1.1 Cellular Foundations All organism Phototrophs Chemotrophs (energy from (energy from chemical light) Autotroph Heterotrophs Heterotroph (carbon from (carbon from (carbon from organic organIc compounds Cyanobacteria Examples Plants ° Purple bacteria Green bacteria L Ort InorganI Examples: Examples Sulfur bacteria Most prokaryotes FIGURE 1-5 Organisms can be classified according to their source All nonphototrophic of energy (sunlight or oxidizable chemical compounds)and their eukaryotes source of carbon for the synthesis of cellular material. domains, sometimes called Archaea and Bacteria. All eu- atoms exclusively from CO2(that is, no chemotrophs karyotic organisms, which make up the third domain, are autotrophs), but the chemotrophs may be further karya, evolved from the same branch that gave rise classified according to a different criterion whether the to the Archaea; archaebacteria are therefore more fuels they oxidize are inorganic (lithotrophs )oror- closely related to eukaryotes than to eubacteria ganic (organotrophs) Within the domains of archaea and bacteria are sub Most known organisms fall within one of these four groups distinguished by the habitats in which they live. broad categories-autotrophs or heterotrophs among the In aerobie habitats with a plentiful supply of oxygen, photosynthesizers, lithotrophs or organotrophs among some resident organisms derive energy from the trans- the chemical oxidizers. The prokaryotes have several gen- fer of electrons from fuel molecules to oxygen. Other eral modes of obtaining carbon and energy. Escherich environments are anaerobic, virtually devoid of oxy- coli, for example, is a chemoorganoheterotroph; it re- gen, and microorganisms adapted to these environments quires organic compounds from its environment as fuel obtain energy by transferring electrons to nitrate(form- and as a source of carbon. Cyanobacteria are photo- ing N2), sulfate(forming HS), or CO2(forming CHa) lithoautotrophs; they use sunlight as an energy source Many organisms that have evolved in anaerobic envi- and convert CO2 into biomolecules. We humans, like E ronments are obligate anaerobes: they die when ex- coli, are chemoorganoheterotrophs posed to oxygen. We can classify organisms according to how they obtain the energy and carbon they need for synthesiz Escherichia coli Is the Most-Studied Prokaryotic Cell ng cellular material (as summarized in Fig. 1-5. There Bacterial cells share certain common structural fea- are two broad categories based on energy sources: pho- tures, but also show group-specific specializations (fig totrophs (Greek trophe, "nourishment") trap and use 1-6). E. coli is a usually harmless inhabitant of the hu- sunlight, and chemotrophs derive their energy from man intestinal tract. The E coli cell is about 2 um long oxidation of a fuel. All chemotrophs require a source of and a little less than I um in diameter. It has a protec- organic nutrients; they cannot fix CO2 into organic com- tive outer membrane and an inner plasma membrane pounds. The phototrophs can be further divided into that encloses the cytoplasm and the nucleoid. Between those that can obtain all needed carbon from CO2(au- the inner and outer membranes is a thin but strong layer totrophs) and those that require organic nutrients of polymers called peptidoglycans, which gives the cell heterotrophs). No chemotroph can get its carbon its shape and rigidity. The plasma membrane and the
domains, sometimes called Archaea and Bacteria. All eukaryotic organisms, which make up the third domain, Eukarya, evolved from the same branch that gave rise to the Archaea; archaebacteria are therefore more closely related to eukaryotes than to eubacteria. Within the domains of Archaea and Bacteria are subgroups distinguished by the habitats in which they live. In aerobic habitats with a plentiful supply of oxygen, some resident organisms derive energy from the transfer of electrons from fuel molecules to oxygen. Other environments are anaerobic, virtually devoid of oxygen, and microorganisms adapted to these environments obtain energy by transferring electrons to nitrate (forming N2), sulfate (forming H2S), or CO2 (forming CH4). Many organisms that have evolved in anaerobic environments are obligate anaerobes: they die when exposed to oxygen. We can classify organisms according to how they obtain the energy and carbon they need for synthesizing cellular material (as summarized in Fig. 1–5). There are