Chapter 2
Cells
Cells are the structural and functional units of all living organisms. The smallest organisms consist of single cells and are microscopic, whereas larger organisms are multicellular. The human body, for example, contains at least 1014 cells. Unicellular organisms are found in great variety throughout virtually every environment from Antarctica to hot springs to the inner recesses of larger organisms. Multicellular organisms contain many different types of cells, which vary in size, shape, and specialized function. Yet no matter how large and complex the organism, each of its cells retains some individuality and independence.
nucleus (eukaryotes) or nucleoid (bacteria) contains genetic material – DNA and associated proteins, nucleus is membrane-bounded, plasma membrane, tough, flexible bilayer, selectively permeable to polar substances, includes membrane proteins that function in transport, in signal reception, and as enzymes, cytoplasm, aqueous cell contents and suspended particles and organelles, centrifuge at 150,000 g, supernatant: cytosol, concentrated solution of enzymes, RNA, building block molecules, metabolites, inorganic ions, pellet: particles and organelles, ribosomes, storage granules, mitochondria, chloroplasts, lysosomes, endoplasmic reticulum
Figure 2–1  The universal features of all living cells: a nucleus or nucleoid, a plasma membrane, and cytoplasm. The cytosol is that portion of the cytoplasm that remains in the supernatant after centrifugation of a cell extract of 150,000 g for 1 h.
Despite their many differences, cells of all kinds share certain structural features (Fig. 2–1). The plasma membrane defines the periphery of the cell, separating its contents from the surroundings. It is composed of enormous numbers of lipids and protein molecules, held together primarily by noncovalent hydrophobic interactions (p. 18), forming a thin, tough, pliable, hydrophobic bilayer around the cell. The membrane is a barrier to the free passage of inorganic ions and most other charged or polar compounds; transport proteins in the plasma membrane allow the passage of certain ions and molecules. Other membrane proteins are receptors that transmit signals from the outside to the inside of the cell, or are enzymes that participate in membrane-associated reaction pathways.
Because the individual lipid and protein subunits of the plasma membrane are not covalently linked, the entire structure is remarkably flexible, allowing changes in the shape and size of the cell. As a cell grows, newly made lipid and protein molecules are inserted into its plasma membrane; cell division produces two cells, each with its own membrane. Growth and fission occur without loss of membrane integrity. In a reversal of the fission process, two separate membrane surfaces can fuse, also without loss of integrity. Membrane fusion and fission are central to mechanisms of transport known as endocytosis and exocytosis.
The internal volume bounded by the plasma membrane, the cytoplasm, is composed of an aqueous solution, the cytosol, and a variety of insoluble, suspended particles (Fig. 2–1). The cytosol is not simply a dilute aqueous solution; it has a complex composition and gel-like consistency. Dissolved in the cytosol are many enzymes and the RNA molecules that encode them; the monomeric subunits (amino acids and nucleotides) from which these macromolecules are assembled; hundreds of small organic molecules called metabolites, intermediates in biosynthetic and degradative pathways; coenzymes, compounds of
intermediate molecular weight (Mr 200 to 1,000) that are essential participants in many enzyme-catalyzed reactions; and inorganic ions.
Among the particles suspended in the cytosol are supramolecular complexes and, in higher organisms but not in bacteria, a variety of membrane-bounded organelles in which specialized metabolic machinery is localized. Ribosomes, complexes of over 50 different protein and RNA molecules, are small particles, 18 to 22 nm in diameter. Ribosomes are the enzymatic machines on which protein synthesis occurs; they often occur in clusters called polysomes (polyribosomes) held together by a strand of messenger RNA. Also present in the cytoplasm of many cells are granules containing stored nutrients such as starch and fat. Nearly all living cells have either a nucleus or a nucleoid, in which the genome (the complete set of genes, composed of DNA) is stored and replicated. The DNA molecules are always very much longer than the cells themselves, and are tightly folded and packed within the nucleus or nucleoid as supramolecular complexes of DNA with specific proteins. The bacterial nucleoid is not separated from the cytoplasm by a membrane, but in higher organisms, the nuclear material is enclosed within a double membrane, the nuclear envelope. Cells with nuclear envelopes are called eukaryotes (Greek eu, “true”, and karyon, “nucleus”); those without nuclear envelopes – bacterial cells – are prokaryotes (Greek pro, “before”). Unlike bacteria, eukaryotes have a variety of other membrane-bounded organelles in their cytoplasm, including mitochondria, lysosomes, endoplasmic reticulum, Golgi complexes, and, in photosynthetic cells, chloroplasts.
In this chapter we review briefly the evolutionary relationships among some commonly studied cells and organisms, and the structural features that distinguish cells of various types. Our main focus is on eukaryotic cells. Also discussed in brief are the cellular parasites known as viruses.
Most cells are of microscopic size. Animal and plant cells are typically 10 to 30 μm in diameter, and many bacteria are only 1 to 2 μm long.
20 μm, 10 μm, 5 μm, number of cubes, n, 1, 8, 64, length of a side, l (μm), 20, 10, 5, total surface area, l2 × 6n (μm2), 2,400, 4,800, 9,600, total volume, l3 × n (μm3), 8,000, 8,000, 8,000, surface area:volume ratio, 6/l, 0.3, 0.6, 1.2
What limits the dimensions of a cell? The lower limit is probably set by the minimum number of each of the different biomolecules required by the cell. The smallest complete cells, certain bacteria known collectively as mycoplasma, are 300 nm in diameter and have a volume of about 10-14 mL. A single ribosome is about 20 nm in its longest dimension, so a few ribosomes take up a substantial fraction of the cell’s volume. In a cell of this size, a 1 μM solution of a compound represents only 6,000 molecules.
The upper limit of cell size is set by the rate of diffusion of solute molecules in aqueous systems. The availability of fuels and essential nutrients from the surrounding medium is sometimes limited by the rate of their diffusion to all regions of the cell. A bacterial cell that depends upon oxygen-consuming reactions for energy production (an aerobic cell) must obtain molecular oxygen (O2) from the surrounding medium by diffusion through its plasma membrane. The cell is so small, and the ratio of its surface area to its volume is so large, that every part of its cytoplasm is easily reached by O2 diffusing into the cell. As the size of a cell increases, its surface-to-volume ratio decreases (Fig. 2–2), until metabolism consumes O2 faster than diffusion can
Figure 2–2  Smaller cells have larger ratios of surface area to volume, and their interiors are therefore more accessible to substances diffusing into the cell through the surface. When the large cube (representing a large cell) is subdivided into many smaller cubes (cells), the total surface area increases greatly without a change in the total volume, and the surface-to-volume ratio increases accordingly.
supply it. Aerobic metabolism thus becomes impossible as cell size increases beyond a certain point, placing a theoretical upper limit on the size of the aerobic cell.
There are interesting exceptions to this generalization that cells must be small. The giant alga Nitella has cells several centimeters long. To assure the delivery of nutrients, metabolites, and genetic information (RNA) to all of its parts, each cell is vigorously “stirred” by active cytoplasmic streaming (p. 43). The shape of a cell can also help to compensate for its large size. A smooth sphere has the smallest surface-to-volume ratio possible for a given volume. Many large cells, although roughly spherical, have highly convoluted surfaces (Fig. 2–3a), creating larger surface areas for the same volume and thus facilitating the uptake of fuels and nutrients and release of waste products to the surrounding medium. Other large cells (neurons, for example) have large surface-to-volume ratios because they are long and thin, star-shaped, or highly branched (Fig. 2–3b), rather than spherical.

Figure 2–3  Convolutions of the plasma membrane, or long, thin extensions of the cytoplasm, increase the surface-to-volume ratio of cells. (a) Cells of the intestinal mucosa (the inner lining of the small intestine) are covered with microvilli, increasing the area for absorption of nutrients from the intestine. (b) Neurons of the hippocampus of the rat brain are several millimeters long, but the long extensions (axons) are only about 10 nm wide.
Because all living cells have evolved from the same progenitors, they share certain fundamental similarities. Careful biochemical study of just a few cells, however different in biochemical details and varied in superficial appearance, ought to yield general principles applicable to all cells and organisms. The burgeoning knowledge in biology in the past 150 years has supported these propositions over and over again. Certain cells, tissues, and organisms have proved more amenable to experimental studies than others. Knowledge in biochemistry, and much of the information in this book, continues to be derived from a few representative tissues and organisms, such as the bacterium Escherichia coli, the yeast Saccharomyces, photosynthetic algae, spinach leaves, the rat liver, and the skeletal muscle of several different vertebrates.
A dividing Escherichia coli cell.
Saccharomyces cerevisiae (baker’s yeast).
In the isolation of enzymes and other cellular components, it is ideal if the experimenter can begin with a plentiful and homogeneous source of the material. The component of interest (such as an enzyme or nucleic acid) often represents only a miniscule fraction of the total material, and many grams of starting material are needed to obtain a few micrograms of the purified component. Certain types of physical and chemical studies of biomolecules are precluded if only microgram quantities of the pure substance are available. A homogeneous source of an enzyme or nucleic acid, in which all of the cells are genetically and biochemically identical, leaves no doubt about which cell type yielded the purified component, and makes it safer to extrapolate the results of in vitro studies to the situation in vivo. A large culture of bacterial or protistan cells (E. coli, Saccharomyces, or Chlamydomonas, for example), all derived by division from the same parent and therefore genetically identical, meets the requirement for a plentiful and homogeneous source. Individual tissues from laboratory animals (rat liver, pig brain, rabbit muscle) are plentiful sources of similar, though not identical; cells. Some animal and plant cells proliferate in cell culture, producing populations of identical (cloned) cells in quantities suitable for biochemical analysis.
