rim of electron-dense filaments referred to nucleolar genes. In a remarkable electron mi¬ as the pars fibrosa or dense fibrillar component of crograph of such a preparation (Fig. 1-17), the nucleolus. The size of the nucleolus and one can see multiple RNA-precursor mole¬ the pattern of its nucleolonema may differ cules radiating from the isolated segment of considerably from one cell type to another the genome in a Christmas-tree-like configu¬ and in different functional states of the same ration, with the transcripts quite short near cell type. It is largest in cells that are very the start and steadily progressing to full- active in protein synthesis. length completed molecules at the other end. Nucleolar genes code for ribosomal RNA and The nucleolus disappears during cell divi¬ it appears that their transcription occurs in sion and is reformed in the daughter cells the fibrillar centers of the nucleolus, whereas during reconstruction of their nuclei. Nucleoli the early steps in the processing of RNA-pre- initially develop from each of several, nucleo¬ cursor molecules and RNA-protein assembly lus-organizing regions in the set of chromo¬ takes place in the surrounding dense fibrillar somes, but their subsequent coalescence usu¬ component. The resulting ribonucleoprotein ally reduces the number of nucleoli in the particles accumulate in the pars granulosa, interphase nucleus to one or two, but larger where later steps of maturation of the ribo¬ numbers may be found in polyploid cells. somal subunits are carried out. The ribosomal The protein synthetic activities of the cyto¬ proteins are believed to be synthesized in the plasm are dependent on the functional integ¬ cytoplasm and targeted to their intranuclear rity of the nucleolus. Its destruction by a laser site of assembly by nuclear-localization-signal beam results in cessation of incorporation of sequences on the molecules. The completed RNA precursors into the ribosomes on which ribosomal subunits are then transported protein synthesis depends. In a mutant of the through the nuclear pores into the cytoplasm African clawed fog Xenopus, which lacks where they carry out their function. nucleoli, embryonic development is arrested  16 THE CELL rRNA molecules Nucleolar V gene. y tAf-n Start Figure 1-17. Micrograph of a dissociated amphibian nucleolar core, showing rRNA-precursor molecules radiating from nucleolar genes. (Micrograph courtesy of O. Miller and C. Beatty. 1969. Science 164:164). at a very early stage due to the inability of the filaments. However, it is possible that the net¬ cells to synthesize rRNA. work revealed may be a product of the proce¬ dures required for its demonstration and not a true reflection of the architecture of the nu¬ NUCLEAR MATRIX cleoplasm in vivo. The notion of an internal karyoskeleton anchored to the fibrous lamina Considerable progress has been made in at the periphery of the nucleus has not gained studying isolated condensed chromosomes, general acceptance. but relatively little is known about the form and disposition of the chromosomes in the interphase nucleus. It has been suggested that CYTOPLASM there may be a karyoskeletal network of fibrils in the interior of the nucleus on which the The principal metabolic activities and the chromosomes are arranged. After deoxyribo¬ various specialized functions of the cell are nuclease digestion of the chromatin from res¬ carried out in its extranuclear portion, the inless sections, and extraction with nonionic cytoplasm, which contains several kinds of cell detergents, a network of relatively thick fibrils, organelles that carry out different functions coated with adherent residues of the nucleo¬ that are essential to cell metabolism. The ma¬ plasm, has been reported. Further treatment jority of the organelles are membrane- with high-ionic strength salt solutions strips bounded elements that have a highly charac¬ away adherent material and leaves behind a teristic form and internal structure. They are network of thin, branching, 8-10-nm fila¬ suspended in a semifluid cytoplasmic matrix ments composed of RNA and protein. The called the cytosol. A coarse network, consisting view has been advanced that the chromo¬ of bundles of fine filaments that traverse the somes, and other components of the nucleo¬ cytoplasm and attach to the cell membrane, plasm, may be organized around these core provide internal support and help to maintain  THE CELL 17 the normal shape of the cell. These, together face of its limiting membrane; and the smooth with slender straight microtubules, constitute endoplasmic reticulum, which lacks adherent the cytoskeleton. We proceed now to a descrip¬ particles. The two forms are continuous, but tion of the cell organelles. their relative proportions vary in different cell types. The rough or granular