two broad categories based on energy sources: phototrophs (Greek trophe- , “nourishment”) trap and use sunlight, and chemotrophs derive their energy from oxidation of a fuel. All chemotrophs require a source of organic nutrients; they cannot fix CO2 into organic compounds. The phototrophs can be further divided into those that can obtain all needed carbon from CO2 (autotrophs) and those that require organic nutrients (heterotrophs). No chemotroph can get its carbon atoms exclusively from CO2 (that is, no chemotrophs are autotrophs), but the chemotrophs may be further classified according to a different criterion: whether the fuels they oxidize are inorganic (lithotrophs) or organic (organotrophs). Most known organisms fall within one of these four broad categories—autotrophs or heterotrophs among the photosynthesizers, lithotrophs or organotrophs among the chemical oxidizers. The prokaryotes have several general modes of obtaining carbon and energy. Escherichia coli, for example, is a chemoorganoheterotroph; it requires organic compounds from its environment as fuel and as a source of carbon. Cyanobacteria are photolithoautotrophs; they use sunlight as an energy source and convert CO2 into biomolecules. We humans, like E. coli, are chemoorganoheterotrophs. Escherichia coli Is the Most-Studied Prokaryotic Cell Bacterial cells share certain common structural features, but also show group-specific specializations (Fig. 1–6). E. coli is a usually harmless inhabitant of the human intestinal tract. The E. coli cell is about 2 m long and a little less than 1 m 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 polymers called peptidoglycans, which gives the cell its shape and rigidity. The plasma membrane and the 1.1 Cellular Foundations 5 Heterotrophs (carbon from organic compounds) Examples: •Purple bacteria •Green bacteria Autotrophs (carbon from CO2) Examples: •Cyanobacteria •Plants Heterotrophs (carbon from organic compounds) Phototrophs (energy from light) Chemotrophs (energy from chemical compounds) All organisms Lithotrophs (energy from inorganic compounds) Examples: •Sulfur bacteria •Hydrogen bacteria Organotrophs (energy from organic compounds) Examples: •Most prokaryotes •All nonphototrophic eukaryotes FIGURE 1–5 Organisms can be classified according to their source of energy (sunlight or oxidizable chemical compounds) and their source of carbon for the synthesis of cellular material. 8885d_c01_005 12/20/03 7:04 AM Page 5 mac76 mac76:385_reb:
Chapter 1 The Foundations of Biochemistry Ribosomes bacterial ribosomes are smaller than FIGURE 1-6 Common structural features of bacterial cells. because eukaryotic ribosomes, but serve the same function- protein synthesis from an RNA message. of differences in the cell envelope structure, some eubacteria (gram positive bacteria) retain Grams stain, and others (gram-negative Nucleoid Contains a single bacteria) do not. E. coli is gram-negative. Cyanobacteria are also simple, long circular DNA eubacteria but are distinguished by their extensive internal membrane tystem, in which photosynthetic pigments are localized. Although the Pili provide similar under the electron microscope, the structures of the membrane lipids and the polysaccharides of the cell envelope are distinctly dif- Flagella layers outside it constitute the cell envelope. In the Archaea, rigidity is conferred by a different type of poly- hrough its ∴ rounding mer(pseudopeptidoglycan). 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 strikingly from those of the eubacteria The cytoplasm of E. coli contains about 15,000 ribosomes, thousands of copies each of about 1, 000 Cell envelope different enzymes, numerous metabolites and cofac tructure varies tors, and a variety of inorganic ions. The nucleoid with type of contains a single, circular molecule of DNA, and the cytoplasm (like that of most bacteria) contains one or more smaller, circular segments of DNA called plas- mids. In nature, some plasmids confer resistance to toxins and antibiotics in the environment in the labo ratory, these DNA segments are especially amenable to experimental manipulation and are extremely use- Most bacteria (including E coli) lead existences as individual cells, but in some bacterial species cells tend Outer membrane to associate in clusters or filaments, and a few(the Peptidoglycan layer Inner memb myxobacteria, for example) demonstrate simple social behavior Eukaryotic Cells Have a Variety of