Genetic mutants, in which a defect in a single gene produces a specific functional defect in the cell or organism, are extremely useful in establishing that a certain cellular component is essential to a particular cellular function. Because it is technically much simpler to produce and detect mutants in bacteria and yeast, these organisms (E. coli and Saccharomyces cerevisiae, for example) have been favorite experimental targets for biochemical geneticists.
An organism that is easy to culture in the laboratory, with a short generation time, offers significant advantages to the research biochemist. An organism that requires only a few simple precursor molecules in its growth medium can be cultured in the presence of a radioisotopically labeled precursor, and the metabolic fate of that precursor can then be conveniently traced by following the incorporation of the radioactive atoms into its metabolic products. The short generation time (minutes or hours) of microorganisms allows the investigator to follow a labeled precursor or a genetic defect through many generations in a few days. In higher organisms with generation times of months or years, this is virtually impossible.
Some highly specialized tissues of multicellular organisms are
remarkably enriched in some particular component related to their specialized function. Vertebrate skeletal muscle is a rich source of actin and myosin; pancreatic secretory cells contain high concentrations of rough endoplasmic reticulum; sperm cells are rich in DNA and in flagellar proteins; liver (the major biosynthetic organ of vertebrates) contains high concentrations of many enzymes of biosynthetic pathways; spinach leaves contain large numbers of chloroplasts; and so on. For studies on such specific components or processes, biochemists commonly choose a specialized tissue for their experimental systems.
Sometimes simplicity of structure or function makes a particular cell or organism attractive as an experimental system. For studies of plasma membrane structure and function, the mature erythrocyte (red blood cell) has been a favorite; it has no internal membranes to complicate purification of the plasma membrane. Some bacterial viruses (bacteriophages) have few genes. Their DNA molecules are therefore smaller and much simpler than those of humans or corn plants, and it has proved easier to study replication in these viruses than in human or corn chromosomes.
The biochemical description of living cells in this book is a composite, based on studies of many types of cells. The biochemist must always exercise caution in generalizing from results obtained in studies of selected cells, tissues, and organisms, and in relating what is observed in vitro to what happens within the living cell.
Nostoc sp., a photosynthetic cyanobacterium.
All of the organisms alive today are believed to have evolved from ancient, unicellular progenitors. Two large groups of extant prokaryotes evolved from these early forms: archaebacteria (Greek, arché, “origin”) and eubacteria. Eubacteria inhabit the soil, surface waters, and the tissues of other living or decaying organisms. Most common and well-studied bacteria, including E. coli and the cyanobacteria (formerly called blue–green algae), are eubacteria. The archaebacteria are more recently discovered and less well studied. They inhabit more extreme environments – salt brines, hot acid springs, bogs, and the deep regions of the ocean.
Within each of these two large groups of bacteria are subgroups distinguished by the habitats to which they are best adapted. In some habitats there is a plentiful supply of oxygen, and the resident organisms live by aerobic metabolism; their catabolic processes ultimately result in the transfer of electrons from fuel molecules to oxygen. Other environments are virtually devoid of oxygen, forcing resident organisms to conduct their catabolic business without it. Many of the organisms that have evolved in these anaerobic environments are obligate anaerobes; they die when exposed to oxygen.
All organisms, including bacteria, can be classified as either chemotrophs (those obtaining their energy from a chemical fuel) or phototrophs (those using sunlight as their primary energy source). Certain organisms can synthesize some or all of their monomeric subunits, metabolic intermediates, and macromolecules from very simple starting materials such as CO2 and NH3; these are the autotrophs. Others must acquire some of their nutrients from the environment preformed (by autotrophic organisms, for example); these are heterotrophs. There are therefore four general modes of obtaining fuel and energy, and four general groups of organisms distinguished by these
chemoheterotrophs, organic compounds, O2 → CO2, cells, chemoautotroph, CO2, H2S, Fe2+ → S, Fe3+, cells, photoheterotroph, organic compounds, light → cells, photoautotroph, CO2, light → O2, cells
Figure 2–4  Organisms can be classified according to their source of energy (shaded red) and the form in which they obtain carbon atoms (shaded blue) for the synthesis of cellular material. Organic compounds are both energy source and carbon source for chemoheterotrophs such as ourselves. Some, but not all, chemoheterotrophs consume O2 and produce CO2, and some photoautotrophs produce O2 (shaded green).
modes: chemoheterotrophs, chemoautotrophs, photoheterotrophs, and photoautotrophs (Fig. 2–4).
As shown in Figure 2–5, the earliest cells probably arose about 3.5 billion (3.5 × 109) years ago in the rich mixture of organic compounds, the “primordial soup”, of prebiotic times; they were almost certainly chemoheterotrophs. The organic compounds were originally synthesized from such components of the early earth’s atmosphere as CO, CO2, N2, and CH4 by the nonbiological actions of volcanic heat and lightning (Chapter 3). Primitive heterotrophs gradually acquired the capability to derive energy from certain compounds in their environment and to use that energy to synthesize more and more of their own precursor molecules, thereby becoming less dependent on outside sources of these molecules – less extremely heterotrophic. A very significant evolutionary event was the development of pigments capable of capturing visible light from the sun and using the energy to reduce or “fix” CO2 into more complex organic compounds. The original electron (hydrogen) donor for these photosynthetic organisms was probably H2S, yielding elemental sulfur as the byproduct, but at some point cells developed the enzymatic capacity to use H2O as the electron donor in photosynthetic reactions, producing O2. The cyanobacteria are the modern descendants of these early photosynthetic O2 producers.
The atmosphere of the earth in the earliest stages of biological evolution was nearly devoid of O2, and the earliest cells were therefore anaerobic. With the rise of O2-producing photosynthetic cells, the earth’s atmosphere became progressively richer in O2, allowing the evolution of aerobic organisms, which obtained energy by passing electrons from fuel molecules to O2 (that is, by oxidizing organic compounds). Because electron transfers involving O2 yield energy (they are very exergonic; see Chapter 1), aerobic organisms enjoyed an energetic advantage over their anaerobic counterparts when both competed in an environment containing O2. This advantage translated into the predominance of aerobic organisms in O2-rich environments.
millions of years ago, 0, diversification of large eukaryotes, 500, multicellular eukaryotes, 1,500, eukaryotes, aerobic bacteria, development of O2-rich atmosphere, 2,500, photosynthetic O2-producing cyanobacteria, nonphotosynthetic sulfur bacteria, 3,500, anaerobic photosynthetic sulfur bacteria, anaerobic methanogens, formation of oceans and continents, 4,500, formation of the earth
Modern bacteria inhabit almost every ecological niche in the biosphere, and there are bacterial species capable of using virtually every type of organic compound as a source of carbon and energy. Perhaps three-fourths of all the living matter on the earth consists of microscopic organisms, most of them bacteria.
Bacteria play an important role in the biological exchanges of matter and energy. Photosynthetic bacteria in both fresh and marine waters trap solar energy and use it to generate carbohydrates and other cell materials, which are in turn used as food by other forms of life. Some bacteria can capture molecular nitrogen (N2) from the atmosphere and use it to form biologically useful nitrogenous compounds, a process known as nitrogen fixation. Because animals and most plants cannot do this, bacteria form the starting point of many food chains in the biosphere. They also participate as ultimate consumers, degrading the organic structures of dead plants and animals and recycling the end products to the environment.
Figure 2–5  Landmarks in the evolution of life on earth.
ribosomes, bacterial ribosomes are smaller than eukaryotic ribosomes, but serve the same function – protein synthesis from an RNA message, nucleoid, contains a single, simple, long circular DNA molecule, pili, provide points of adhesion to surface of animal cells, flagella, propel cell through its surroundings, cell envelope, structure varies with different types of bacteria, gram-negative bacteria, outer membrane and peptidoglycan layer, outer membrane, peptidoglycan layer, inner membrane, gram-positive bacteria, thicker peptidoglycan layer, outer membrane absent, peptidoglycan layer, inner membrane, cyanobacteria, type og gram-negative bacteria with tougher peptidoglycan layer and extensive internal membrane system containing photosynthetic pigments, archaebacteria, no peptidoglycan layer
Figure 2–6  Common structural features of bacterial cells. Because of differences in cell envelope structure, some eubacteria (gram-positive bacteria) retain Gram’s stain, and others (gram-negative bacteria) do not. E. coli is gram-negative. Cyanobacteria are also eubacteria, but are distinguished by their extensive internal membrane system, in which photosynthetic pigments are localized.
Bacterial cells share certain common structural features, but also show group-specific specializations (Fig. 2–6). E. coli is a usually harmless inhabitant of the intestinal tract of human beings and many other mammals. The E. coli cell is about 2 μm long and a little less than 1 μm in diameter. It has a protective outer membrane and an inner plasma membrane that encloses the cytoplasm and the nucleoid. Between the inner and outer membranes is a thin but strong layer of peptidoglycans (sugar polymers cross-linked by amino acids), which gives the cell its shape and rigidity. The plasma membrane and the layers outside it constitute the cell envelope. Differences in the cell envelope account for the different affinities for the dye Gentian violet, which is the basis for Gram’s stain; gram-positive bacteria retain the dye, and gram-negative bacteria do not. The outer membrane of E. coli, like that of other gram-negative eubacteria, is similar to the plasma membrane in structure but is different in composition. In gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus, for example) there is no outer membrane, and the peptidoglycan layer surrounding the plasma membrane is much thicker than that in gram-negative bacteria. The plasma membranes of eubacteria consist of a thin bilayer of lipid molecules penetrated by proteins. Archaebacterial membranes have a similar architecture, although their lipids differ from those of the eubacteria.