reticulum is most abundant in glandular cells that secrete ENDOPLASMIC RETICULUM protein. The 20—25-nm particles associated with the rough form of endoplasmic reticu¬ In the cytoplasm of nearly all cell types, lum are called ribosomes. Despite their small there is an extensive system of membrane- size, they are complex structures consisting of bounded canaliculi called the endoplasmic retic¬ ribonucleic acid and 20 or more proteins. At ulum. This organelle is not ordinarily visible high magnifications, a larger and a smaller in histological sections, but if intact cells in subunit can be resolved in each ribosome, and tissue culture are stained with a lipophilic, it is the larger subunit that is bound to the nonionic, fluorescent dye, it appears as a lace¬ membrane of the reticulum. Clusters of ribo¬ like network throughout the cytoplasm (Fig. somes are also found free in the cytoplasmic 1-18). Its continuity is less evident in electron matrix. Both bound and free ribosomes are micrographs of thin sections, where it is repre¬ sites where amino acids are assembled in the sented by branching tubular profiles of vary¬ synthesis of proteins. To carry out this func¬ ing length. The tubules may be locally ex¬ tion, the ribosomes must be associated with panded into broad flat saccules called cisternae a molecule of messenger RNA (mRNA) which (Fig. 1-19). These are often closely spaced in contains the information determining the se¬ parallel arrays (Fig. 1-20). quence in which the amino acids are assem¬ Two morphologically and functionally dis¬ bled to form polypeptides. Ribosomes usually tinct regional differentiations of the organelle occur in clusters of 10 or more linked together are identified: the rough endoplasmic reticulum by their common attachment to a long mole¬ bearing small dense particles on the outer sur¬ cule of mRNA. Such units of several ribo- Figure 1-18. The continuity of the endoplasmic reticulum is not apparent in thin sections. This is a photomicrograph of a portion of a thin intact tissue culture cell stained with a lipophilic cationic fluorescent dye. The reticulum appears as a lace-like network of tubular elements. (Photomicrograph courtesy of M. Teresaki.)  18 THE CELL Polyribosomes Tubules Cisternae Figure 1-19. Drawing of the three-dimensional configuration of the rough endoplasmic reticulum. It consists of branching and anastomosing tubular elements and expanded saccules called cisternae. Polyribosomes occur in spirals or rosettes on the outer surface of its limiting membrane. somes bound to the same strand of mRNA homogenized cells. The cell fraction princi¬ are called polyribosomes or polysomes. As amino pally involved was found to be the microsome acids are added to their ends, the forming fraction, which consisted of vesicular frag¬ polypeptide chains elongate vectorially from ments of the rough endoplasmic reticulum the larger subunit of each ribosome, ex¬ that was broken up during cell homogenation. tending through the underlying membrane If the microsome fraction was treated with of the rough endoplasmic reticulum and into lipid solvents to remove the membranes, fur¬ its lumen. When the polypeptides have at¬ ther centrifugation at high speeds yielded a tained their full length, they are released from ribosome fraction, and protein synthesis could their respective ribosomes and are then free be induced, in vitro, by the addition of mes¬ within the rough endoplasmic reticulum senger RNA and cofactors to this fraction. (RER). Small vesicles containing the newly In the late 1800s, cytologists described synthesized protein pinch off from the endo¬ coarse clumps of material in the basal cyto¬ plasmic reticulum and are transported to a plasm of secretory epithelial cells that stained second organelle, the Golgi complex, where intensely with basic dyes. Tlie amount of this they are concentrated and packaged into se¬ material, which they called the ergastoplasm, cretory granules for export from the cell. All changed in different phases of the secretory intrinsic proteins of the cytoplasm and nucleo¬ cycle. A strongly basophilic cytoplasm and a plasm are also synthesized on polyribosomes prominent nucleolus came to be regarded as either on the reticulum or free in the cyto¬ defining features of secretory cells producing plasmic matrix. The intracellular pathway a product rich in protein. With the develop¬ taken by secretory proteins will be considered ment of suitable histochemical staining meth¬ in greater detail in a later chapter, Glands and ods and ultraviolet absorption techniques, the Secretion. basophilic material of such cells was identified Most of our knowledge of the mechanisms as ribonucleoprotein. In the 1940s, the elec¬ of protein synthesis has been based on bio¬ tron microscope revealed an abundance of chemical studies on centrifugal fractions of rough endoplasmic reticulum in secretory  THE CELL 19 Figure 1-20. Micrograph of several cisternae of rough endoplasmic reticulum, bearing ribosomes on its limiting membrane and products of protein synthesis in its lumen. (Micrograph courtesy of H. Warshawsky.) cells and isolated ribosomes were found to fibrils of the myocytes. Its principal function is bind basic dyes. It then became clear that ag¬ the sequestration of calcium ions that control gregations of rough endoplasmic reticulum muscle contraction. correspond to the ergastoplasm of classical cy- tologists and that the basophilia of the cyto¬ plasm of cells, in general, is largely attribut¬ ANNULATE LAMELLAE able to their content of ribosomes. The smooth endoplasmic reticulum is less The term annulate lamellae describes an or¬ extensive than the rough, in most cell types, ganelle that is relatively uncommon. It consists and usually takes the form of a close-meshed of stacks of parallel lamellae or cisternae con¬ network of branching tubules (Fig. 1-21). taining many pores that are similar to those of Smooth-surfaced cisternae are rarely ob¬ the nuclear envelope (Fig. 1-22). The cisternae served. The smooth reticulum is involved in are spaced 80-100 nm apart and are often con¬ the synthesis of fatty acids and other lipids. tinuous, at their ends, with tubules or cisternae It is found in greatest abundance in cells of of the rough endoplasmic reticulum. In sur¬ steroid-secreting endocrine glands. In the face view, the pores are 70-80 nm in diameter liver, it plays an important role in the synthesis and closely spaced in hexagonal array. The in- of the lipid component of very-low-density li¬ terlamellar cytoplasm often contains small poproteins that are carriers of cholesterol in densities that have a fibrillar substructure. the blood. It is also the principal site of detoxi¬ Annulate lamellae tend to occur in rapidly fication and metabolism of lipid-soluble exog¬ dividing cells, especially germ cells, and in a enous drugs. Chronic administration of such few other cell types in the early stages of their drugs induces a marked hypertrophy of the differentiation. Because they contain pores smooth endoplasmic reticulum of the liver. resembling those of the nuclear envelope, Striated muscle contains a specialized form of they were formerly thought to arise by delami¬ smooth reticulum, the sarcoplasmic reticulum, nation from the nuclear membrane. It is now which forms networks around all of the myo¬ considered more likely that they arise as pre-  20 THE CELL Figure 1-21. Micrograph of a small area of liver cell cytoplasm showing a tight-meshed network of tubular elements of smooth endoplasmic reticulum. Some contain small droplets of very-low-density lipoprotein. (Microqraph courtesy of R Bolender.) cursors of the nuclear envelope in cells pre¬ processing, concentration, and packaging in paring to divide. If present in excess, those secretory granules for discharge from the cell. not incorporated in the nuclear envelope dur¬ In addition to its function in the processing of ing the next cell division may persist for some secretory proteins, the Golgi complex control time and then break down. the traffic in small vesicles involved in the re¬ Despite their morphological similarity to the cycling of membrane between organelles, and nuclear envelope, the possibility remains that from the cytoplasm to the surface for renewal the two structures are unrelated. Annulate la¬ of the cell membrane. mellae do not react positively with labeled anti¬ The Golgi complex is not seen in routine his¬ body to the lamins that form the fibrous lamina tological preparations but it can be revealed by of the nuclear envelope. Conversely, an anti¬ silver or osmium impregnation in classical body believed to be specific for annulate lamel¬ staining methods developed in the 1800s. It lae fails to cross-react with the nuclear enve¬ can also be identified with histochemical proce¬ lope. The origin and functional significance of dures for localizing some of its enzymes. In annulate lamellae remain obscure. electron micrographs, it appears as stack of 4 to 10 parallel cisternae (Figs. 1-23 and 1-24). The lumen of these cisternae is narrow GOLGI COMPLEX throughout most of their length but tends to be slightly expanded at their ends. Although The Golgi complex (Golgi apparatus) is a ma¬ the complex is quite variable in form, its two or jor organelle found in nearly all cells. It is an three stacks of cisternae are often curved, with essential organelle in the secretory pathway a convex outer surface and a concave inner sur¬ and is most conspicuous in those cell types that face. These correspond to the arciform struc¬ produce a large volume of secretion. Proteins tures called dictyosomes by early cytologists. synthesized in the endoplasmic reticulum are Functional polarity is evident in the organi¬ transported to the Golgi complex for further zation of the Golgi complex. The convex sur-  THE CELL 21 tated protein. The terminal cisterna on that side of the organelle often has distended seg¬ ments forming condensing vacuoles that are pre¬ cursors of secretory granules. Other portions of the terminal cisterna are highly fenestrated, forming a specialized region of the organelle, called the trans-Golgi network (Fig. 1—23). Based on the distribution of enzymes re¬ vealed by histochemical staining methods, the Golgi complex is thought to have three func¬ tionally distinct compartments through which proteins pass in sequence. The highly fenes¬ trated initial cisterna has the unique property of being heavily stained by prolonged expo¬ sure to osmium and is designated the cis-com- partment. The next few cisternae that react positively for nicotinamide adenine dinucleo¬ tide phosphatase (NADPase), and A/-acetyl glucosamine transferase (AGT), form the in¬ termediate compartment, and a number of suc¬ ceeding cisternae that stain for thiamine pyro¬ phosphatase (TTPase), sialyl transferase (ST), and galactosyl transferase (GT) constitute the trans-compartment. In most secretory cells, gly¬ coproteins synthesized in the endoplasmic re¬ ticulum undergo only minor posttranslational modification in the Golgi, involving removal Figure 1-22. Micrograph of annulate lamellae. Note their of mannose groups from certain oligosaccha¬ close resemblance to the nuclear envelope at the left of rides and the addition of A-acetylglucosamine the figure (Nuc). The lamellae are continuous above with in the intermediate compartment, followed by cisternae of rough endoplasmic reticulum. (Micrograph the addition of galactose and sialic acid in the courtesy of S. Ito.) trans-compartment. However, in cartilage cells producing matrix proteoglycans, the Golgi complex has a major synthetic role, add¬ face, to which secretory protein is transported ing to the core protein up to 10 times its weight from the endoplasmic reticulum, is called the in glycosaminoglycan polysaccharides. cis-face of the organelle, and the opposite side is The endoplasmic reticulum synthesizes a the trans-face. There is considerable difference very great number of different proteins. Some in the cisternae along the cis—trans axis. The of these are for export as secretory products, first element at the cis-face is highly fenestrated but many others are destined to be incorpo¬ and, in reconstructions from serial sections, it rated into structural components of the cell. appears as a network of anastomosing tubules, One of the more challenging problems in cell rendering somewhat inappropriate its de¬ biology has been to discover how the various scription as a flat saccule or cisterna. The suc¬ proteins are sorted and delivered to their re¬ ceeding elements are true cisternae, but they spective destinations. It is now known that spe¬ may have a central fenestration in register with cific chemical groups are added to the proteins similar opening in the overlying and underly¬ in the reticulum and modified in their journey ing cisternae. The resulting discontinuity in through the Golgi complex, and these groups the stack of cisternae may contain a few small label the proteins for separate packaging and vesicles. Similar vesicles are commonly found serve to “address” them to the appropriate around the periphery of the stacks of cisternae. sites within the cell. The sorting takes place The transport of material from the cis- to the at the trans-face of the Golgi complex. There, trans-face of the organelle is believed to de¬ certain proteins are condensed into secretory pend on vesicles budding off from one cisterna granules, others are packaged in smooth-sur¬ and fusing with the next in the stack. Near the faced small vesicles, and still others are en¬ trans-side of the Golgi, the lumen of the cistei - closed in vesicles having a bristle-like coating. nae tends to be wider and may contain precipi¬ The majority of the vesicles appear to arise  22 THE CELL Figure 1-23. Schematic representation of the components of the Golgi complex and its relation to the rough endoplasmic reticulum. It is considered to have three compartments, the cis-, intermediate, and trans-compartments. from the trans-Golgi network. The secretory rich in mannose are phosphorylated in the 6- granules move to the cell surface for exo- carbon position. This modification evidently cytosis. There is suggestive evidence that the serves as a label, enabling these enzymes to coated vesicles transport newly synthesized bind to mannose-6-phosphate receptors. enzymes to late endosomes and then to lyso- These receptors and their ligands are found somes. The smooth vesicles may be involved to be concentrated in the trans-Golgi network either in constitutive secretion or in mem¬ where they are packaged in small vesicles that brane recycling. fuse specifically with the membrane of devel- The network of tubules associated with the oping lysosomes. The signals and the sorting terminal cisternae of the Golgi complex was mechanisms are not yet known for the many identified some 30 years ago as a potential other proteins that are processed in the Golgi site of biogenesis of the lysosomes and was complex. assigned the acronym GERL to indicate its close relation to the Golgi (G), its continuity with the endoplasmic reticulum (ER), and its MITOCHONDRIA role in lysosome formation (L). In later stud¬ ies, its continuity with the reticulum was not Slender rods 0.4—0.8 ^u.m in diameter and confirmed. This network of anastomosing tu¬ 4-9 fxm in length can be seen in thin living bules is now considered to be an integral part cells examined with the phase contrast micro¬ of the Golgi complex and a major site of exit scope. These mitochondria are quite flexible, of materials from it. The acronym GERL has changing shape as they slowly move about in now fallen into disfavor and has been replaced the cytoplasm. Their function is to provide by the term trans-Golgi network. the energy required for the biosynthetic and There is strong evidence that, after newly motor activities of the cell. Mitochondria may synthesized lysosomal enzymes have been be randomly distributed in the cytoplasm or transferred from the endoplasmic reticulum concentrated near sites of high energy utiliza¬ to the Golgi, specific oligosaccharide chains tion. Their numbers range from a few to sev-  THE CELL 23 Figure 1-24. Micrograph of the Golgi complex of a cell in the epithelium of the vas deferens. (Micrograph courtesy of D. Friend.) eral hundred depending on the size and en¬ the intracristal spaces, but they are not function¬ ergy needs of the various cell types. ally distinct from the rest of the membrane In electron micrographs, a mitochondrion space. The membrane space is only 10—20 nm is limited by a smooth-contoured outer mem¬ across and appears empty, for it apparently brane about 7 nm thick and a slightly thinner contains no protein precipitable by chemical inner membrane. The inner membrane runs fixatives. The larger intercristal space is occu¬ parallel to the outer membrane, except where pied by a moderately electron-dense mitochon¬ it forms thin folds that project into the interior drial matrix. In negatively stained preparations of the organelle. These are called the cristae of mitochondria that have been isolated and mitochondnales (Fig. 1—25). This plication of broken open, the inner membrane is studded the inner membrane is a device for increasing with numerous minute particles called the in¬ the area of this enzyme-rich membrane. The ner membrane subunits. These consist of a globu¬ number of cristae per mitochondrion is much lar head, about 10 nm in diameter, connected greater in cells with high-energy requirements to the membrane by a slender stem 3 to 4 nm than in those having a lower rate of metabo¬ thick and about 5 nm long. In conventional lism. In a few cell types, the cristae mitochon- electron micrographs, these subunits are not driales are tubular instead of slender folds of visible and the inner and outer membranes the inner membrane (Fig. 1—26). look alike. However, they differ in chemical The mitochondrial membranes bound two composition and physiological properties. compartments: a large intercristal space, com¬ The outer membrane contains a protein that prising all of the area within the inner mem¬ forms transmembrane channels that are brane, and a smaller membrane space, consisting freely permeable to small particles. The inner of the narrow cleft between the outer and membrane is relatively impermeable and has inner membranes and extending inward be¬ a higher content of protein than any other tween the two leaves of the cristae. These lat¬ membrane in the cell. ter extensions are sometimes referred to as Dense, more-or-less spherical matrix gran- Figure 1-25. Electron micrograph of a typical mitochondrion from the pancreas of a bat, showing the cristae, matrix, and matrix granules. (Micrograph courtesy of K.R. Porter.) Figure 1 26. Electron micrograph of mitochondria in a cell of the hamster adrenal cortex These mitochondria are unusual in containing large numbers of cristae that are tubular instead of lamellar. 