Membranous 医」0 Organelles, Which Can Be Isolated for Study Gram-negative bacteria Gram-positive bacteria Typical eukaryotic cells (Fig. 1-7 are much larger than Outer membrane No outer membrane prokaryotic cells--commonly 5 to 100 um in diameter thicker peptidoglycan layer with cell volumes a thousand to a million times larger than those of bacteria. The distinguishing characteristics of eukaryotes are the nucleus and a variety of membrane- bounded organelles with specific functions: mitochondria endoplasmic reticulum, Golgi complexes, and lysosomes Plant cells also contain vacuoles and chloroplasts(Fig 1-7. Also present in the cytoplasm of many cells are granules or droplets containing stored nutrients such as starch and fat Cyanobacteria Gram-negative; tougher In a major advance in biochemistry, Albert Claude peptidoglycan layer; Christian de duve, and George Palade developed meth- extensive internal ods for separating organelles from the cytosol and from embrane system with each other-an essential step in isolating biomolecules and larger cell components and investigating their
layers outside it constitute the cell envelope. In the Archaea, rigidity is conferred by a different type of polymer (pseudopeptidoglycan). 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 strikingly from those of the eubacteria. The cytoplasm of E. coli contains about 15,000 ribosomes, thousands of copies each of about 1,000 different enzymes, numerous metabolites and cofactors, and a variety of inorganic ions. The nucleoid contains a single, circular molecule of DNA, and the cytoplasm (like that of most bacteria) contains one or more smaller, circular segments of DNA called plasmids. In nature, some plasmids confer resistance to toxins and antibiotics in the environment. In the laboratory, these DNA segments are especially amenable to experimental manipulation and are extremely useful to molecular geneticists. Most bacteria (including E. coli) lead existences as individual cells, but in some bacterial species cells tend to associate in clusters or filaments, and a few (the myxobacteria, for example) demonstrate simple social behavior. Eukaryotic Cells Have a Variety of Membranous Organelles, Which Can Be Isolated for Study Typical eukaryotic cells (Fig. 1–7) are much larger than prokaryotic cells—commonly 5 to 100 m in diameter, with cell volumes a thousand to a million times larger than those of bacteria. The distinguishing characteristics of eukaryotes are the nucleus and a variety of membranebounded organelles with specific functions: mitochondria, endoplasmic reticulum, Golgi complexes, and lysosomes. Plant cells also contain vacuoles and chloroplasts (Fig. 1–7). Also present in the cytoplasm of many cells are granules or droplets containing stored nutrients such as starch and fat. In a major advance in biochemistry, Albert Claude, Christian de Duve, and George Palade developed methods for separating organelles from the cytosol and from each other—an essential step in isolating biomolecules and larger cell components and investigating their 6 Chapter 1 The Foundations of Biochemistry 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 other cells. Flagella Propel cell through its surroundings. Cell envelope Structure varies with type of bacteria. Gram-negative bacteria Outer membrane; peptidoglycan layer Outer membrane Peptidoglycan layer Inner membrane Gram-positive bacteria No outer membrane; thicker peptidoglycan layer Cyanobacteria Gram-negative; tougher peptidoglycan layer; extensive internal membrane system with photosynthetic pigments Archaebacteria No outer membrane; peptidoglycan layer outside plasma membrane Peptidoglycan layer Inner membrane FIGURE 1–6 Common structural features of bacterial cells. Because of differences in the cell envelope structure, some eubacteria (grampositive 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. Although the cell envelopes of archaebacteria and gram-positive eubacteria look similar under the electron microscope, the structures of the membrane lipids and the polysaccharides of the cell envelope are distinctly different in these organisms. 8885d_c01_006 11/3/03 1:39 PM Page 6 mac76 mac76:385_reb:
1.1 Cellular Foundations 7 (a)Animal cell Ribosomes are protein synthesizing machines Peroxisome destroys peroxides Lysosome degrades intracellular debris Transport vesicle shuttles lipids etween ER, golgi Golgi complex processes packages, and targets proteins to other organelles or for export mooth endoplasmic reticulum (SER)is site of lipid synthesis and drug metabolism Nuclear envelope segregates Nucleolus is site of ribosome hromatin (DNA+ protein) RNA synthesis Rough endoplasmic reticulum Nucleus contains the (RER)is site of much protein genes(chromatin Ribosomes Cytoskeleton out of cell Mitochondrion oxidizes fuels to produce ATP olgi complex Chloroplast harves roduces atp and Starch granule temporarily stores carbohydrate products of Thylakoids are site of light- driven ATP synthesis m Vacuole degrades and recycles macromolecules, stores Cell wall of adjacent cell between two plant cell Glyoxysome contains enzymes of FIGURE 1-7 Eu c cell structure. Schematic illustrations of the (b)Plant cell two major types of eukaryotic cell: (a) a representative animal cell and (b) a representative plant cell. Plant cells are usually 10 to 00 um in diameter-larger than animal cells, which typically range from 5 to 30 um. Structures labeled in red are unique to either animal or plant cells
1.1 Cellular Foundations 7 Ribosomes are proteinsynthesizing machines Peroxisome destroys peroxides Lysosome degrades intracellular debris Transport vesicle shuttles lipids and proteins between ER, Golgi, and plasma membrane Golgi complex processes, packages, and targets proteins to other organelles or for export Smooth endoplasmic reticulum (SER) is site of lipid synthesis and drug metabolism Nucleus contains the genes (chromatin) Ribosomes Cytoskeleton Cytoskeleton supports cell, aids in movement of organells Golgi complex Nucleolus is site of ribosomal RNA synthesis Rough endoplasmic reticulum (RER) is site of much protein synthesis Mitochondrion oxidizes fuels to produce ATP Plasma membrane separates cell from environment, regulates movement of materials into and out of cell Chloroplast harvests sunlight, produces ATP and carbohydrates Starch granule temporarily stores carbohydrate products of photosynthesis Thylakoids are site of lightdriven ATP synthesis Cell wall provides shape and rigidity; protects cell from osmotic swelling Cell wall of adjacent cell Plasmodesma provides path between two plant cells Nuclear envelope segregates chromatin (DNA protein) from cytoplasm Vacuole degrades and recycles macromolecules, stores metabolites (a) Animal cell (b) Plant cell Glyoxysome contains enzymes of the glyoxylate cycle FIGURE 1–7 Eukaryotic cell structure. Schematic illustrations of the two major types of eukaryotic cell: (a) a representative animal cell and (b) a representative plant cell. Plant cells are usually 10 to 100 m in diameter—larger than animal cells, which typically range from 5 to 30 m. Structures labeled in red are unique to either animal or plant cells. 8885d_c01_007 1/15/04 3:28 PM Page 7 mac76 mac76:385_reb:
Chapter 1 The Foundations of Biochemistry structures and functions. In a typical cell fractionation Differential centrifugation results in a rough fraction- (Fig. 1-8), cells or tissues in solution are disrupted by ation of the cytoplasmic contents, which may be further gentle homogenization. This treatment ruptures the purified by isopycnic (same density)centrifugation In plasma membrane but leaves most of the organelles in- his procedure, organelles of different buoyant densities tact. The homogenate is then centrifuged; organelles (the result of different ratios of lipid and protein in each such as nuclei, mitochondria, and lysosomes differ in type of organelle) are separated on a density gradient. By size and therefore sediment at different rates. They alse carefully removing material from each region of the gra differ in specific gravity, and they float "at different dient and observing it with a microscope, the biochemist levels in a density gradient can establish the sedimentation position of each organelle FIGURE 1-8 Subcellular fractionation of tissue. A tissue such as liver is first mechanically homogenized to break cells and disperse thei contents in an aqueous buffer. The sucrose medium has an osmotic pressure similar to that in organelles, thus preventing diffusion of w ter into the organelles, which would swell and burst. (a)The large and small particles in the suspension can be separated by centrifugation at different speeds, or(b)particles of different density can be sepa- rated by isopycnic centrifugation In isopycnic centrifugation, a cen- trifuge tube is filled with a solution, the density of which increases (a) Differential from top to bottom; a solute such as sucrose is dissolved at different centrifugation concentrations to produce the 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 thei buoyant density exactly matches that in the gradient. Each layer can be collected separately. (1,000g,10min) upernatant subjected to (b) Isopycnic (20,000g,20min) centrifugation Tissue ::5. g,1h) Centrifugation Supernatant subjected to (150,000g,3h) whole cells, 4 membranes Pellet Less dense (fragments of er), small vesicles Fractionation More dens ribosomes, large macromolecules 87654321
❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ structures and functions. In a typical cell fractionation (Fig. 1–8), cells or tissues in solution are disrupted by gentle homogenization. This treatment ruptures the plasma membrane but leaves most of the organelles intact. The homogenate is then centrifuged; organelles such as nuclei, mitochondria, and lysosomes differ in size and therefore sediment at different rates. They also differ in specific gravity, and they “float” at different levels in a density gradient. Differential centrifugation results in a rough fractionation of the cytoplasmic contents, which may be further purified by isopycnic (“same density”) 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 sedimentation position of each organelle 8 Chapter 1 The Foundations of Biochemistry ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ Centrifugation Fractionation Sample Less dense component More dense component Sucrose gradient 8765 3 4 21 ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ Isopycnic (sucrose-density) centrifugation ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ (b) ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ▲ Low-speed centrifugation (1,000 g, 10 min) Supernatant subjected to medium-speed centrifugation (20,000 g, 20 min) Supernatant subjected to high-speed centrifugation (80,000 g, 1 h) Supernatant subjected to very high-speed centrifugation (150,000 g, 3 h) Differential centrifugation Tissue homogenization Tissue homogenate Pellet contains mitochondria, lysosomes, peroxisomes Pellet contains microsomes (fragments of ER), small vesicles Pellet contains ribosomes, large macromolecules Pellet contains whole cells, nuclei, cytoskeletons, plasma membranes Supernatant contains soluble proteins ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ▲ ❚ ❚ ❚ (a) ▲ ▲ ▲ ▲▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ▲ ▲▲ ▲ ▲ ▲▲▲ ▲ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ ❚ FIGURE 1–8 Subcellular fractionation of tissue. A tissue such as liver is first mechanically homogenized to break cells and disperse their contents in an aqueous buffer. The sucrose medium has an osmotic pressure similar to that in organelles, thus preventing diffusion of water into the organelles, which would swell and burst. (a) The large and small particles in the suspension can be separated by centrifugation at different speeds, or (b) particles of different density can be separated by isopycnic centrifugation. In isopycnic centrifugation, a centrifuge tube is filled with a solution, the density of which increases from top to bottom; a solute such as sucrose is dissolved at different concentrations to produce the 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. 8885d_c01_01-46 10/27/03 7:48 AM Page 8 mac76 mac76:385_reb:
1.1 Cellular Foundations and obtain purified organelles for further study. For into their protein subunits and reassembly into fila- example, these methods were used to establish that ments. Their locations in cells are not rigidly fixed but lysosomes contain degradative enzymes, mitochondria may change dramatically with mitosis, cytokinesis contain oxidative enzymes, and chloroplasts contain amoeboid motion, or changes in cell shape. The assem- photosynthetic pigments. The isolation of an organelle en- bly, disassembly, and location of all types of filaments riched in a certain enzyme is often the first step in the are regulated by other proteins, which serve to link or purification of that enzyne bundle the filaments or to move cytoplasmic organelles long the filaments The Cytoplasm Is Organized by the Cytoskeleton The picture that emerges from this brief survey and Is Highly Dynamic of cell structure is that of a eukaryotic cell with a meshwork of structural fibers and a complex system of Electron microscopy reveals several types of protein fila- membrane-bounded compartments(Fig. 