The plasma membrane contains proteins capable of transporting certain ions and compounds into the cell and carrying products and waste out. Also in the plasma membrane of most eubacteria are electron-carrying proteins (cytochromes) essential in the formation of ATP from ADP (Chapter 1). In the photosynthetic bacteria, internal membranes derived from the plasma membrane contain chlorophyll and other light-trapping pigments.
From the outer membrane of E. coli cells and some other eubacteria protrude short, hairlike structures called pili, by which cells adhere to the surfaces of other cells. Strains of E. coli and other motile bacteria have one or more long flagella, which can propel the bacterium through its aqueous surroundings. Bacterial flagella are thin, rigid, helical rods, 10 to 20 nm thick. They are attached to a protein structure that spins in the plane of the cell surface, rotating the flagellum.
The cytoplasm of E. coli contains about 15,000 ribosomes, thousands of copies of each of several thousand different enzymes, numerous metabolites and cofactors, and a variety of inorganic ions. Under some conditions, granules of polysaccharides or droplets of lipid accumulate. The nucleoid contains a single, circular molecule of DNA. Although the DNA molecule of an E. coli cell is almost 1,000 times longer
than the cell itself, it is packaged with proteins and tightly folded into the nucleoid, which is less than 1 μm in its longest dimension. As in all bacteria, no membrane surrounds the genetic material. In addition to the DNA in the nucleoid, the cytoplasm of most bacteria contains many smaller, circular segments of DNA called plasmids. These nonessential segments of DNA are especially amenable to experimental manipulation and are extremely useful to the molecular geneticist. In nature, some plasmids confer resistance to toxins and antibiotics in the environment.
There is a primitive division of labor within the bacterial cell. The cell envelope regulates the flow of materials into and out of the cell, and protects the cell from noxious environmental agents. The plasma membrane and the cytoplasm contain a variety of enzymes essential to energy metabolism and the synthesis of precursor molecules; the ribosomes manufacture proteins; and the nucleoid stores and transmits genetic information. Most bacteria lead existences that are nearly independent of other cells, but some bacterial species tend to associate in clusters or filaments, and a few (the myxobacteria, for example) demonstrate primitive social behavior. Only eukaryotic cells, however, form true multicellular organisms with a division of labor among cell types.
Fossils older than 1.5 billion years are limited to those from small and relatively simple organisms, similar in size and shape to modern prokaryotes. Starting about 1.5 billion years ago, the fossil record begins to show evidence of larger and more complex organisms, probably the earliest eukaryotic cells (see Fig. 2–5). Details of the evolutionary path from prokaryotes to eukaryotes cannot be deduced from the fossil record alone, but morphological and biochemical comparison of modern organisms has suggested a reasonable sequence of events consistent with the fossil evidence.
membrane-bounded nucleus, plasma membrane, membrane-bounded organelles, ribosomes, protists, fungi, animals, plants, primitive anaerobic eukaryote, mitochondria, chloroplasts, cyanobacteria, heterotrophic anaerobes, other eubacteria, archaebacteria, ancestral prokaryote, nucleoid, ribosomes, plasma membrane, time
Three major changes must have occurred as prokaryotes gave rise to eukaryotes (Fig. 2–7). First, as cells acquired more DNA (Table 2–1), mechanisms evolved to fold it compactly into discrete complexes with specific proteins and to divide it equally between daughter cells at cell division. These DNA-protein complexes, chromosomes, (Greek chroma, “color” and soma, “body”), become especially compact at the time of cell division, when they can be visualized with the light microscope as threads of chromatin. Second, as cells became larger, a system of intracellular membranes developed, including a double membrane surrounding the DNA. This membrane segregated the nuclear process of RNA synthesis using a DNA template from the cytoplasmic process of protein synthesis on ribosomes. Finally, primitive eukaryotic cells, which were incapable of photosynthesis or of aerobic metabolism, pooled their assets with those of aerobic bacteria or photosynthetic bacteria to form symbiotic associations that became permanent. Some aerobic bacteria evolved into the mitochondria of modern eukaryotes, and some photosynthetic cyanobacteria became the chloroplasts of modern plant cells. Prokaryotic and eukaryotic cells are compared in Table 2–2.
Figure 2–7  One view of how modern plants, animals, fungi, protists, and bacteria share a common evolutionary precursor.
Table 2–1  DNA content and genome complexity
Genome size Relative genome size   Length of DNA
                      (nucleotide pairs)   (E. coli = 1) (mm)
Viruses
  SV40 5 × 103 0.00125                 0.0017
  T7 4 × 104 0.01   0.014
  T2 2 × 105 0.05   0.068
Prokaryotes
  Mycoplasma           3 × 105 0.075   0.10
  Bacillus 3 × 106 0.75   1.02
  E. coli 4 × 106 1.00   1.36
Fungi
  Yeast 2 × 107 5   6.8
Animals
  Fruit fly 2 × 108 50   68
  Chicken 2 × 109 500   680
  Human 5 × 109 1,250   1,700
Plants
  Peas 9 × 109 2,250   3,100
  Trillium 1 × 1011                30,000   34,000       

Source: From Becker, W.M. & Deamer, D.W. (1991) The World of the Cell, 2nd edn, p. 363, The
Benjamin/Cummings Publishing Company, Menlo Park, CA.
Table 2–2  Comparison of prokaryotic and eukaryotic cells
Characteristic          Prokaryotic cell                                Eukaryotic cell                                             
Size Generally small (1–10 μm) Generally large (10–100 μm)
Genome DNA with nonhistone protein; DNA complexed with histone and nonhistone
  genome in nucleoid,   proteins in chromosomes; chromosomes in
  not surrounded by membrane   nucleus with membranous envelope
Cell division Fission or budding; no mitosis Mitosis including mitotic spindle; centrioles in
    many
Membrane- Absent Mitochondria, chloroplasts (in plants),
  bounded     endoplasmic reticulum, Golgi complexes,
  organelles     lysosomes, etc.
Nutrition Absorption; some photosynthesis Absorption, ingestion; photosynthesis by some
Energy metabolism No mitochondria; oxydative enzymes Oxydative enzymes packaged in mitochondria;
    bound to plasma membrane; great   more unified pattern of oxidative metabolism
    variation in metabolic pattern
Cytoskeleton None Complex, with microtubules, intermediate
    filaments, actin filaments
Intracellular None Cytoplasmic streaming, endocytosis,
  movement     phagocytosis, mitosis, axonal transport

Source: Modified from Hickman, C.P., Roberts, L.S., & Hickman, F.M. (1990) Biology of Animals, 5th edn, p. 30, Mosby–Yearbook, Inc.
St. Louis, MO.
With the rise of primitive eukaryotic cells, further evolution led to a tremendous diversity of unicellular eukaryotic organisms (protists). Some of these (those with chloroplasts) resembled modern photosynthetic protists such as Euglena and Chlamydomonas; other, nonphotosynthetic protists were more like Paramecium or Dictyostelium. Unicellular eukaryotes are abundant, and the cells of all multicellular animals, plants, and fungi are eukaryotic; there are only a few thousand prokaryotic species, but millions of species of eukaryotic organisms.
Typical eukaryotic cells (Fig. 2–8) are much larger than prokaryotic cells – commonly 10 to 30 μm in diameter, with cell volumes 1,000 to 10,000 times larger than those of bacteria. The distinguishing characteristic of eukaryotes is the nucleus with a complex internal structure, surrounded by a double membrane. The other striking difference between eukaryotes and prokaryotes is that eukaryotes contain a number of other membrane-bounded organelles. The following sections describe the structures and roles of the components of eukaryotic cells in more detail.
Figure 2–8  Schematic illustration of the two types of eukaryotic cell: a representative animal cell (a) and a representative plant cell (b).
ribosomes, peroxisome, cytoskeleton, lysosome, transport vesicle, Golgi complex, centrioles, nuclear envelope, plasma membrane, mitochondrion, rough endoplasmic reticulum, nucleolus, nucleus, smooth endoplasmic reticulum, ribosomes, cytoskeleton, Golgi complex, chloroplast, starch granule, thylakoids, cell wall, cell wall of adjacent cell, vacuole, plasmodesma
The external surface of a cell is in contact with other cells, the extracellular fluid, and the solutes, nutrient molecules, hormones, neurotransmitters, and antigens in that fluid. The plasma membranes of all cells contain a variety of transporters, proteins that span the width of the membrane and carry nutrients into and waste products out of the cell. Cells also have surface membrane proteins (signal receptors) that present highly specific binding sites for extracellular signaling molecules (receptor ligands). When an external ligand binds to its specific receptor, the receptor protein transduces the signal carried by that ligand into an intracellular message (Fig. 2–9). For example, some surface receptors are associated with ion channels that open when the receptor is occupied; others span the membrane and activate or inhibit cellular enzymes on the inner membrane surface. Whatever the mode of signal transduction, surface receptors characteristically act as signal amplifiers – a single ligand molecule bound to a single receptor may cause the flux of thousands of ions through an opened channel, or the synthesis of thousands of molecules of an intracellular messenger molecule by an activated enzyme.
transporter, nutrient → nutrient, signal receptor, ligands, substrate → product, ion channel, ions → ions, (intracellular signals)
Figure 2–9  Proteins in the plasma membrane serve as transporters, signal receptors, and ion channels. Extracellular signals are amplified by receptors, because binding of a single ligand molecule to the surface receptor causes many molecules of an intracellular signal molecule to be formed, or many ions to flow through the opened channel. Transporters carry substances into and out of the cell, but do not act as signal amplifiers.
Some surface receptors recognize ligands of low molecular weight, and others recognize macromolecules. For example, binding of acetylcholine (Mr 146) to its receptor begins a cascade of cellular events that underlie the transmission of signals for muscle contraction. Blood proteins (Mr > 20,000) that carry lipids (lipoproteins) are recognized by specific cell surface receptors and then transported into the cells. Antigens (proteins, viruses, or bacteria, recognized by the immune system as foreign) bind to specific receptors and trigger the production of antibodies. During the development of multicellular organisms, neighboring cells influence each other’s developmental paths, as signal molecules from one cell type react with receptors of other cells. Thus the surface membrane of a cell is a complex mosaic of different kinds of highly specific “molecular antennae” through which cells receive, amplify, and react to external signals.
Most cells of higher plants have a cell wall outside the plasma membrane (Fig. 2–8b), which serves as a rigid, protective shell. The cell wall, composed of cellulose and other carbohydrate polymers, is thick but porous. It allows water and small molecules to pass readily, but swelling of the cell due to the accumulation of water is resisted by the rigidity of the wall.
Endocytosis is a mechanism for transporting components of the surrounding medium deep into the cytoplasm. In this process (Fig. 2–10), a region of the plasma membrane invaginates, enclosing a small volume of extracellular fluid within a bud that pinches off inside the cell by membrane fission. The resulting small vesicle (endosome) can move into the interior of the cell, delivering its contents to another organelle bounded by a single membrane (a lysosome, for example; see p. 34) by fusion of the two membranes. The endosome thus serves as an intracellular extension of the plasma membrane, effectively allowing intimate contact between components of the extracellular medium and regions deep within the cytoplasm, which could not be reached by diffusion alone. Phagocytosis is a special case of endocytosis, in which the material carried into the cell (within a phagosome) is particulate, such as a cell fragment or even another, smaller cell. The inverse of endocytosis is exocytosis (Fig. 2–10), in which a vesicle in the cytoplasm moves to the inside surface of the plasma membrane and fuses with it, releasing the vesicular contents outside the membrane. Many proteins destined for secretion into the extracellular space are released by exocytosis after being packaged into secretory vesicles.
The small transport vesicles moving to and from the plasma membrane in exocytosis and endocytosis are parts of a dynamic system of intracellular membranes (Fig. 2–10), which includes the endoplasmic reticulum, the Golgi complexes, the nuclear envelope, and a variety of small vesicles such as lysosomes and peroxisomes. Although generally represented as discrete and static elements, these structures are in fact in constant flux, with membrane vesicles continually budding from one of the structures and moving to and merging with another.
nucleus, rough endoplasmic reticulum, proteins synthesized for export, transport vesicle, smooth endoplasmic reticulum, Golgi complex, cis side, trans side, secretory vesicles, exocytosis of secretory products, proteins, polysaccharides, etc., endocytosis or phagocytosis of bacteria, debris, etc., phagosome/endosome, lysosome
The endoplasmic reticulum is a highly convoluted, three-dimensional network of membrane-enclosed spaces extending throughout the cytoplasm and enclosing a subcellular compartment (the lumen of the endoplasmic reticulum) separate from the cytoplasm. The many flattened branches (cisternae) of this compartment are continuous with each other and with the nuclear envelope. In cells specialized for the secretion of proteins into the extracellular space, such as the pancreatic cells that secrete the hormone insulin, the endoplasmic reticulum is particularly prominent. The ribosomes that synthesize proteins destined for export attach to the outer (cytoplasmic) surface of the endoplasmic reticulum, and the secretory proteins are passed through the membrane into the lumen as they are synthesized. Proteins destined for sequestration within lysosomes, or for insertion into the nuclear or plasma membranes, are also synthesized on ribosomes attached to the endoplasmic reticulum. By contrast, proteins that will remain and function within the cytosol are synthesized on cytoplasmic ribosomes unassociated with the endoplasmic reticulum.
The attachment of thousands of ribosomes (usually in regions of large cisternae) gives the rough endoplasmic reticulum its granular appearance (Fig. 2–10) and thus its name. In other regions of the cell, the endoplasmic reticulum is free of ribosomes. This smooth endoplasmic reticulum, which is physically continuous with the rough
endoplasmic reticulum, is the site of lipid biosynthesis and of a variety of other important processes, including the metabolism of certain drugs and toxic compounds. Smooth endoplasmic reticulum is generally tubular, in contrast to the long, flattened cisternae typical of rough endoplasmic reticulum. In some tissues (skeletal muscle, for example) the endoplasmic reticulum is specialized for the storage and rapid release of calcium ions. Ca2+ release is the trigger for many cellular events, including muscle contraction.
Figure 2–10  The endomembrane system includes the nuclear envelope, endoplasmic reticulum, Golgi complex, and several types of small vesicles. This system encloses a compartment (the lumen) distinct from the cytosol. Contents of the lumen move from one region of the endomembrane system to another as small transport vesicles bud from one component and fuse with another. High-magnification electron micrographs of a sectioned cell show rough endoplasmic reticulum, studded with ribosomes, smooth endoplasmic reticulum, and the Golgi complex.
     The endomembrane system is dynamic; newly synthesized proteins move into the lumen of the rough endoplasmic reticulum and thus to the smooth endoplasmic reticulum, then to the Golgi complex via transport vesicles. In the Golgi complex, molecular “addresses” are added to specific proteins to direct them to the cell surface, lysosomes, or secretory vesicles. The contents of secretory vesicles are released from the cell by exocytosis. Endocytosis and phagocytosis bring extracellular materials into the cell. Fusion of endosomes (or phagosomes) with lysosomes, which are full of digestive enzymes, results in the degradation of the extracellular materials.
Nearly all eukaryotic cells have characteristic clusters of membrane vesicles called dictyosomes. Several connected dictyosomes constitute a Golgi complex. A Golgi complex (also called Golgi apparatus) is most commonly seen as a stack of flattened membrane vesicles (cisternae) (Fig. 2–10). Near the ends of these cisternae are numerous, much smaller, spherical vesicles (transport vesicles) that bud off the edges of the cisternae.
The Golgi complex is asymmetric, structurally and functionally. The cis side faces the rough endoplasmic reticulum, and the trans side, the plasma membrane; between these are the medial elements. Proteins, during their synthesis on ribosomes bound to the rough endoplasmic reticulum, are inserted into the interior (lumen) of the cisternae. Small membrane vesicles containing the newly synthesized proteins bud from the endoplasmic reticulum and move to the Golgi complex, fusing with the cis side. As the proteins pass through the Golgi complex to the trans side, enzymes in the complex modify the protein molecules by adding sulfate, carbohydrate, or lipid moieties to side chains of certain amino acids. One of the functions of this modification of a newly synthesized protein is to “address” it to its proper destination as it leaves the Golgi complex in a transport vesicle budding from the trans side. Certain proteins are enclosed in secretory vesicles, eventually to be released from the cell by exocytosis. Others are targeted for intracellular organelles such as lysosomes, or for incorporation into the plasma membrane during cell growth.
Lysosomes, found in the cytoplasm of animal cells, are spherical vesicles bounded by a single membrane. They are usually about 1 μm in diameter, about the size of a small bacterium (Fig. 2–10). Lysosomes contain enzymes capable of digesting proteins, polysaccharides, nucleic acids, and lipids. They function as cellular recycling centers for complex molecules brought into the cell by endocytosis, fragments of foreign cells brought in by phagocytosis, or worn-out organelles from the cell’s own cytoplasm. These materials selectively enter the lysosomes by fusion of the lysosomal membrane with endosomes, phagosomes, or defective organelles, and are then degraded to their simple components (amino acids, monosaccharides, fatty acids, etc.), which are released into the cytosol to be recycled into new cellular components or further catabolized.
The degradative enzymes within lysosomes would be harmful if not confined by the lysosomal membrane; they would be free to act on all cellular components. The lysosomal compartment is more acidic (pH ≤ 5) than the cytoplasm (pH ≈ 7); the acidity is due to the action of an ATP-fueled proton pump in the lysosomal membrane. Lysosomal enzymes are much less active at pH 7 than at pH ≤ 5, which provides a second line of defense against destruction of cytosolic macromolecules, should these enzymes escape into the cytosol.
Plant cells do not have organelles identical to lysosomes, but their vacuoles carry out similar degradative reactions as well as other functions
cell wall, mitochondrion, vacuole, H2O, turgor pressure, cytosol, tonoplast (vacuole membrane), chloroplast
Figure 2–11  The vacuole of a plant cell contains high concentrations of a variety of stored compounds and waste products. Water enters the vacuole by osmosis and increases the vacuolar volume. The resulting turgor pressure forces the cytoplasm out against the cell wall. The rigidity of the cell wall prevents expansion and rupture of the plasma membrane.
not found in animal cells. Growing plant cells contain several small vacuoles, vesicles bounded by a single membrane, which fuse and become one large vacuole in the center of the mature cell (Fig. 2–11; see also Fig. 2–8b). The surrounding membrane, the tonoplast, regulates the entry into the vacuole of ions, metabolites, and cellular structures destined for degradation. In the mature cell, the vacuole may represent as much as 90% of the total cell volume, pressing the cytoplasm into a thin layer between the tonoplast and the plasma membrane. The liquid within the vacuole, the cell sap, contains digestive enzymes that degrade and recycle macromolecular components no longer useful to the cell. In some plant cells, the vacuole contains high concentrations of pigments (anthocyanins) that give the deep purple and red colors to the flowers of roses and geraniums and the fruits of grapes and plums. Like the contents of lysosomes, the cell sap is generally more acidic than the surrounding cytosol. In addition to its role in storage and degradation of cellular components, the vacuole also provides physical support to the plant cell. Water passes into the vacuole by osmosis because of the high solute concentration of the cell sap, creating outward pressure on the cytosol and the cell wall. This turgor pressure within cells stiffens the plant tissue (Fig. 2–11).
Some of the oxidative reactions in the breakdown of amino acids and fats produce free radicals and hydrogen peroxide (H2O2), very reactive chemical species that could damage cellular machinery. To protect the cell from these destructive byproducts, such reactions are segregated within small membrane-bounded vesicles called peroxisomes. The hydrogen peroxide is degraded by catalase, an enzyme present in large quantities in peroxisomes and glyoxysomes; it catalyzes the reaction 2H2O2 → 2H2O + O2.
Glyoxysomes are specialized peroxisomes found in certain plan cells. They contain high concentrations of the enzymes of the glyoxylate cycle, a metabolic pathway unique to plants that allows the conversion of stored fats into carbohydrates during seed germination. Lysosomes, peroxisomes, and glyoxysomes are sometimes referred to collectively as microbodies.
nucleolus – transcription of ribosomal RNA, chromatin – tight complex of DNA and histone proteins, nuclear pores – specific transport of RNA and proteins, paired membranes of nuclear envelope, rough endoplasmic reticulum, ribosomes
The eukaryotic nucleus is very complex in both its structure and its biological activity, compared with the relatively simple nucleoid of prokaryotes. The nucleus contains nearly all of the cell’s DNA, typically 1,000 times more than is present in a bacterial cell; a small amount of DNA is also present in mitochondria and chloroplasts. The nucleus is surrounded by a nuclear envelope, composed of two membranes separated by a narrow space and continuous with the rough endoplasmic reticulum (Fig. 2–12; see also Fig. 2–10). At intervals the two nuclear membranes are pinched together around openings (nuclear pores), which have a diameter of about 90 nm. Associated with the pores are protein structures (nuclear pore complexes), specific macromolecule transporters that allow only certain molecules to pass between the cytoplasm and the aqueous phase of the nucleus (the nucleoplasm), such as enzymes synthesized in the cytoplasm and required in the nucleoplasm for DNA replication, transcription, or repair. Messenger RNA precursors and associated proteins also pass out of the nucleus through the nuclear pore complexes, to be translated on ribosomes in the cytoplasm; the nucleoplasm contains no ribosomes.
Figure 2–12  The nucleus and nuclear envelope. (a) Scanning electron micrograph of the surface of the nuclear envelope, showing numerous nuclear pores. (b) Electron micrograph of the nucleus of the alga Chlamydomonas. The dark body in the center of the nucleus is the nucleolus, and the granular material that fills the rest of the nucleus is chromatin. The nuclear envelope has paired membranes with nuclear pores; two are shown by arrows.
mitotic chromosome, chromatid (≈600 nm in diameter), chromatin fiber (30 nm in diameter), nucleosomes (10 nm in diameter), histones, DNA
Inside the nucleus is the nucleolus, which appears dense in electron micrographs (Fig. 2–12b) because of its high content of RNA. The nucleolus is a specific region of the nucleus, in which the DNA contains many copies of the genes encoding ribosomal RNA. To produce the large number of ribosomes needed by the cell, these genes are continually copied into RNA (transcribed). The nucleolus is the visible evidence of the transcriptional machinery and the RNA product. Ribosomal RNA produced in the nucleolus passes into the cytoplasm through the nuclear pores. The rest of the nucleus contains chromatin, so called because early microscopists found that it stained brightly with certain dyes. Chromatin consists of DNA and proteins bound tightly to the DNA, and represents the chromosomes, which are decondensed in the interphase (nondividing) nucleus and not individually visible.
Before division of the cell (cytokinesis), nuclear division (mitosis) occurs. The chromatin condenses into discrete bodies, the chromosomes (Fig. 2–13). Cells of each species have a characteristic number of chromosomes with specific sizes and shapes. The protist Tetrahymena has 4; cabbage has 20, humans have 46, and the plant Ophioglossum, about 1,250! Usually each cell has two copies of each chromosome; such cells are called diploid. Gametes (egg and sperm, for example) produced by meiosis (Chapter 24) have only one copy of each chromosome and are called haploid. During sexual reproduction, two haploid gametes combine to regenerate a diploid cell in which each chromosome pair consists of a maternal and a paternal chromosome.
Chromosomes and chromatin are composed of DNA and a family of positively charged proteins, histones, which associate strongly with DNA by ionic interactions with its many negatively charged phosphate groups. About half of the mass of chromatin is DNA and half is histones. When DNA replicates prior to cell division, large quantities of histones are also synthesized to maintain this 1:1 ratio. The histones and DNA associate in complexes called nucleosomes, in which the DNA strand winds around a core of histone molecules (Fig. 2–13). The DNA of a single human chromosome forms about a million nucleosomes; nucleosomes associate to form very regular and compact supramolecular complexes. The resulting chromatin fibers, about 30 nm in diameter, condense further by forming a series of looped regions, which cluster with adjacent looped regions to form the chromosomes visible during cell division. This tight packing of DNA into nucleosomes achieves a remarkable condensation of the DNA molecules. The DNA in the chromosomes of a single diploid human cell would have a combined length of about 2 m if fully stretched as a DNA double helix, but the combined length of all 46 chromosomes is only about 200 nm.
Figure 2–13  Chromosomes are visible in the electron microscope during mitosis. Shown here is one of the 46 human chromosomes. Every chromosome is composed of two chromatids, each consisting of tightly folded chromatin fibers. Each chromatin fiber is in turn formed by the packaging of a DNA molecule wrapped about histone proteins to form a series of nucleosomes.  (Adapted from Becker, W.M. & Deamer, D.W. (1991) The World of the Cell, 2nd edn, Fig. 13–20, The Benjamin/Cummings Publishing Company, Menlo Park, CA.)
→ late interphase, centrioles, nuclear envelope → early prophase, plasma membrane → late prophase, mitotic spindle, sister chromatids, nuclear envelope fragmenting, centrosome (pair of centrioles) → metaphase, paired chromatids, spindle fibers attached to daughter chromosomes at centromeres → anaphase, nuclear envelope reforming → telophase → early interphase
Before the beginning of mitosis, each chromosome is duplicated to form paired, identical chromatids, each of which is a double helix of DNA. During mitosis (Fig. 2–14), the two chromatids move to opposite ends (poles) of the cell, each becoming a new chromosome. Small cylindrical particles called centrioles, composed of the protein tubulin, provide the spatial organization for the migration of chromatids to opposite ends of the dividing cell. To allow the separation of chromatids, the nuclear envelope breaks down, dispersing into membrane vesicles. When the separation of the two sets of chromosomes is complete, a nuclear envelope derived from the endoplasmic reticulum re-forms around each set. Finally, the two halves of the cell are separated by cytokinesis, and each daughter cell has a complete diploid complement of chromosomes. After mitosis is complete the chromosomes decondense to form dispersed chromatin, and the nucleoli, which disappeared early in mitosis, reappear.
Figure 2–14  Mitosis and cell division in animal cells. In the interphase (nondividing) nucleus (a), the chromosomes are in the form of dispersed chromatin. As mitosis begins (b), chromatin condenses into chromosomes and the mitotic spindle begins to form; centrosomes, which typically contain centriole pairs, dictate the orientation of the spindle. The nuclear envelope disintegrates and the nucleolus disappears (c), and the chromosomes align at the center of the cell (d). The chromatids of each chromosome move to opposite poles of the cell, pulled by spindle fibers attached to their centromeres (e), and a nuclear envelope forms around each new set of chromosomes (f). Finally, two daughter cells form by cell division (cytokinesis) (g). Although the same basic process occurs in all eukaryotes, there are differences in details of mitosis in plants, fungi, and protists.
Mitochondria (singular, mitochondrion) are very conspicuous in the cytoplasm of most eukaryotic cells (Fig. 2–15). These membrane-bounded organelles vary in size, but typically have a diameter of about 1 μm, similar to that of bacterial cells. Mitochondria also vary widely in shape, number, and location, depending on the cell type or tissue function. Most plant and animal cells contain several hundred to a thousand mitochondria. Generally, cells in more metabolically active tissues devote a larger proportion of their volume to mitochondria.
DNA, crista, matrix, ribosomes, inner membrane, outer membrane
Each mitochondrion has two membranes. The outer membrane is unwrinkled and completely surrounds the organelle. The inner membrane has infoldings called cristae, which give it a large surface area. The inner compartment of mitochondria, the matrix, is a very concentrated aqueous solution of many enzymes and chemical intermediates involved in energy-yielding metabolism. Mitochondria contain many enzymes that together catalyze the oxidation of organic nutrients by molecular oxygen (O2); some of these enzymes are in the matrix and some are embedded in the inner membrane. The chemical energy released in mitochondrial oxidations is used to generate ATP, the major energy-carrying molecule of cells. In aerobic cells, mitochondria are the
principal producers of ATP, which diffuses to all parts of the cell and provides the energy for cellular work.
Unlike other membranous structures such as lysosomes, Golgi complexes, and the nuclear envelope, mitochondria are produced only by division of previously existing mitochondria; each mitochondrion contains its own DNA, RNA, and ribosomes. Mitochondrial DNA codes for certain proteins specific to the mitochondrial inner membrane, but other mitochondrial proteins are encoded in nuclear DNA. This and other evidence supports the theory that mitochondria are the descendants of aerobic bacteria that lived symbiotically with early eukaryotic cells.
Figure 2–15  Structure of a mitochondrion. This electron micrograph of a mitochondrion shows the smooth outer membrane and the numerous infoldings of the inner membrane, called cristae. (Note the extensive rough endoplasmic reticulum surrounding the mitochondrion.)
outer membrane, inner membrane, DNA, ribosomes, thylakoids
Plastids are specialized organelles in the cytoplasm of plants; they have two surrounding membranes. Most conspicuous of the plastids and characteristically present in all green plant cells and eukaryotic algae are the chloroplasts (Fig. 2–16). Like mitochondria, the chloroplasts may be considered power plants, with the important difference that chloroplasts use solar energy, whereas mitochondria use the chemical energy of oxidizable molecules. Pigment molecules in chloroplasts absorb the energy of light and use it to make ATP and, ultimately, to reduce carbon dioxide to form carbohydrates such as starch and sucrose. The photosynthetic process in eukaryotes and in cyanobacteria produces O2 as a byproduct of the light-capturing reactions. Photosynthetic plant cells contain both chloroplasts and mitochondria. Chloroplasts transduce energy only in the light, but mitochondria function independently of light, oxidizing carbohydrates generated by photosynthesis during daylight hours.
Figure 2–16  A chloroplast in a photosynthetic cell. The thylakoids are flattened membranous sacs that contain chlorophyll, the light-harvesting pigment.
Chloroplasts are generally larger (diameter 5 μm) than mitochondria and occur in many different shapes. Because chloroplasts contain a high concentration of the pigment chlorophyll, photosynthetic cells are usually green, but their color depends on the relative amounts of other pigments present. These pigment molecules, which together can absorb light energy over much of the visible spectrum, are localized in the internal membranes of the chloroplast, which form stacks of closed cisternae known as thylakoids (Fig. 2–16). Like mitochondria, chloroplasts contain DNA, RNA, and ribosomes. Chloroplasts appear to have had their evolutionary origin in symbiotic ancestors of the cyanobacteria.
ancestral anaerobe, anaerobic metabolism is inefficient because fuel is not completely oxidized, aerobic bacterium, aerobic metabolism is efficient because fuel is oxidized to CO2 → host cell with aerobic endosymbionts, the endo symbiont and the host cell share materials, to the advantage of both, cyanobacterium, uses the energy of light to synthesize cellular sytructures and fuels from CO2 and H2O → modern photosynthetic eukryotic cell, nucleus, chloroplast, mitochondrion, symbiosis allows further specialization of photosynthetic membranes, cell oxidizes fuel efficiently and can obtain energy from sunlight
Figure 2–17  A plausible theory for the evolutionary origin of mitochondria and chloroplasts. It is based on a number of striking biochemical and genetic similarities between certain aerobic bacteria and mitochondria, and between certain cyanobacteria and chloroplasts. During the evolution of eukaryotic cells, the invading bacteria became symbiotic with the host cell. Ultimately the cytoplasmic bacteria became the mitochondria and chloroplasts of modern cells.
Several independent lines of evidence suggest that the mitochondria and chloroplasts of modern eukaryotes were derived during evolution from aerobic bacteria and cyanobacteria that took up endosymbiotic residence in early eukaryotic cells (Fig. 2–17; see also Fig. 2–7). Mitochondria are always derived from preexisting mitochondria, and chloroplasts from chloroplasts, by simple fission, just as bacteria multiply by fission. Mitochondria and chloroplasts are in fact semiautonomous; they contain DNA, ribosomes, and the enzymatic machinery to synthesize proteins encoded in their DNA. Sequences in mitochondrial DNA are strikingly similar to sequences in certain aerobic bacteria, and chloroplast DNA shows strong sequence homology with the DNA of certain cyanobacteria. The ribosomes found in mitochondria and chloroplasts are more similar in size, overall structure, and ribosomal RNA sequences to those of bacteria than to those in the cytoplasm of the eukaryotic cell. The enzymes that catalyze protein synthesis in these organelles also resemble those of the bacteria more closely.
If mitochondria and chloroplasts are the descendants of early bacterial endosymbionts, some of the genes present in the original freeliving bacteria must have been transferred into the nuclear DNA of the host eukaryote over the course of evolution. Neither mitochondria nor chloroplasts contain all of the genes necessary to specify all of their proteins. Most of the proteins of both organelles are encoded in nuclear genes, translated on cytoplasmic ribosomes, and subsequently imported into the organelles.
Several types of protein filaments visible with the electron microscope crisscross the eukaryotic cell, forming an interlocking three-dimensional meshwork throughout the cytoplasm, the cytoskeleton. There are three general types of cytoplasmic filaments: actin filaments, microtubules, and intermediate filaments (Fig. 2–18). They differ in width (from about 6 to 22 nm), composition, and specific function, but all apparently provide structure and organization to the cytoplasm and shape to the cell. Actin filaments and microtubules also help to produce the motion of organelles or of the whole cell.
Each of the cytoskeletal components is composed of simple protein subunits that polymerize to form filaments of uniform thickness. These filaments are not permanent structures; they undergo constant disassembly into their monomeric subunits and reassembly into filaments. Their locations in cells are not rigidly fixed, but may change dramatically with mitosis, cytokinesis, or changes in cell shape. All types of filaments associate with other proteins that cross-link filaments to themselves or to other filaments, influence assembly or disassembly, or move cytoplasmic organelles along the filaments.
Figure 2–18  The three types of cytoplasmic filaments. The upper panels show epithelial cells photographed after treatment with antibodies that bind to and specifically stain (a) actin filaments bundled together to form “stress fibers”, (b) microtubules radiating from the cell center, and (c) intermediate filaments, extending throughout the cytoplasm. For these experiments, antibodies that specifically recognize actin, tubulin, or intermediate filament proteins are covalently attached to a fluorescent compound. When the cell is viewed with a fluorescence microscope, only the stained structures are visible. The lower panels show each type of filament as visualized by electron microscopy.
Actin is a protein present in virtually all eukaryotes, from the protists to the vertebrates. In the presence of ATP, the monomeric protein spontaneously associates into linear, helical polymers, 6 to 7 nm in diameter, called actin filaments or microfilaments (Fig. 2–19).
actin subunits, ATP → actin (thin) filaments, 6–7 nm, + fodrin, + filamin
Figure 2–19  Individual subunits of actin polymerize to form actin filaments. The protein filamin holds two filaments together where they cross at right angles. The filaments are cross-linked by another protein, fodrin, to form side-by-side aggregates or bundles.
The importance of actin polymerization and depolymerization is clear from the effects of cytochalasins, compounds that bind to actin and block polymerization. Cells treated with a cytochalasin lose actin filaments and their ability to carry out cytokinesis, phagocytosis, and amoeboid movement. However, chromatid separation at mitosis is not affected, ruling out an essential role for actin in this process. Compounds such as cytochalasins, which are naturally occurring poisons or specific toxins, are often very helpful in experimental studies in pinpointing the important participants in a biological process.
Cells contain proteins that bind to actin monomers or filaments and influence the state of actin aggregation (Fig. 2–19). Filamin and fodrin cross-link actin filaments to each other, stabilizing the meshwork and greatly increasing the viscosity of the medium in which the filaments are suspended; a concentrated solution of actin in the presence of filamin is a gel too viscous to pour. Large numbers of actin filaments bound to specific plasma membrane proteins lie just beneath and more or less parallel to the plasma membrane, conferring shape and rigidity on the cell surface.
Actin filaments bind to a family of proteins called myosins, enzymes that use the energy of ATP breakdown to move themselves along the actin filament in one direction. The simplest members of this family, such as myosin I, have a globular head and a short tail (Fig. 2–20). The
actin filament, myosin, ATP → ADP + PO43−, cytoplasmic vesicle or organelle, actin filaments, streaming cytoplasm with organelles and vesicles, vacuole, chloroplasts in stationary cytoplasm
head binds to and moves along an actin filament, driven by the breakdown of ATP. The tail region binds to the membrane of a cytoplasmic organelle, dragging the organelle behind as the myosin head moves along the actin filament. It appears likely that myosins of this type bind to various organelles, providing specific transport systems to move each type of organelle through the cytoplasm. This motion is readily seen in living cells such as the giant green alga Nitella; endoplasmic reticulum, as well as mitochondria, nucleus, and other membrane-bound organelles and vesicles, move uniformly around the cell at 50 to 75 μm/s in a process called cytoplasmic streaming (Fig. 2–20). This motion has the effect of mixing the cytoplasmic contents of the enormous algal cell much more efficiently than would occur by diffusion alone.
Figure 2–20  Myosin molecules move along actin filaments using energy from ATP. Cytoplasmic streaming is produced in the giant green alga Nitella as myosin pulls organelles around a track of actin filaments. The chloroplasts of Nitella are located in the layer of stationary cytoplasm that lies between the actin filaments and the cell membrane.
A larger form of myosin is found in muscle cells, and also in the cytoplasm of many nonmuscle cells. This type of myosin also has a globular head that binds to and moves along actin filaments in an ATP-driven reaction, but it has a longer tail, which permits myosin molecules to associate side by side to form thick filaments (see Fig. 7–31). Contractile systems composed of actin and myosin occur in a wide variety of organisms, from slime molds to humans. Actin–myosin complexes form the contractile ring that squeezes the cytoplasm in two during cytokinesis in all eukaryotes. In multicellular animals, muscle cells are filled with highly organized arrays of actin (thin) filaments and myosin (thick) filaments, which produce a coordinated contractile force by ATP-driven sliding of actin filaments past stationary myosin filaments.
Like actin filaments, microtubules form spontaneously from their monomeric subunits, but the polymeric structure of microtubules is slightly more complex. Dimers of α- and β-tubulin form linear polymers (protofilaments), 13 of which associate side by side to form the hollow microtubule, about 22 nm in diameter (Fig. 2–21). Most microtubules undergo continual polymerization and depolymerization in cells by addition of tubulin subunits primarily at one end and dissociation at the other. Microtubules are present throughout the cytoplasm, but are concentrated in specific regions at certain times. For example, when sister chromatids move to opposite poles of a dividing cell during mitosis, a highly organized array of microtubules (the mitotic spindle; Fig. 2–14) provides the framework and probably the motive force for the separation of chromatids. Colchicine, a poisonous alkaloid from meadow saffron, prevents tubulin polymerization. Colchicine treatment reversibly blocks the movement of chromatids during mitosis, demonstrating that microtubules are required for this process.
Figure 2–21  Microtubules are formed from dimers of the proteins α- and β-tubulin. Colchicine blocks the assembly of microtubules, and can be used to arrest mitosis in cells.
tubulin subunits, β subunit, α subunit ⇌ α,β-tubulin dimers ⇌ microtubule, tubulin, α, β, 8 nm, 22 nm, colchicine blocks polymerization
cytoplasmic vesicle or organelle, kinesin, ATP → ADP + PO43−, dynein, ATP → ADP + PO43−, microtubule
Microtubules, like actin filaments, associate with a variety of proteins that move along them, form cross-bridges, or influence their state of polymerization. Kinesin and cytoplasmic dynein, proteins found in the cytoplasm of many cells, bind to microtubules and move along them using the energy of ATP to drive their motion (Fig. 2–22). Each protein is capable of associating with specific organelles and pulling them along the microtubule over long distances at rates of about 1μm/s. The beating motion of cilia and eukaryotic flagella also involves dynein and microtubules.
Figure 2–22  Kinesin and dynein are ATP-driven molecular engines that move along microtubular “rails”.
cilium, plasma membrane, microtubule doublet, microtubule doublet, radial spoke, dynein arms, 0.1 μm
Cilia and flagella, motile structures extending from the surface of many protists and certain cells of animals and plants, are all constructed on the same microtubule-based architectural plan (Fig. 2–23). (Although they bear the same name, the flagella of bacteria (p. 28) are completely different in structure and in action from the flagella of eukaryotes.) Eukaryotic cilia and flagella, which are sheathed in an extension of the plasma membrane, contain nine fused pairs of microtubules arranged around two central microtubules (the 9 + 2 arrangement; Fig. 2–23). Ciliary and flagellar motion results from the coordinated sliding of outer doublet microtubules relative to their neighbors, driven by ATP. The motions of cilia and flagella propel protists through their surrounding medium, in search of food, or light, or some condition essential to their survival. Sperm are also propelled by flagellar beating. Ciliated cells in tissues such as the trachea and oviduct move extracellular fluids past the surface of the ciliated tissue.
The contraction of skeletal muscle, the propelling action of cilia and flagella, and the intracellular transport of organelles all rely on the same fundamental mechanism: the splitting of ATP by proteins such as kinesin, myosin, and dynein drives sliding motion along microfilaments or microtubules.
Figure 2–23  Cilia and eukaryotic flagella have the same architecture: nine microtubule doublets surround a central pair of microtubules. Cross section of cilia shows the 9 + 2 arrangement of microtubules.
The third type of cytoplasmic filament is a family of structures with dimensions (diameter 8 to 10 nm) intermediate between actin filaments and microtubules. Several different types of monomeric protein subunits form intermediate filaments. Some cells contain large amounts of one type; some types of intermediate filament are absent from certain cells; and some cell types apparently lack intermediate filaments altogether. As is the case for actin filaments and microtubules, intermediate filament formation is reversible, and the cytoplasmic distribution of these structures is subject to regulated changes.
The function of intermediate filaments is probably to provide internal mechanical support for the cell and to position its organelles. Vimentin (Mr 57,000) is the monomeric subunit of the intermediate filaments found in the endothelial cells that line blood vessels, and in adipocytes (fat cells). Vimentin fibers appear to anchor the nucleus and fat droplets in specific cellular locations. Intermediate filaments composed of desmin (Mr 55,000) hold the Z disks of striated muscle tissue in place. Neurofilaments are constructed of three different protein subunits (Mr 70,000, 150,000, and 210,000), and provide rigidity to the long extensions (axons) of neurons. In the glial cells that surround neurons, intermediate filaments are constructed from glial fibrillary acidic protein (Mr 50,000).
The intermediate filaments composed of keratins, a family of structural proteins, are particularly prominent in certain epidermal cells of vertebrates, and form covalently cross-linked meshworks that persist even after the cell dies. Hair, fingernails, and feathers are among the structures composed primarily of keratins.
The picture that emerges from this brief survey is of a eukaryotic cell with a cytoplasm crisscrossed by a meshwork of structural fibers, throughout which extends a complex system of membrane-bounded compartments (see Fig. 2–8). Both the filaments and the organelles are dynamic: the filaments disassemble and reassemble elsewhere; membranous vesicles bud from one organelle, move to and join another. Transport vesicles, mitochondria, chloroplasts, and other organelles move through the cytoplasm along protein filaments, drawn by kinesin, cytoplasmic dynein, myosin, and perhaps other similar proteins. Exocytosis and endocytosis provide paths between the cell interior and the surrounding medium, allowing for the secretion of proteins and other components produced within the cell and the uptake of extracellular components. The intracellular membrane systems segregate specific metabolic processes, and provide surfaces on which certain enzyme-catalyzed reactions occur.
Although complex, this organization of the cytoplasm is far from random. The motion and positioning of organelles and cytoskeletal elements are under tight regulation, and at certain stages in a eukaryotic cell’s life, dramatic, finely orchestrated reorganizations occur, such as spindle formation, chromatid migration to the poles, and nuclear envelope disintegration and re-formation during mitosis. The interactions between the cytoskeleton and organelles are noncovalent, reversible, and subject to regulation in response to various intracellular and extracellular signals. Cytoskeletal rearrangements are modulated by Ca2+ and by a variety of proteins.
A major advance in the biochemical study of cells was the development of methods for separating organelles from the cytosol and from each other. In a typical cellular fractionation, cells or tissues are disrupted by gentle homogenization in a medium containing sucrose (about 0.2 M). This treatment ruptures the plasma membrane but leaves most of the organelles intact. (The sucrose creates a medium with an osmotic pressure similar to that within organelles; this prevents diffusion of water into the organelles, which would cause them to swell, burst, and spill their contents.)
Organelles such as nuclei, mitochondria, and lysosomes differ in size and therefore sediment at different rates during centrifugation. They also differ in specific gravity, and they “float” at different levels in a density gradient (Fig. 2–24). Differential centrifugation results in a rough fractionation of the cytoplasmic contents, which may be further purified by isopycnic centrifugation. In this procedure, organelles of different buoyant densities (the result of different ratios of lipid and protein in each type of organelle) are separated on a density gradient. By carefully removing material from each region of the gradient and observing it with a microscope, the biochemist can establish the position of each organelle and obtain purified organelles for further study. In this way it was established, for example, that lysosomes contain degradative enzymes, mitochondria contain oxidative enzymes, and chloroplasts contain photosynthetic pigments. The isolation of an organelle enriched in a certain enzyme is often the first step in the purification of that enzyme.
Figure 2–24  A tissue such as liver is mechanically homogenized to break cells and disperse their contents in an aqueous buffer. The large and small particles in this suspension can be separated by centrifugation at different speeds (a), or particles of different density can be separated by isopycnic centrifugation (b). In isopycnic centrifugation, a centrifuge tube is filled with a solution, the density of which increases from top to bottom; some solute such as sucrose is dissolved at different concentrations to produce this density gradient. When a mixture of organelles is layered on top of the density gradient and the tube is centrifuged at high speed, individual organelles sediment until their buoyant density exactly matches that in the gradient. Each layer can be collected separately.
differential centrifugation, tissue homogenization, tissue homogenate, low-speed centrifugation (1,000 g, 10 min) → pellet contains whole cells, nuclei, cytoskeletons, plasma membranes, supernatant subjected to medium-speed centrifugation (20,000 g, 20 min) → pellet contains mitochondria, lysosomes, peroxisomes, supernatant subjected to high-speed centrifugation (80,000 g, 1 h) → pellet contains microsomes, small vesicles, supernatant subjected to very high-speed centrifugation (150,000 g, 3 h) → pellet contains ribosomes, viruses, large macromolecules, supernatant contains soluble proteins; isopycnic (sucrose-density) centrifugation, sample, centrifugation → sucrose gradient, less dense component, more dense component, fractionation
One of the most effective approaches to understanding a biological process is to study purified individual molecules such as enzymes, nucleic acids, or structural proteins. The purified components are amenable to detailed characterization in vitro; their physical properties and catalytic activities can be studied without “interference” from other molecules present in the intact cell. Although this approach has been remarkably revealing, it must always be remembered that the inside of a
cell is quite different from the inside of a test tube. The “interfering” components eliminated by purification may be critical to the biological function or regulation of the molecule purified. In vitro studies of pure enzymes are commonly done at very low enzyme concentrations in thoroughly stirred aqueous solutions. In the cell, an enzyme is dissolved or suspended in a gel-like cytosol with thousands of other proteins, some of which bind to that enzyme and influence its activity. Within cells, some enzymes are parts of multienzyme complexes in which reactants are channeled from one enzyme to another without ever entering the bulk solvent. Diffusion is hindered in the gel-like cytosol, and the cytosolic composition varies in different regions of the cell. In short, a given molecule may function somewhat differently within the cell than it does in vitro. One of the central challenges of biochemistry is to understand the influences of cellular organization and macromolecular associations on the function of individual enzymes – to understand function in vivo as well as in vitro.
All modern unicellular eukaryotes – the protists – contain the organelles and mechanisms that we have described, indicating that these organelles and mechanisms must have evolved relatively early. The protists are extraordinarily versatile. The ciliated protist Paramecium, for example, moves rapidly through its aqueous surroundings by beating its cilia; senses mechanical, chemical, and thermal stimuli from its environment, and responds by changing its path; finds, engulfs, and digests a variety of food organisms, and excretes the indigestible fragments; eliminates excess water that leaks through its membrane; and finds and mates with sexual partners. Nonetheless, being unicellular has its disadvantages. Paramecia probably live out their lives in a very small region of the pond in which they began life, because their motility is limited by the small thrust of their microscopic cilia, and their ability to detect a better environment at a distance is limited by the short range of their sensory apparatus.
Figure 2–25  A gallery of differentiated cells. (a) Secretory cells of the pancreas, with an extensive endoplasmic reticulum. (b) Portion of a skeletal muscle cell, with organized actin and myosin filaments. (c) Collenchyma cells of a plant stem. (d) Rabbit sperm cells, with long flagella for motility. (e) Human erythrocyte. (f) Human embryo at the two-celled stage.
At some later stage of evolution, unicellular organisms found it advantageous to cluster together, thereby acquiring greater motility, efficiency, or reproductive success than their free-living single-celled competitors. Further evolution of such clustered organisms led to permanent associations among individual cells and eventually to specialization within the colony – to cellular differentiation.

The advantages of cellular specialization led to the evolution of ever more complex and highly differentiated organisms, in which some cells carried out the sensory functions, others the digestive, photosynthetic, or reproductive functions. Many modern multicellular organisms contain hundreds of different cell types, each specialized for some function that supports the entire organism. Fundamental mechanisms that evolved early have been further refined and embellished through evolution. The simple mechanism responsible for the motion of myosin along actin filaments in slime molds has been conserved and elaborated in vertebrate muscle cells, which are literally filled with actin, myosin, and associated proteins that regulate muscle contraction. The same basic structure and mechanism that underlie the beating motion of cilia in Paramecium and flagella in Chlamydomonas are employed by the highly differentiated vertebrate sperm cell. Figure 2–25 illustrates the range of cellular specializations encountered in multicellular organisms.
The individual cells of a multicellular organism remain delimited by their plasma membranes, but they have developed specialized surface structures for attachment to and communication with each other (Fig. 2–26). At tight junctions, the plasma membranes of adjacent cells are closely apposed, with no extracellular fluid separating them. Desmosomes (occurring only in plant cells) hold two cells together; the small extracellular space between them is filled with fibrous, presumably adhesive, material. Gap junctions provide small, reinforced openings between adjacent cells, through which electric currents, ions, and small molecules can pass. In higher plants, plasmodesmata form channels resembling gap junctions; they provide a path through the cell wall for the movement of small molecules between adjacent cells. Each of these junctions is reinforced by membrane proteins or cytoskeletal filaments. The type of junction(s) between neighboring cells varies from tissue to tissue.
cell 1, cell 2, plasma membrane, cytoplasm, tight junction, cytoskeletal filaments, glycoprotein filaments, desmosome, extracellular space, plasma membranes of two adjacent cells, gap junction
Figure 2–26  Three types of junctions between cells. (a) Tight junctions produce a seal between adjacent cells. (b) Desmosomes, typical of plant cells, weld adjacent cells together and are reinforced by various cytoskeletal elements. (c) Gap junctions allow ions and electric currents to flow between adjacent cells.
bacterial virus (bacteriophage) injects its DNA through cell envelope, animal virus enters host cell by endocytosis, viral genome → replication → transcription → translation → assembly and packaging, exit by breakdown of cell envelope, exit by outward budding
Viruses are supramolecular complexes that can replicate themselves in appropriate host cells. They consist of a nucleic acid (DNA or RNA) molecule surrounded by a protective shell, or capsid, made up of protein molecules and, in some cases, a membranous envelope. Viruses exist in two states. Outside the host cells that formed them, viruses are simply nonliving particles called virions, which are regular in size, shape, and composition and can be crystallized. Once a virus or its nucleic acid component gains entry into a specific host cell, it becomes an intracellular parasite. The viral nucleic acid carries the genetic message specifying the structure of the intact virion. It diverts the host cell’s enzymes and ribosomes from their normal cellular roles to the manufacture of many new daughter viral particles. As a result, hundreds of progeny viruses may arise from the single virion that infected the host cell (Fig. 2–27). In some host–virus systems, the progeny virions escape through the host cell’s plasma membrane. Other viruses cause cell lysis (membrane breakdown and host cell death) as they are released.
A different type of response results from some viral infections, in which viral DNA becomes integrated into the host’s chromosome and is replicated with the host’s own genes. Integrated viral genes may have little or no effect on the host’s survival, but they often cause profound changes in the host cell’s appearance and activity.
Hundreds of different viruses are known, each more or less specific for a host cell (Table 2–3), which may be an animal, plant, or bacterial cell. Viruses specific for bacteria are known as bacteriophages, or simply phages (Greek phagein, “to eat”). Some viruses contain only one kind of protein in their capsid – the tobacco mosaic virus, for example, a simple plant virus and the first to be crystallized. Other viruses contain dozens or hundreds of different kinds of proteins. Even some of these large and complex viruses have been crystallized, and their detailed molecular structures are known (Fig. 2–28). Viruses differ greatly in size. Bacteriophage ΦX174, one of the smallest, has a diameter of 18 nm. Vaccinia virus is one of the largest; its virions are almost as large as the smallest bacteria. Viruses also differ in shape and complexity of structure. The human immunodeficiency virus (HIV) (Fig. 2–29) is relatively simple in structure, but devastating in action; it causes AIDS.
Table 2–3 summarizes the type and size of the nucleic acid components of a number of viruses. Some viruses are highly pathogenic in humans; for example, those causing poliomyelitis, influenza, herpes, hepatitis, AIDS, the common cold, infectious mononucleosis, shingles, and certain types of cancer.
Biochemistry has profited enormously from the study of viruses, which has provided new information about the structure of the genome, the enzymatic mechanisms of nucleic acid synthesis, and the regulation of the flow of genetic information.
Figure 2–27  Infection of a bacterial cell by a bacteriophage (left), and of an animal cell by a virus (right) results in the formation of many copies of the infecting virus.
Figure 2–28  The structures of several viruses, viewed with the electron microscope. Turnip yellow mosaic virus (small, spherical particles), tobacco mosaic virus (long cylinders), and bacteriophage T4 (shaped like a hand mirror).
Table 2–3  Some well-studied animal viruses
  Genomic   Genome size
Virus                                       Known hosts                   material (kilobases)*
Adenoviruses Vertebrates DNA 36
SV40 Primates DNA 5
Herpes Vertebrates DNA 150
Vaccinia Vertebrates DNA 200
Parvoviruses Vertebrates DNA 1–2
Retroviruses Vertebrates and (?) RNA/DNA   5–8
Reoviruses Vertebrates RNA 1.2–4.0†
Influenza Mammals RNA   1.0–3.3†  
Vesicular stomatitis Vertebrates RNA 12
Sindbis Insects and vertebrates    RNA 10
Poliomyelitis Primates RNA 7
Human immunodeficiency (HIV)    Primates RNA 9.7

Source: From Darnell, J., Lodish, H., & Baltimore D. (1990) Molecular Cell Biology, 2nd edn, p. 183,
Scientific American Books, Inc., New York.

* Size is given in kilobases (1 kilobase = 1,000 nucleotides) for single-stranded nucleic acids, or
kilobase pairs for double-stranded molecules.

† Reoviruses have ten double-stranded RNA segments, and influenza has eight single-stranded
RNA segments; the length of each segment is in the range indicated.
Figure 2–29  Human immunodeficiency viruses (HIV), the causative agent of AIDS, leaving an infected T lymphocyte of the immune system.
Chapter R
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2.1 t: Figure 2–2       __________________________________________ 35,459
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m: Figure 2–3       __________________________________________ 40,011
2.2 d: A dividing E. coli cell       ____________________________ 19,552
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2.3 d: A photosynthetic cyanobacterium        ______________________ 30,891
t: Figure 2–4       __________________________________________ 31,420
t: Figure 2–5       __________________________________________ 33,352
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2.5 –: Figure 2–8       __________________________________________ 111,349
2.5.1 t: Figure 2–9       __________________________________________ 18,462
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2.5.6 r: Figure 2–11       _________________________________________ 85,452
2.5.8 t: Figure 2–12       _________________________________________ 43,837
t: Figure 2–13       _________________________________________ 54,731
t: Figure 2–14       _________________________________________ 45,442
2.5.9 t: Figure 2–15       _________________________________________ 35,989
2.5.10 r: Figure 2–16       _________________________________________ 44,374
2.5.11 t: Figure 2–17       _________________________________________ 44,832
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2.5.13 r: Figure 2–19       _________________________________________ 63,224
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