24  THE CELL 25 ules, 30 to 50 nm in diameter may be found Mitochondria possess their own DN A and ri- anywhere in the intercristal space but are of¬ bosomal, transfer, and messenger RNAs. At ten located near the cristae. Their density very high magnifications, their DNA can be tends to obscure their internal structure, but seen as lose aggregations of slender filaments in very thin sections, they appear to contain in electron-lucent areas of the mitochondrial multiple minute compartments separated by matrix. When isolated from centrifugal frac¬ relatively thick septa. Their composition and tions of mitochondria and examined in nega¬ function continue to be subjects of contro¬ tively stained preparations, it closely resembles versy. When calcium or other divalent cations the DNA of bacteria, consisting of a double he¬ are present in high concentration in the fluid lix of naked DNA in the form of a circle with a bathing isolated mitochondria, the size and circumference of about 5.5 /i,m (Fig. 1-28). It density of the matrix granules are increased. differs from DNA of the nucleus in its circular This led to the suggestion that they may be form and much lower molecular weight. Al¬ involved in regulation of the internal concen¬ though mitochondria have their own genome, tration of ions in the matrix. This interpreta¬ they are not self-sufficient. Their DNA con¬ tion has been weakened by X-ray microanaly¬ tains only about 15,000 nucleotides and, thus, sis which failed to reveal calcium in the matrix has a very limited coding capacity. It encodes granules of mitochondria in most tissues. the RNA of mitochondrial ribosomes, which There is, at present, no convincing evidence are visible as 12-nm granules distributed that mitochondrial matrix granules, in tissues throughout the matrix, and for transfer RNAs, other than cartilage and bone, are involved in but its messenger RNAs code for only a few calcium sequestration in vivo. Their ultra¬ components of the respiratory enzyme com¬ structure, and their affinity for osmium, sug¬ plexes of the inner membrane. Nuclear DNA gest that their major component is lipid and encodes the rest of the inner membrane pro¬ biochemical analysis of centrifugal fractions teins, those of the outer membrane and those enriched in matrix granules indicates that of the matrix. These proteins are all synthe¬ they are composed of phospholipoprotein. At sized in the cytoplasm and imported into the present, their function is purely conjectural. mitochondria. All enzymes needed to replicate Mitochondria are self-replicating organ¬ DNA and transcribe it into RNA also depend elles, with a limited life span, that maintain on nuclear genes. Thus, in evolving from free- their numbers by a form of division that re¬ living bacteria, mitochondria have given over sembles the binary fission of bacteria. Indeed, to the nucleus and cytoplasm of the host cell the mitochondria are believed to have evolved coding and synthesis of most of their proteins. from symbiotic bacteria very early in the evo¬ However, it is interesting that mitochondrial lution of unicellular organisms, at a time when protein synthesis is blocked by antibacterial an¬ the Earth’s atmosphere was poor in oxygen tibiotics that do not affect protein synthesis and most of the organisms depended on an¬ elsewhere in the cell. aerobic fermentation of organic molecules. It The principal biochemical activity of mito¬ is speculated that a small bacterium that had chondria is oxidative phosphorylation—the oxi¬ evolved the ability to utilize oxygen, invaded dation, by molecular oxygen, of metabolites of a larger anaerobic cell type and a symbiotic the nutrients (glucose and fatty acids) that the relationship developed, in which the aerobic cell receives from the blood. The energy thus symbiont generated energy in return for pro¬ generated is used to synthesize adenosine tri¬ tection and nutrients supplied by the larger phosphate (ATP) from adenosine diphosphate host cell. With the passage of time, the interde¬ (ADP) and inorganic phosphate. ATP released pendence of the two increased and the para¬ from the mitochondria, into the cytoplasm, is site became an indispensable cell organelle, an ubiquitous store of energy that is needed for passed on by the host cell to its progeny. Mito¬ transport across membranes for all synthetic chondria retain a mode of division in which processes and for the mechanical work in¬ elongation of a centrally situated crista forms volved in motor activities of the cell. On de¬ a partition across the organelle and opposing mand, a high-energy phosphate bond in ATP folds of the outer membrane then extend be¬ is split, instantly releasing energy and con¬ tween the leaves of the partition to meet and verting ATP to ADP. ATP is then regenerated fuse, completing the separation of the two from ADP, by the mitochondria, using phos¬ halves—a mode of division resembling that of phoric acid and energy derived from cell nutri¬ the ancestral symbiotic bacterium and of many ents. Mitochondria can be regarded as the pow¬ modern bacteria (Fig. 1—27). erhouses of the cell.  26 THE CELL Figure 1-27. Micrographs of successive stages (A-C) in the division of a mitochondrion. (Micrograph courtesy of T. Kanaseki.) LYSOSOMES microscopic images. They are so heteroge¬ neous that no single description encompasses Lysosomes range in number from a few to all of their variations, but, in general, they are several hundred per cell, in different cell round, ovoid, or highly irregular, electron- types. They were not recognized as a true cell dense bodies 0.25—0.8 p,m in diameter (Fig. organelle by classical cytologists because they 1-29). Their interior may appear homoge¬ do not have consistent form or tinctorial prop¬ neous or may consist of dense granules of erties with traditional staining methods. At¬ varying size in a less-dense matrix. They may tention was drawn to them by histochemical contain crystals or concentric systems of la¬ reactions for acid hydrolases and by electron mellae, interpreted as myelin forms of phos- / A'-**YY:: ' v is*V-*. V L- S-. v [: W-rw&MZ&H':--' RNP rranules? mm&kmmM.—— (B)DNATs2“aS “rShhoSr2,'aaxeCnoL0l AJ" 2? "*'*(Micro,,aph courtesy of H : conlou, length. (Micrograph courtesy of I.B. Dawid and wSenholilieJ * °CCU,S '0rm °' circles 5-6  THE CELL 27 struation. In these normal events, the lyso¬ somal membranes are altered permitting es¬ cape of acid hydrolases that break down the cells no longer needed. Lysosomes play their most important role in the defense of the organism against bacterial invasion. Lysosomes are abundant in poly¬ morphonuclear leukocytes of the blood and in tissue macrophages which are cell types spe¬ cialized for phagocytosis —the ingestion and in¬ tracellular digestion of bacteria and other for¬ eign particulate matter. When bacteria are engulfed by these cells they are taken into the cytoplasm in a membrane-bounded phagocyto¬ sis vacuole or phagosome. Lysosomes then gather around this vacuole and their mem¬ branes fuse with its membrane, releasing into its interior hydrolytic enzymes that destroy the ingested bacterium (Fig. 1-30). The lysosomes of the cell are also involved in the elimination of cell organelles in reorga¬ nizations of the cytoplasm associated with changes in physiological activity. Mitochon¬ dria or endoplasmic reticulum that are pres¬ ent in excess are enveloped in a membrane to form an autophagic vacuole. Lysosomes then Figure 1-29. Micrograph of a group of lysosomes in the fuse with this vacuole releasing enzymes that Golgi region of a cell of the adrenal cortex. digest its contents (Fig. 1—31). This process of controlled degradation of organelles in a healthy cell is called autophagy to distinguish pholipid. Lysosomes cannot be confidently it from heterophagy which is the digestion of identified, as such, by morphological criteria exogenous material taken into the cell. alone. Histochemical demonstration of acid Owing to the substrate diversity of the lyso¬ phosphatase or other hydrolases in their inte¬ somal enzymes, digestion of the content of rior is required for verification. autophagic and heterophagic vacuoles is usu¬ Forty or more hydrolytic enzymes have now ally quite complete; however, undigestible res¬ been reported to occur in lysosomes. These idues, in small amounts, may persist in mem¬ include proteases, glycosidases, nucleases, brane-bounded residual bodies. These may phosphatases, phospholipases, and sulfatases. coalesce into larger masses that are variously The pH optima of nearly all of these enzymes designated as wear-and-tear pigment, lipochrome are in the acid range. The lysosomes are now pigment, or lipofuchsin pigment. Where abun¬ regarded as an intracellular digestive system dant, these may be visible with the light micro¬ capable of degrading nearly all naturally oc¬ scope as irregularly shaped, yellow or brown curring chemical constituents of cells. In the masses in the cytoplasm. The development normal cell, these potentially damaging en¬ of the concept of lysosomes as intracellular zymes are safely contained within the limiting digestive organelles has led to a proliferation membrane of the lysosome. However, in cer¬ of redundant terms. To simplify the terminol¬ tain pathological conditions, the permeability ogy, it has been suggested that primary lysosome of this membrane may be altered, allowing be used to describe those that have not yet enzymes to escape and digest or lyse the cell. become engaged in digestive activity and that Cell digestion by lysosomes is not limited to the term secondary lysosome be applied to any disease states. Programmed death of certain vacuolar structure that is the site of current cells is a normal event in the embryonic devel¬ or past digestive activity. The latter term opment of some organs. In postnatal life, cer¬ would thus encompass autophagic and heter¬ tain organs may undergo a massive regres¬ ophagic vacuoles and the enzymatically inac¬ sion, as in the mammary gland after weaning tive residual bodies and lipofuchsin pigment or in the endometrium of the uterus in men¬ deposits. 28 THE CELL Figure 1-30. Schematic depiction of the role of lysosomes in heterophagy (lower right) and in autophagy (upper left). Bacteria are taken up by phagocytosis in endocytic vacuoles. Primary lysosomes fuse with these vacuoles and their enzymes digest their contents. In autophagy, membranes may be formed around excess organelles or inclusions and lysosomes then fuse with these vacuoles. The end products of both processes may be recognized as residual bodies or lipofuchsin pigment deposits. The origin of lysosomes has long been con¬ may escape, destroying collagen and elastin troversial, but recent research indicates that in the surrounding connective tissue. It is now lysosomal enzymes are synthesized, together realized that some of the damage to kidneys, with secretory and other proteins, in the endo¬ joints, and lungs following protracted in¬ plasmic reticulum and transported in vesicles flammation is a consequence of this leakage to the Golgi complex. There mannose resi¬ of acid hydrolases from activated phagocytic dues on enzymes destined to be incorporated cells that were mobilized for defense against in lysosomes are phosphorylated. They then invading bacteria. bind to mannose-6-phosphate receptors in the Clinical interest in lysosomes has been stim¬ membrane of the trans-Golgi network, from ulated by the discovery that a number of un¬ which they are transported in small vesicles common “storage diseases” of children are targeted to developing lysosomes. Uncer¬ due to an inherited inability to synthesize cer¬ tainty persists as to the origin of the structures tain lysosomal enzymes. In the absence of a with which these vesicles fuse to form lyso¬ particular lysosomal enzyme, its normal sub¬ somes. This will be discussed later in this chap¬ strate accumulates in large membrane- ter in the section on endocytosis. bounded inclusions, which are, in effect, au¬ Although lysosomes usually carry out their tophagic vacuoles that are unable to digest digestive function intracellularly, there are a their content. Massive accumulations of such few situations in which lysosomal enzymes are material in the cells of the liver, and other released from cells. Osteoclasts, cells special¬ organs, seriously affects their functions and ized for the remodeling of bone, secrete en¬ leads to early death. zymes into a sealed cavity between the cell and the underlying bone to digest the bone matrix. Also, at sites of acute inflammation, where PEROXISOMES leukocytes are actively phagocydzing bacteria, lysosomes may fuse with a forming phago¬ In early electron microscopic studies of the some before its complete closure and enzymes liver, 0.2-1.0-p,m membrane-limited bodies  THE CELL 29 body temperature. The metabolic interaction of reactions within the peroxisomes with path¬ ways in the surrounding cytoplasm is exten¬ sive, necessitating the transport of a variety of metabolites across their membrane. The functions of peroxisomes in cell metabolism are still incompletely understood. Peroxisomes are present in nearly all cell types and they number in the hundreds in metabolically active cells such as those of the liver. In electron micrographs of cells in sev¬ eral animal species, there is a denser region of the peroxisome, called the nucleoid, which is eccentrically situated in an otherwise homo¬ geneous gray matrix. The nucleoid is a para- crystalline array of minute tubules of urate oxidase. Peroxisomes of birds and primates lack this enzyme and do not have a nucleoid. Clinical interest in peroxisomes rests on the finding that there are several inherited dis¬ eases in which there is either deficiency of a single enzyme (X-linked adrenoleukodystro- phy) or defects in peroxisome formation and deficiency of several enzymes (Zellweger syn¬ drome). Figure 1-31. Examples of autophagic vacuoles. (A) A per¬ oxisome and a mitochondrion enclosed in the same vacu¬ CENTROSOME AND CENTRIOLES ole. (B) Elements of the endoplasmic reticulum in two adja¬ cent autophagic vacuoles. In suitably stained cells, one can usually find a centrosome, a small, more-or-less spherical area with a texture differing slightly from that of lower density than lysosomes were observed of the surrounding cytoplasm. In its center and tentatively called microbodies. A decade are two short rods, the centrioles. In addition later, with the development of centrifugation to the centrioles, it may also contain a variable procedures of improved resolution, it was pos¬ number of small dense bodies called centriolar sible to separate these from a crude lysosomal satellites. In some epithelia, the centrosome fraction and to study their biochemical prop¬ and its pair of c