1-0. The fila- ments crisscrossing the eukaryotic cell, forming an inter ments disassemble and then reassemble elsewhere. Mem- locking three-dimensional meshwork, the cytoskeleton. branous vesicles bud from one organelle and fuse with There are three general types of cytoplasmic filaments- another Organelles move through the cytoplasm alon actin filaments, microtubules, and intermediate filaments protein filaments, their motion powered by energy de- (Fig. 1-9)--differing in width(from about 6 to 22 nm), pendent motor proteins. The endomembrane system composition, and specific function. All types provide segregates specific metabolic processes and provides structure and organization to the cytoplasm and shape surfaces on which certain enzyme-catalyzed reactions to the cell Actin filaments and microtubules also help to occur. Exocytosis and endocytosis, mechanisms of produce the motion of organelles or of the whole cell ransport (out of and into cells, respectively)that involve o o: Each type of cytoskeletal component is composed membrane fusion and fission, provide paths between the simple protein subunits that polymerize to form fila- cytoplasm and surrounding medium, allowing for secre- ments of uniform thickness. These filaments are not per- tion of substances produced within the cell and uptake manent structures; they undergo constant disassembly of extracellular materials Actin stress fibers Microtubules Intermediate filaments b) URE 1-9 The three types of cytoskeletal filaments. The up lin,or intermediate filament proteins are covalently attached to a els show epithelial cells photographed after treatment with fluorescent compound. When the cell is viewed with a fluorescence that bind to and specifically stain(a)actin filaments bundled microscope, only the stained structures are visible. The lower panels form"stress fibers, "(b)microtubules radiating from the cell center, show each type of filament as visualized by (a, b) transmission or and(c)intermediate filaments extending throughout the cytoplasm. For (c) scanning electron microscopy. these experiments, antibodies that specifically recognize actin, tubu-
and obtain purified organelles for further study. For example, these methods were used to establish 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. The Cytoplasm Is Organized by the Cytoskeleton and Is Highly Dynamic Electron microscopy reveals several types of protein filaments crisscrossing the eukaryotic cell, forming an interlocking three-dimensional meshwork, the cytoskeleton. There are three general types of cytoplasmic filaments— actin filaments, microtubules, and intermediate filaments (Fig. 1–9)—differing in width (from about 6 to 22 nm), composition, and specific function. All types 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 type of cytoskeletal component 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 protein subunits and reassembly into filaments. Their locations in cells are not rigidly fixed but may change dramatically with mitosis, cytokinesis, amoeboid motion, or changes in cell shape. The assembly, disassembly, and location of all types of filaments are regulated by other proteins, which serve to link or bundle the filaments or to move cytoplasmic organelles along the filaments. The picture that emerges from this brief survey of cell structure is that of a eukaryotic cell with a meshwork of structural fibers and a complex system of membrane-bounded compartments (Fig. 1–7). The filaments disassemble and then reassemble elsewhere. Membranous vesicles bud from one organelle and fuse with another. Organelles move through the cytoplasm along protein filaments, their motion powered by energy dependent motor proteins. The endomembrane system segregates specific metabolic processes and provides surfaces on which certain enzyme-catalyzed reactions occur. Exocytosis and endocytosis, mechanisms of transport (out of and into cells, respectively) that involve membrane fusion and fission, provide paths between the cytoplasm and surrounding medium, allowing for secretion of substances produced within the cell and uptake of extracellular materials. 1.1 Cellular Foundations 9 Actin stress fibers (a) Microtubules (b) Intermediate filaments (c) FIGURE 1–9 The three types of cytoskeletal 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 (a, b) transmission or (c) scanning electron microscopy. 8885d_c01_009 12/20/03 7:04 AM Page 9 mac76 mac76:385_reb: