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Main Cell Biology and Histology
Cell Biology and HistologyLeslie Gartner, James L. Hiatt PhD, Judy M. Strum
Univ. of Maryland, Baltimore. Exam review of cell biology and history for students studying for the USMLE Step 1 and for course exams. Outline format. Softcover. Previous edition: c1993. DNLM: Histology--outlines.
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CELL BIOLOGY AND HISTOLOGY this is for non-commerda[ use by Russian-speakers only ! if you are not a Russian-speaker delete this file irnrnediate[y ! aHHblH CKaH npe/HO3HaleH IHLUb IFI : 1. pyccKHx nob3OBaTeeH- BpaHeH H yHeHblX, B Oco6eHHOCTH 2. CBO60HOrO, HeKoMMepecKoro H BECflATHOFO pacnpocTpaHeHH CKaHHpOBaHO nOTOM H KpOBb lvC T OT.py I Plasma Membrane I. Overview--The Plasma Membrane (plasmalemma; cell membrane) A. Structure. The plasma membrane is about 7.5 nanometers (nm) thick and consists of a lipid bilayer and associated proteins. 1. The inner leaflet of the plasma membrane faces the cytoplasm and the outer leaflet faces the extracellular environment. 2. The plasma membrane displays a trilaminar (unit membrane) structure when examined by transmission electron microscopy (TEM). B. Function 1. The plasma membrane envelops the cell and maintains its structural and functional integrity. 2. It acts as a semipermeable membrane between the cytoplasm and the external environment. 3. It permits the cell to recognize (and be recognized by) other cells and macromolecules. II. Fluid Mosaic Model of the Plasma Membrane A. The lipid bilayer (Figure 1.1) is freely permeable to small, lipid-soluble, nonpolar molecules but is impermeable to charged ions. 1o Molecular structure. The lipid bilayer is composed of phospholipids, glycolipids, and cholesterol. --'------------ a. PhospholiIids are amphipathic, consisting of one polar {hydro- phillic) head and two nonpolar (hydrophobic) fatty acyl tails. (1) The polar head of each molecule faces the membrane surface, whereas the tails project into the interior of the membrane. (2) The tails of the two leaflets face each other, forming weak bonds that attach the leaflets to each other. b. Glycolipids are restricted to the outer leaflet. Polar carbohydrate residues of glycolipids extend from the outer leaflet into the extracellu- lar space and form part of the glycocalyx. 1 2 ] Cell Biology and Histology Figure 1.1. Diagrammatic representation of the plasma membrane showing the outer (top) and inner (bottom) leaflets of the unit membrane. The hydrophobic fatty acyl tails and the polar heads of the phospholipids constitute the lipid bilayer. Integral proteins are embedded in the lipid bilayer. Peripheral proteins are located primarily on the cytoplasmic aspect of the inner leaflet and are attached by noncovalent interactions to integral proteins. c. Cholesterol constitutes 2% of plasmalemma lipids, is present in both leaflets, and helps maintain the structural integrity of the membrane. Fluidity of the lipid bilayer is crucial to exocytosis, endocytosis, membrane trafficking, and membrane biogenesis. a. Fluidity increases with increased temperature and with decreased saturation of the fatty acyl tails. b. Fluidity decreases with an increase in the membrane's cholesterol content. B. Membrane proteins (see Figure 1.1) include integral proteins and peripheral proteins. 1. Integral proteins are dissolved in the lipid bilayer. a. Transmembrane proteins span the entire plasma membrane and function as membrane receptors and transport proteins. (1) Most transmembrane proteins are glycoproteins. (2) Transmembrane proteins are amphipathic and contain hydro- philic and hydrophobic amino acids, some of which interact with the hydrocarbon tails of the membrane phospholipids. b. Integral proteins may also be anchored to the inner (or occasionally outer) leaflet via fatty acyl or prenyl groups. c. In freeze-fracture preparations, integral proteins remain preferentially attached to the P-face, the external surface of the inner leaflet, rather than the E-face (Figure 1.2). 2. Peripheral proteins do not extend into the lipid bflayer. a. These proteins are located on the cytoplasmic aspect of the inner leaflet. b. The outer leaflets of some cells possess covalently linked glycolipids to which peripheral proteins are anchored; these peripheral proteins thus project into the extracellular space. Plasma Membrane ] 3 Figure 1.2. Freeze-fracturing cleaves the plasma membrane (5). The impressions (2) of the transmembrane proteins are evident on the E-face, between the inner (3) and outer leaflets (4). The integral proteins (1) remain preferentially attached to the P-face (A), the external surface of the inner leaflet; fewer proteins remain assodated with the E-face (B), the internal surface of the outer leaflet. The arrowhead indicates a transmembrane protein attached to both E-face and P-face.(Reprinted with permission from Krstic RV: Ultrastruktur der Saugertierzelle. Beriin, Spdnger Veriag, 1976, p 177.) c. Peripheral proteins bond to the phospholipid polar groups or integral proteins of the membrane via noncovalent interactions. d. They usually function as part of the cytoskeleton or as part of an intracellular second messenger system. 3. Functional characteristics of membrane proteins a. The lipid-to-protein ratio in plasma membranes ranges from 1:1 (by weight) in most cells to 4__'.1 in myelin. b. Some membrane proteins diffuse laterally in the lipid bilayer; others are immobile and are held in place by cytoskeletal components. C. Glycocalyx (cell coat) is the sugar coat located on the outer surface of the outer leaflet of the plasmalemms_ When examined by TEM, it varies in appearance (fuzziness) and thickness (up to 50 nm). 1. Composition. The glycocalyx consists of_polar oligosaccharide side chains linked covalently to most proteins and soe lipids (glylipids) of the plas ----E- alemma. It also contains prote____o lglglglgc_ans (glycoscans integral proteins). 2. Function a. The glycocalyx aids in attachment of ceells (e.g., fibroblasts but not epithelial cells) to extracellular 'atrix components:- -- b. It binds antigens andenzymes to the cell surface. c. It facilitates cell-cell recognition and i 4 [ Cell Biology and Histology Exterior interior 0 Simple diffusion Ion channel- Carrier protein- mediated mediated diffusion diffusion Figure 1.3. Passive transport of molecules across plasrna membranes may occur by simple diffusion (left) and by either of the two types of facilitated diffusion mediated by ion channel proteins (center) and carrier proteins (right). III. Plasma Membrane Transport Processes. These processes include transport of a single molecule rt) or cotranspo_rt_0wo different mole- cules in the same (symport) or opposite (antiport) direction. A. Passive transport (Figure 1.3) includes simple and facilitated diffusion. Neither of these processes requires energy because molecules move across the plasma membrane down a concentration or electrochemical gradient. 1. Simple diffusion transports small nonpolar molecules (e.g., O and N_ and small, uncharged, polar molecules (e.g., HO, CO, and glrol). It exhibits little specificity, and the diffusion rate is proportional to the concen- tration gradient of the diffusing molecule. 2. Facilitated diffusion occurs via ion channel and/or carrier proteins, structures that exhibit specificity for the transported molecules. It is faster than simple diffusion; ions and large polar molecules are thus capable of traversing membranes that would otherwise be impermeable to them a. Ion channel proteins are highly folded transmembrane proteins that form small aqueous pores across membranes through which specific small water-soluble molecules and ions pass down an electrochemical gradient. b. Carrier proteins are highly folded transmembrane proteins that un- de.Lgo reversible conformational change, thus transporting specific mole- cules across the membrane; these proteins function in both passive transport and active transport. B. Active transport is an.energy_ -requiring process which transports a molecule against an electrochemical gradient via carrier proteins. 1. Naч-K ч pump a. Mechanism. The Na+-K + pump involves the antiport transport of Na + and K + ions mediated by the carrier protein,N-K ATPase. "(1) .Te_eN__* ions are pumped out of the cell and two K ч ions are pumped int_o t__he c___e (2) The T P molecule byes reqmre(1 co r_ansport( ons. Plasma Membrane b. Function (1) The primary function is to maintain constant cell volume by decreasing the intracellularon concentration (and thus the osmotic re2F_C_sre) and ncrea_sln tg_he 'excellular 1on concentration, thus ecreas_ing _the flow of_water into the cell. (2) The Na+-K ч pump also plays a minor role in the maintenance of a potential difference across the plasma membrane. Glucose transport involves the symport movement ofglucose across -an epithelium (transepithelial transport). Transport is frequently pow- .ered by an electrochemical Na + gradient, which drives carrier proteins located at specific reions of th cell surface. C. Facilitated diffusion of ions can occur via ion channel proteins or iono- phores. 1. Selective ion channel proteins permit only certain ions to traverse them. a. K ч leak channels are the most common ion channels. These channels a-e tmated a-'eak K +, the ions most responsible for establishing a poten--n-fi-dTfference across the piasm. b. Gated ion channels open only transiently in response to various stimuli. They include the following types: (1) Voltage-gated channels ppep whe__n_ t__he_ poten_tj_aldifference_ across the membrane changes (e.g., voltage-gated Na + channels wLic-h-funi-on in the generation of action potentials; see Chapter 9 VIII B 1 e). (2) Mechanically gated channels open in response to a mechanical e s*(e.g., the tactile resp0ns of the hair cells in th inner ear.- (3) Li. gand-gated channels open in response to the binding of a signaling molecule or on. These channels include neurotrans- mitter-gated channels, nucleotide-gated channe!s,, and G-protein- gated K ч channels of cardiac muscle cells. 2. Ionophores are molecules that can insert into the lipid bilayer and increase membrane permeability to specific ions. IV. Cell-to-Cell Communication Signaling molecules are secreted by cells and function in cell-to-cell commu- nication. Examples include veurotransmitters, which are released into the synaptic cleft (see Chapter 8 IV A 1 b; Chapter 9 IV B 5); endocrine hormones, which are carried in the bloodstream and act on distant target ce-I-and hormones released into the intercellular spacee which act_on nearby._cells (para- crin-- hormones)-leasn'-cell (,autocrine hormones_) 1. Lipid-soluble signaling molecules jenetrate the plasma membrane and bind to r.P.cDtors in the cytoplasm or nuceus activa lular messengers. Examples include hormies that influence gene tran- scription. Cell Biology and Histology 2. Hydrophilic signaling molecules bind to and activate cell-s_ urf. ace receptors (as do some lipid-soluble signaling molecules) and have diverse p yiogic effects (see Chapter 13). Examples include neurotransmitters and numerous hopjmnes (e.g., serotonin, thoid-stimulating hormone., in- sulin). Membrane receptors are lrimarily gl:fco_proteins. They are located on the ell surface, and specific signaling molecules bind to them. 1. Function a. Membrane receptors control plasmalemma permeability by regu- late conformation_of_ip.n_can_n__pr_oe]ns___: b. They regnlate the entry, of_molecules into the cell (e.g., the delivery of cholesterol via low-density liDoprotdin receptors). c. They bind extracellnlar matrix molecules to the c.ytoskeleton via integrins, which are essential fo cell-cell contact and cell-matrix inter- actions. - d. They act as trans__ducers to transfer extracellular events intracellu- lar to second messenger systems. e. They permit pathogens that mimic normal ligands to enter cells. Types of membrane receptors a. Channel-Hnked receptors bind a signaling molecule that temporar- ily opens or closes the gate, permitting or inhibiting the movement of ions across the cell membrane. Examples include nicotinic acetylcho- line receptors on the muscle-cell sarcolemma at the myoneural junc- tion (see Chapter 8 IV A). b. Catalytic receptors are single-pass transmembrane proteins. (1) Their extracellular moiety is a receptor and their cytoplasmic aspect is a protein kinase. (2) Some catalytic receptors lack an extracytoplasmic moiety and as a result are continuously activated; such defective receptors are coded for by some oncogenes. (3) Examples include: (a) Insulin, which binds to its receptor, which autophosphory- lates. The cell then ta es upthe insu -recepr compex by endocytosis, enabling the complex to function within the cell. (b) Growth factors (e.g., epidermal growth factor, platelet-de- rived growth factor), which bind to some catalytic receptors - and induce mitosis c. G-protein-linked receptors are transmembrane proteins associ- ated with an ion channel or with an enzyme bound to the cyto- plasmic surface of the cell membrane. Plasma Membrane [ 7 (1) These receptors interact with heterotrimeric G-protein (GTP- binding regulatory protein) after binding of a signaling molecule. This interaction results in the activation of intracellular second messengers, the most common of which are cyclic AMP (cAMP) and Ca ч. (2) Examples include: (a) Heterotrimeric G proteins (Table 1.1), which include stimu- latory G protein (Gs) [Figure 1A], inhibitory G protein phospholipase C activator G protein (Gp), olfactory-specific G protein (Go If), and transducin (Gt) Table 1.1. Functions and Examples of Heterotrimeric G Proteins Type Function Result - Gs Activates adenylate cyclase Activation of protein leading to formation of kinases cAMP Gi Inhibits adenylate cyclase Protein kinases remain preventing formation of inactive cAMP Gp Activates phospholipase C Influx of Ca 2+ into cytosol leading to formation of and activation of protein inositel triphosphate and kinase C diacylglycerol Go Activates adenylate cyclase Opens cAMP-gated Na + ion in olfactory neurons channels Gt Activates cGMP Hyperpolarization of rod- phosphodiesterase in rod cell membrane cell membranes leading to hydrolysis of cGMP Examples Binding of epinephrine to I-adrenergic receptors increases cAMP levels in cytosol Binding of epinephrine to 2-adrenergic receptors decreases cAMP levels in cytosol Binding of antigen to membrane-bound IgE causes the release of histamine by mast cells Binding of odorant to G protein linked receptors initiates generation of nerve impulse Photon activation of rhodopsin causes rod cells to fire cAMP = cyclic adenosine monophosphate; cGMP = cyclic guanosine monophosphate. Signaling molecule Exterior = _jReceptor <cAcleansY reate '1(1"-- Activation t c ATP Figure 1.4. Functioning of G,-proteln-Ilnked receptors. The signaling molecule binds to the receptor, which causes the alpha subunlt of the G, protein to bind GTP as well as dissociate from the beta and gamma subunlts. Activation of adenylate cyclase by the GTP---subunlt complex stimulates synthesis of cAMP, one of the most common Intrecellular messengers. 8 [ Cell Biology and Histology (b) Monomeric G proteins (low-molecular weight G pro- teins), which are small single-chain proteins that also function in signal transduction (i) Various subtypes resemble Ras, Rho, Rab, and ARF pro- teins. (ii) These proteins are involved in pathways that regulate cell proliferation and differentiation, protein synthesis, attach- ment of cells to the extracellular matrix, exocytosis, and vesicular traffic. Plasmalemma-Cytoskeleton Association. The plasmalemma and coskeleton associate through "te. The extracellular domain of_integrins binds to extracellular matrix components and -he intracellular dmain binds to cytoskeletal ComponentsZIntegrins stabilize the plaalemna a e__eermine and maintain cell shape. A. Red blood cells (Figure 1.5A) have integrins, called band 3 pr_oteins, which are located in the plasmalemma. The cytoskeleto of a red blood cell consists mainly of spectrin, actin, band 4.1 protein, and ankz. A B Integrin Plasrna • rnmbrane Tal,n Figure 1.5. Plasrnalemma--cytoskeleton association in red blood cells (A) and nonerythroid cells (B). (Adapted with permission from Widnell CC and Pfenninger KH: Essential Cell Biology. Baltimore, Williams & Wilkins, 1990, p 82.) Plasma Membrane ] 9 Vie 1.Spectrin is a long, flexible protein (about 110 nm long) which forms tetra- mers and provide aor structural reinforcement. 2. Actin attaches to binding sites on the spectrin tetramers and holds them together, thus aiding in the ormatlon of a spectnn latticework. 3.._Ban____d 4.1 protein binds to and stabilizes spectrin-actin complexes. 4. Ankyrin is linked to both band 3 proteins and spectrin tetramers, thus -attacIiingthe spectrin-actin complex to transmembrane proteins. B. The cytoskeleton of nonerythroid cells (Figure 1.5B) consists of the follow- ing major components: 1. Actin (and perhaps fodrin, which serves as a nonerythroid spectrin) 2. -Actinin, which crosslinks actin filaments to form a meshwork 3. Vinculin, which binds to a-actinin and to another protein, called talin, which in turn atta-es to tle integrin in the lJlasma membrane Clinical Considerations Bo Cystinurea is a hereditary condition caused by abnormal carrier proteins that are unable to remove cystine from the urine, resulting in the tbrmation of kidney stones. --------- Cholera toxin is an exotoxin produced by the bacterium Vibrio cholerae. ts 1. The cholera toxin alters Gs protein so that it is unable to 1 GTP molecule. " ..... 2. As a result, cAMP levels increase in the surface absor tpe cells of the intesti______ne, leading to excessive los ytes and water and severe diarrhea. C. Venoms such as those of some poisonous snakes, inactivate acetylcholine eceptors of skeletal muscle sarcolemma at neuromuscular junctions. D. Autoimmune diseases may lead to the production ofibode that specifi- .--t nl a-ivate certain plasma membreptors. A example is Graves' disease (hyperthyroidism) [See Chapter 13 VIII B]. So Genetic defects 1. _D__ef.ec__t_iye e pr_o_ tein_s_may lead to.mental retardation, d'minished growth and sexual development, and decreased responses to certain hormones. 2. Her__editar. sphero__cy_tosis results from a defective spectrin that has a decreased ability to bind to band 4.1 protein. Tle Osease s characterized by fragile, mi'sshapen re od-]-cel___ls, Or spierocytes; destruction of these spherocytes in the spleen leads to anemia. Review Test Directions: Each of the numbered items or incomplete statements in this section is followed by answers or by completions of the statement. Select the ONE lettered answer or completion that is BEST in each case. 1. A herpetologist is bitten by a poisonous snake and is brought into the emergency room with progressive muscle paralysis. The venom is prob- ably incapacitating his (A) Na * channels (B) Ca 2* channels (C) phospholipids 6. Symport refers to the process of transporting (A) a molecule into the cell (B) a molecule out of the cell (C) two different molecules in the opposite di- rection (D) two different molecules in the same direction (E) a molecule between the cytoplasm and the (D) acetylcholine receptors .nucleus (E) spectrin {.7 _One of the ways that cells communicate with 2. Cholesterol functions in the plasmalemma to each other is by secretion of various molecules. The secreted molecule is known as (A) increase fluidity of the lipid bilayer (B) decrease fluidity of the lipid bilayer (C) facilitate the diffusion of ions through the lipid bilayer (D) assist in the transport of hormones across the lipid bilayer (E) bind extracellular matrix molecules (A) a receptor molecule (B) a signaling molecule (C) a spectrin tetramer (D) an integrin (E) an anticodon 8. The hormone ACTH travels through the 3. The cell membrane consists of various compo- bloodstream, enters connective tissue spaces, nents, including integral proteins. These integral and attaches to specific sites on target-cell mem- proteins branes. These sites are (A) are not attached to the outer leaflet (B) are not attached to the inner leaflet (C) include transmembrane proteins (D) are preferentially attached to the E-face (E) function in the transport of cholesterol- based hormones 4. Which one of the following transport pro- cesses requires energy? (A) Facilitated diffusion (B) Passive transport (C) Active transport (D) Simple diffusion 5. Which one of the following substances is un- able to traverse the plasma membrane by simple diffusion? (A) peripheral proteins (B) signaling molecules (C) G proteins (D) G-protein-linked receptors (E) ribophorins 9. Examination of the blood smear of a young patient reveals misshapen red blood cells, and the pathology report indicates hereditary sphe- rocytosis. Defects in which one of the following proteins causes this condition? (A) Signaling molecules (B) G proteins • (C) Spectrin (D) Hemoglobin (E) Ankyrin (A) 02 (B) N2 (C) Na + (D) Glycerol (E) C02 10 Plasma Membrane [ 1 1 Directions: Each of the numbered items or incomplete statements in this section is negatively phrased, as indicated by a capitalized word such as NOT, LEAST, or EXCEPT. Select the ONE lettered answer or completion that is BEST in each case. 10. All of the following statements concerning plasma membrane components are true, EXCEPT (A) G proteins are composed of three subunits (B) the glycocalyx is usually composed of phos- pholipids (C) channel proteins are not energy-dependent (D) gated channels are open only transiently (E) ankyrin binds to integrins of the red blood cell plasma membrane Directions: Each group of items in this section consists of lettered options followed by a set of numbered items. For each item, select the ONE lettered option that is most closely associated with it. Each lettered option may be selected once, more than once, or not at all. Questions 11-15 Questions 16-20 (A) Naч-K ч purp - (A) G (B)Glucose carrier protein driven by an Na gradient [ .(C) Gp ,(C) K ч leak channels (D) Voltage-gated Na ч channels- -E) Ligand-gated channels ... |Match each characteristic below with the - \tin that fits best. G pro- Match each characteristic below with the or channel that fits best. pump 16 Formation of inositol triphosphate 11. Maintenance of constant cell volume 12, Establishment of a potential difference across the cell membrane 13. Initiation of muscle contraction 14. Formation of action potentials '- 15. A type of symport 17. Hyperpolarization of rod cells 18. Synthesis of cAMP 19. Prevention of cAlVIP synthesis 20. Mental retardation due to decreased re- sponses to certain hormones Answers and Explanations 1-D. Snake venom usually blocks acetylcholine receptors, thus preventing depolarization of the muscle cell. The Na * and Ca ч channels are not incapacitated by snake venoms. 2-B. The fluidity of the lipid bilayer is decreased in three ways (1) by lowering the temperature, (2) by increasing the saturation of the fatty acyl tails of the phospholipid molecules, and (3) by increasing the membrane's cholesterol content. 12 / Cell Biology and Histology 3-C. Integral proteins are not only closely associated with the lipid bilayer but also tightly bound to the membrane. These proteins frequently span the entire thickness of the plasmalemma, and are thus termed transmembrane proteins. 4-C. Active transport requires energy. Facilitated diffusion, which is mediated by membrane pro- teins, and simple diffusion, which involves passage of material directly across the lipid bilayer, are types of passive transport. 5-C. Na ч and other ions require channel (carrier) proteins for their transport across the plasma membrane. The other substances are small, nonpolar molecules and small, uncharged polar molecules. The molecules can traverse the plasma membrane by simple diffusion. 6-D. The coupled transport of two different molecules in the same direction is termed symport. 7-B. Cells can communicate with each other by releasing signaling molecules, which attach to receptor molecules on target cells. 8-D. G-protein-linked receptors are sites where ACTH and some other signaling molecules attach. Binding of ACTH to its receptor causes G protein to activate adenylate cyclase, setting in motion the specific response elicited by the hormone. 9-C. Hereditary spherocytosis is caused by a defect in spectrin that renders the protein incapable of binding to band 4.1 protein, thus destabilizing the spectrin-actin complex of the cytoskeleton. Although defects in hemoglobin (the respiratory protein of erythrocytes) also cause red blood cell anomalies, hereditary spherocytosis is not one of them. 10-Bo The glycocalyx (the sugar coat on the membrane surface) is composed mostly of polar carbohydrate residues. ll-& The main function of the Naч-K ч pump is to maintain constant cell volume by reducing the ion concentration intracellularly and increasing it extracellularly, thus decreasing the flow of water into the cell. 12oC. K ч leak channels are primarily responsible for establishing the potential difference across the plasma membrane. 13-E. Acetylcholine receptors, which are ligand-gated channels, initiate muscle contraction. 14-Do Voltage-gated Na + channels function in the formation of action potentials. I-B. Glucose transport into epithelial cells is mediated by Na+-powered glucose symports. 16-Co Inositol triphosphate is one of the by-products of the hydrolysis of the membrane lipid phosphatidylinositolbisphosphate. The enzyme responsible for this hydrolysis is phospholipase C, which is activated by Gp protein. 17-D. Absorption of a photon by rhodopsin causes it to form a bond with Gt, resulting in activation of cGMP phosphodiesterase, the enzyme that reduces cGMP levels in the rod cytosol. As a result, the rod plasma membrane becomes hyperpolarized, and the rod is no longer able to inhibit the postsynaptic cell. 18-/ G protein activates the enzyme adenylate cyclase, facilitating the formation of cAMP. 19-B. G, protein inhibits adenylate cyclase, preventing the formation of cAMP. 20-/ Hereditary defects in Gs protein can cause decreased responsiveness to certain hormones, resulting in mental retardation, diminished growth, and diminished sexual development. 2 Nucleus I. Overview--The Nucleus (Figure 2.1) A. Structure. The nucleus includes the nuclear envelope, nucleolus, nu- cleoplasm, and chromatin and contains the genetic material encoded in the deoxyribonucleic acid (DNA) of chromosomes. B. Function. The nucleus directs protein synthesis in the cytoplasm via ribo- somal ribonucleic acid (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA). All forms of RNA are synthesized in the nucleus. HC NU EC P P Figure 2.1. Electron micmgraph of the cell nucleus. Note that the nuclear envelope is interrupted by nuclear pores (P). The inactive hetemchmmatin (HC) is dense and mostly confined to the periphery of the nucleus. Euchmmatin (EC), the active form, is less dense and dispersed throughout. The nucleolus (NU) contains fibrillar and granular portions. 13 14 / Cell Biology and Histology Nuclear pore complex Cytoplasmic ring subunit \ /ThiC:ent . OmU:me:nCl:ar Scaffold" XX \\ II // // \ "Inner nuclear ..... / _///// / membrane Nucleoplasmic ring subunit Basket "Transporter subunit Figure 2.2. Diagram of the nudear pore complex. (Adapted with permission from Alberts B, Bray D, Lewis J, et ai: Molecular Biology of the Cell, 3rd ed. New York, Garland Publishing, 1994, p 562.) II. Nuclear Envelope (Figure 2.2). The nuclear envelope surrounds the nuclear material and consists of two parallel membranes separated from each other by a narrow perinuclear cisterna. It is perforated at intervals by openings called nuclear pores. Outer nuclear membrane 1. This membrane is about 6 nanometers (nm) thick. 2. It faces the cytoplasm and is continuous at certain sites with the rough endoplasmic reticulum (RER). 3. A loosely arranged mesh of intermediate filaments (vimentin) surrounds the outer nuclear membrane on its cytoplasmic aspect. 4. Ribosomes stud the cytoplasmic surface of the outer nuclear membrane (see Figure 2.1). These ribosomes synthesize proteins that enter the perinu- clear cisterna. Inner nuclear membrane 1. The inner nuclear membrane is about 6 nm thick. 2. It faces the nuclear material but is separated from it and supported on its inner surface by the nuelear lamina, a fibrous lamina that is 80-300 nm thick and composed primarily of lamins A, B, and C. These intermediate filament proteins help organize the nuclear envelope and perinuclear chro- matin. C. Perinuclear cisterna 1. The perinuclear cisterna is located between the inner and outer nuclear membranes and is 20-40 nm wide. Nucleus [ 15 2. It is continuous with the cisterna of the rough endoplasmic reticulum (RER). 8. It is perforated by nuclear pores at various locations. D. Nuclear pores 1. Nuclear pores average 80 nm in diameter and number from dozens to thousands; they are associated with the nuclear pore complex. 2. They are formed by fusion of the inner and outer nuclear membranes. 8. They permit passage of certain molecules in either direction between the nucleus and cytoplasm via a 9-nm channel opening. E. Nuclear pore complex (NPC) (see Figure 2.2) 1. Structure. The NPC is composed of four protein elements. a. The scaffold surrounds the pore and supports the other elements. It maintains the pore and simple diffusion channels. b. The transporter (central hub) is the central protein ring responsible for transporting proteins into and out of the nucleus via receptor- mediated transport. c. Thick filaments (about 3 nm in diameter) project from the scaffold ring into the cytoplasm and may serve as a staging area prior to protein transport. d. The basket is a structure projecting from the scaffold ring into the nucleoplasm; it is thought to function in RNA transport. 2. Function. The NPC permits passive movement across the nuclear enve- lope via a 9-nm open channel. Most proteins, regardless of size, pass in either direction only by active transport. These proteins have clusters of certain amino acids known as nuclear localization segments (NLS) that act as signals for transport. 3. Transport mechanisms a. Nuclear import (1) Nuclear import is initiated when the NLS of the transported protein binds to the a subunit of importin, a heterodimer receptor located in the cytoplasm, and the subunit of importin docks with the nuclear pore complex. Energy for the transport is provided by hydrolysis of GTP. (2) Once transported, importin dissociates. The subunit stays with the imported protein, whereas the subunit is rapidly returned to the cytosol. (3) The a subunit then dissociates from the substrate and returns to the cytosol in an unknown manner. b. Nuclear export (1) The following describes a possible model. 16 / Cell Biology and Histology III. (2) (3) (a) An intron in HIV-1 (a retrovirus) RNAs produces a Rev re- sponse element (RRE) that permits several Rev molecules to bind at a site containing an "activation"domain, which is the nuclear export signal (NES). The NES is thought to be analogous to the NLS of nuclear import. (b) When coupled to heterologous proteins, the RNAs are ex- ported. Other protein candidates for NESs include human Rev-inter- acting protein (hRip) and Rev activation domain-binding protein (Rab). Several hnRNP proteins also appear to function as shuttling pro- reins (e.g., the M9 portion of hn RNPA1 acts as both NES and NLS signals). Nucleolus Structure. The nucleolus is a well-defined, but not membrane-bound, nuclear inclusion present in cells that are actively synthesizing proteins; more than one nucleolus can be present in the nucleus. It is generally detectable only when the cell is in interphase. It contains nucleolar organizers, regions of some chromosomes (in humans, chromosomes 13, 14, 15, 21, and 22) where rRNA genes are located; these regions are involved in reconstituting the nucleolus during the G1 phase of the cell cycle. The nucleolus contains three distinct regions. 1. Fibrillar centers are composed of inactive ribosomal DNA (rDNA) where DNA is not being transcribed. 2. Fibrillar regions are composed of 5-nm fibrils surrounding the fibrillar centers and contain transcriptionally active rDNA and the rRNA pre- cursors that are being transcribed. 3. Granular regions are composed of 15-nm maturing ribosomal pre- cursor particles. B. Function. The nucleolus is involved in the synthesis of rRNA and its assembly into ribosome precursors. Nucleoplasm. Nucleoplasm is the protoplasm within the nuclear envelope. It consists of a matrix and various types of particles. A. Nuclear matrix acts as a scaffold that aids in organizing the nucleoplasm. 1. Structural components include fibrillar elements, nuclear pore-nuclear lamina complex, residual nucleoli, and a residual ribonucleoprotein (RNP) network. o Functional components are involved in the transcription and processing of mRNA and rRNA, steroid receptor-binding sites, carcinogen-binding sites, heat-shock proteins, DNA viruses, and viral proteins (T antigen). Nucleus B. Nuclear particles 1. Interchromatin granules are clusters of irregularly distributed parti- cles (20-25 nm in diameter) that contain ribonucleoprotein and various enzymes. 2. Periehrmatin granules (see Figure 2.1) are single dense granules (30- 50 nm in diameter) surrounded by a less dense halo. They are located at the periphery ofheterochromatin and exhibit a substructure of 3-nm packed fibrils. a. Perichromatin granules contain 4.78 RNA and two peptides similar to those found in heterogeneous nuclear ribonucleoprotein particles. b. They may represent messenger ribonueleoprotein particles (mRNPs). e. The number of granules increases in liver cells exposed to carcinogens or temperatures above 37°C. 3. Heterogeneous nuclear ribonucleoprotein particles (hnRNPS) are complexes of precursor mRNA (pre-mRNA) and proteins and are involved in processing of pre-mRNA. 4. Small nuclear ribonucleoproteins (snRNPs) are complexes of pro- teins and small RNAs and are involved in hnRNP splicing or in cleavage reactions. Chromatin (see Figure 2.1) A. Structure. Chromatin consists of double-stranded DNA complexed with his- tones and acidic proteins. It resides within the nucleus as heterochromatin and euchromatin. The euchromatin]heterochromatin ratio is higher in malig- nant cells than in normal cells. 1. Heteroehromatin is concentrated at the periphery of the nucleus and around the nucleolus, as well as scattered throughout the nucleoplasm. a. When examined under the light microscope (LM), it appears as baso- philic clumps of nucleoprotein. b. Under the transmission electron microscope (TEM), it appears as dense granular clumps of nucleoprotein that are transcriptionally inac- tive. However, recent evidence indicates that heterochromatin plays a role in interchromosomal interactions and chromosomal segregation during meiosis. c. Heterochromatin corresponds to one of two X chromosomes and is therefore present in nearly all somatic cells of female mammals. During interphase, the inactive X chromosome is visible as a dark-staining body within the nucleus. This structure is called the Barr body, or sex chromatin. 2. Euchromatin is the transcriptionally active form of chromatin that appears in the LM as a lightly stained region of the nucleus. It appears in TEM as electron-lucent regions among heterochromatin. 18 / Cell Biology and Histology DNA ) Extended section Figure 2.3. Diagram showing the packaging of chromatin into the condensed metaphase chromosome. Nucleo- somes contain two copies of histones H2A, H2B, H3, and H4 in extended chromatin. An additional histone, H1, is present in condensed chromatin. (Adapted with permission from Widnell CC and Pfenninger KH: Essential Cell Biology. Baltimore, Williams & Wiikins, 1990, p 47.) B. Function. Chromatin is responsible for RNA synthesis. V/. Chromosomes Structure. Chromosomes consist of chromatin extensively folded into loops; this conformation is maintained by DNA-binding proteins (Figure 2.3). Each chromosome contains a single DNA molecule and associated proteins, assem- bled into nueleosomes, the structural unit of chromatin packaging. Chromo- somes are only visible during mitosis and meiosis when their chromatin con- denses. 1. Extended ehromatin forms the nucleosome core around which the DNA double helix is wrapped two full turns. a. The nucleosome core consists of two copies each of histories It2A, It2B, H3, and H4. Nucleosomes are spaced at intervals of 200 base pairs. When viewed with TEM, extended chromatin resembles "beads on a string"; the beads represent nueleosomes and the string represents linker DNA. 2. Condensed chromatin contains an additional histone, H1, which wraps around groups of nucleosomes forming 30-nm-diameter fibers, the struc- tural unit of the chromosome. Nucleus / 19 G-banding is observed in chromosomes during mitosis after staining with Giemsa, which is specific for DNA sequences rich in adenine (A) and thymine (T). Banding is thought to represent highly folded DNA loops. G-banding is used to identify chromosomes and is characteristic for each species. Karyotype refers to the number and morphology of chromosomes and is characteristic for each species. 1. Haploid number (n) is the number of chromosomes in germ cells (23 in humans). 2. Diploid number (2n) is the number of chromosomes in somatic cells (46 in humans). D. Genome is the total genetic complement of an individual stored in its chromosomes. In humans, the genome consists of 22 pairs of autosomes and 1 pair of sex chromosomes (either XX or XY), totaling 46 chromosomes. VII. DNA. It is a long, double-stranded, linear molecule composed of multiple nucleo- tide sequences. It acts as a template for the synthesis of RNA. A. Nucleotides are composed of a base (purine or pyrimidine), a deoxyribose sugar, and a phosphate group. 1. The purines are adenine (A) and guanine (G). 2. The pyrimidines are cytosine (C) and thymine (T). B. The DNA double helix consists of two complementary DNA strands held together by hydrogen bonds between the base pairs A-T and G-C. C. Exons are regions of the DNA molecule that code for specific RNAs. D. Introns are regions of the DNA molecule, between exons, that do not code for RNAs. E. A codon is a sequence of three bases in the DNA molecule that codes for a single amino acid. F. A gene is a segment of the DNA molecule that is responsible for the formation of a single RNA molecule. RNA. It is a linear molecule similar to DNA except that it is single stranded and contains ribose instead of deoxyribose and uracil (U) instead of thymine. RNA is synthesized by transcription of DNA. Transcription is catalyzed by three RNA polymerases: I for rRNA, II for mRNA, and III for tRNA. A. Messenger RNA carries the genetic code to the cytoplasm to direct protein synthesis (Figure 2.4). 1. This single-stranded molecule consists of hundreds to thousands of nucleo- tides. 20 [ Cell Biology and Histology DNA (' 1 Pre-mRNA NN Nuclear processing binding proteins Ribosomes ' mRNA transport , Translation of mRNA Protein Figure 2.4. Diagram showing steps by which genetic information encoded in DNA is transcribed into mRNA and ultimately converted into proteins in the cytoplasm. (Adapted with permission from Alberts B, Bray D, Lewis J, et al: Molecular Biology of the Cell, 2nd ed. New York, Garland Publishing, 1989, p 482.) 2. mRNA contains codons that are complementary to the DNA codons from which it was transcribed, including one codon (AUG) for initiating protein synthesis and one of three codons (UAA, UAG, or UGA) for terminating protein synthesis. 3. mRNA is synthesized in the following series of steps. a. RNA polymerase II recognizes a promoter on a single strand of the DNA molecule and binds tightly to it. Nucleus ] 2"] IXo b. The DNA helix unwinds about two turns, separating the DNA strands and exposing the codons that act as the template for synthesis of the complementary RNA molecule. c. RNA polymerase II moves along the DNA strand and promotes base pairing between DNA and complementary RNA nucleotides. d. When the RNA polymerase II recognizes a chain-terminator on the DNA molecule, it terminates its association with the DNA and is re- leased to repeat the process of transcription. e. The primary transcript, pre-mRNA, associates with proteins to form an hnRNP. f. Introns are excised and exons are spliced, producing an mRNP. go Proteins are removed as the mRNP enters the cytoplasm, resulting in a functional mRNA. B. Transfer RNA is folded into a cloverleaf shape and contains about 80 nucleo- tides, terminating in adenylic acid (where amino acids attach). 1. Each tRNA combines with a specific amino acid that has been activated by an enzyme. 2. One end of the tRNA molecule possesses an anticodon, a triplet of nucleo- tides that recognizes the complementary codon in mRNA. If recognition occurs, the anticodon insures that the tRNA transfers its activated amino acid molecule in the proper sequence to the growing polypeptide chain. C. Ribosomal RNA associates with many different proteins (including enzymes) to form ribosomes. 1. rRNA associates with mRNA and tRNA during protein synthesis. 2. rRNA synthesis takes place in the nucleolus and is catalyzed by RNA polymerase I. A single 458 precursor rRNA (pre-rRNA) is formed which is then processed to form ribosomes as follows (Figure 2.5). a. Pre-rRNA associates with ribosomal proteins and is cleaved into the three sizes (28S, 18S, and 5.8S) of rRNAs present in ribosomes. bo The RNP containing 28S and 5.8S rRNA then combines with 5S rRNA, which is synthesized outside of the nucleolus, to form the large subunit of the ribosome. c. The RNP containing 18S rRNA forms the small subunit of the ri- bosome. Cell Cycle (Figure 2.6) A. The cell cycle varies in length in different types of cells but is repeated each time a cell divides. 1. It is temporarily suspended in nondividing resting cells (e.g., peripheral lymphocytes), which are in the Go state. Such cells may reenter the cycle and begin to divide again. Cell Biology and Histology O0 0 Ribosomal proteins made in cytoplasm 5S rRNA made outside nuoleolus CYTOPLASM NUCLEUS Immature large subuni Large subunit • 0- Small subunit Transport and activation creates functional ribosomes Large subunit Large subunit of ribosome Small subunit Small subunit of ribosome Figure 2.5. Diagram showing formation of rRNA and its processing into ribosomal subunits, which occurs In the nucleolus. (Adapted with permission from Alberts B, Bray D, Lewis J, et al: Molecular Biology of the Cell, 2rid ed. New York, Garland Publishing, 1989, p 542.) 8 Nucleus ] 23 Nondividing cells Resting cells Figure 2.6. Diagram showing stages of the cell cycle in dividing cells. Differentiated cells that no longer divide have left the cycle, whereas resting cells in the Go state may reenter the cycle and begin dividing again. (Adapted with permission from Widnell CC and Pfenninger KH: Essential Cell Biology. Baltimore, Williams & Wilkins, 1990, p 58.). 2. It is permanently interrupted in differentiated cells that do not divide (e.g., cardiac muscle cells and neurons). B. Two major periods, interphase (interval between cell divisions) and mitosis (M phase, the period of cell division) comprise the cell cycle. 1. Interphase is considerably longer than the M phase and is the period during which the cell doubles in size and DNA content. ao Interphase is divided into three separate phases (G, S, and G2) during which specific cellular functions occur. (1) G phase (gap one phase) lasts from hours to several days. (a) Occurring after mitosis, it is the period during which the cell grows and proteins are synthesized, thus restoring the daugh- ter cells to normal volume and size. (b) Certain "trigger proteins" are synthesized; these proteins en- able the cell to reach a threshold (restriction point) and proceed to the S phase. Cells that fail to reach the restriction point become resting cells and enter the Go state. (2) S phase (synthetic phase) lasts 8-12 hours in most cells. (a} DNA is replicated and proteins are synthesized, resulting in duplication of the chromosomes. (b) Centrioles are also replicated. (3) G2 phase (gap two phase) lasts 2-4 hours. (a) This phase follows the S phase and extends to mitosis. 24 ] Cell Biology and Histology (b) The cell prepares to divide: the centrioles grow to maturity; energy required for the completion of mitosis is stored; and RNA and proteins necessary for mitosis are synthesized. b. Several control factors have been identified. (1) S-phase activator triggers the initiation of DNA synthesis. (2) M-phase delaying factor, which may be identical to S-phase activator, inhibits formation of the M-phase promoting factor and thus prevents the cell from entering the M-phase until all of the DNA has been replicated. (3) M-phase promoting factor induces the cell to enter the M- phase. 2. Mitosis (Figure 2.7) lasts 1-3 hours. It follows the G2 phase and completes the cell cycle. Division of the nucleus (karyokinesis) and cytoplasm (cyto- kinesis) results in the production of two identical daughter cells. It consists of five major stages. a. Prophase begins when the chromosomes condense; during prophase, the nucleolus and nuclear envelope begin to disappear (1) The centrosome contains centrioles and is the principal micro- tubule-organizing center (MTOC) of the cell. Centrioles mi- grate to opposite poles of the cell and give rise to the spindle fibers and astral rays of the mitotic spindle. (2) Chromosomes consist of two parallel sister chromatids (future daughter chromosomes) attached at the centromere, a constric- tion along the chromosome. Kinetochores develop at the centro- mere region and function as MTOCs. entdoles (two pairs) leolus A. Interphase B. Eady prophase C, Late prophase D. Prometaphase E. Metaphase F. Anaphase G. Late anaphase H. Late telophase Figure 2.7. Diagram illustrating events in various phases of mitosis. (Redrawn with permission from Kelly DE, Wood RL, and Enders AC: Bailey's Textbook of Microscopic Anatomy, 18th ed. Baltimore, Williams & Wilkins, 1984, p 89.) Nucleus b. Prometaphase begins when the nuclear envelope disappears. (1) The kinetochores complete development and attach to specific spin- dle microtubules, forming kinetochore microtubules. (2) Spindle microtubules that do not attach to kinetochores are called polar microtubules. c. Metaphase is the phase during which the condensed chromosomes align at the equatorial plate of the mitotic spindle. d. Anaphase begins as the chromatids separate (at the centromere) and daughter chromosomes move to opposite poles of the cell. (1) The spindle elongates. (2) In the later stages of anaphase, a cleavage furrow begins to form around the cell due to contraction of a band of actin filaments called the contractile ring. eo Telophase is characterized by a deepening of the cleavage furrow; the midbody (containing overlapping polar microtubules) is now located between the newly forming daughter cells. (1) Microtubules in the midbody are depolymerized, facilitating cyto- kinesis and formation of two identical daughter cells. (2) The nuclear envelope is reestablished around the condensed chromosomes in the daughter cells and nucleoli reappear. Nucleoli arise from the specific nucleolar organizer regions (called secondary constriction sites), which are carried on five separate chromosomes in humans. (3) The daughter nuclei gradually enlarge and the condensed chromo- somes disperse to form the typical interphase nucleus with hetero- chromatin and euchromatin. X. Meiosis (Figure 2.8) A. Meiosis is a special form of cell division in which the chromosome number is reduced from diploid (2n) to haploid (n). 1o It occurs in developing germ cells in preparation for sexual reproduction. Subsequent fertilization results in diploid zygotes. 2° The DNA content of the original diploid cell is doubled in the S phase preparatory to meiosis. a. This phase is followed by two successive cell divisions that give rise to four haploid cells. b. In addition, recombination of maternal and paternal genes occurs by crossing-over and random assortment, yielding the unique haploid genome of the gamete. B° Stages of meiosis 1. Reductional division (meiosis I) occurs after interphase when the 46 chromosomes are duplicated, giving the cell a 4CDNA content (considered to be the total DNA content of the cell). 26 ] Cell Biology and Histology 4CDNA -- 4CDNA-- 2CDNA-- 2CDNA-- - 1CDN Figure 2.8. Diagram Illustrating meiosis in men and women. Spermatogenesis in the male gives rise to sperm, each containing the haploid number of chromosomes. Oogenesis in the female gives dse to an ovum with the haploid number of chromosomes. Fertilization reconstitutes the diploid number of chromosomes in the resulting zygote. (Adapted with permission from Widnell CC and Pfenninger KH: Essential CelIBiology. Baltimore, Williams & Wilkins, 1990, p 69.) ao Prophase I is divided into five stages (leptotene, zygotene, pachytene, diplotene and diakinesis) which accomplish the following events. (1) Chromatin condenses into the visible chromosomes, each containing two chromatids joined at the centromere. (2) Homologous maternal and paternal chromosomes pair via the syn- aptonemal complex, forming a tetrad. Crossing-over (random exchanging of genes between segments of homologous chromo- somes) occurs at the chiasmata, thus increasing genetic di- versity. (3) The nucleolus and nuclear envelope disappear. Metaphase I (1) Homologous pairs of chromosomes align on the equatorial plate of the spindle in a random arrangement, facilitating genetic mixing. Nucleus (2) Spindle fibers from either pole attach to the kinetochore of any one of the chromosome pairs, thus assuring that genetic mixing occurs. c. Anaphase I (1) This phase is similar to anaphase in mitosis except that each chromo- some consists of two chromatids that remain held together. (2) Chromosomes migrate to the poles. d. Telophase I is similar to telophase in mitosis, in that the nuclear envelope is reestablished and two daughter cells are formed via cytoki- nesis. (1) Each daughter cell now contains 23 chromosomes (n) number, but has a 2CDNA content (the diploid amount). (2) Each chromosome is composed of two similar sister chromatids (not genetically identical). 2. Equatorial division (meiosis II) begins soon after the completion of meiosis I, following a brief interphase without DNA replication. a. The sister chromatids are portioned out among the two daughter cells formed in meiosis I. The two daughter cells then divide, resulting in the distribution of chromosomes into four daughter cells, each con- taining its own unique recombined genetic material (1CDNA;n). b. The stages of meiosis II are similar to those of mitosis; thus the stages are named similarly (prophase II, metaphase II, anaphase II, and telo- phase II). c. Meiosis II occurs more rapidly than mitosis. XI. Clinical Considerations A. Aneuploidy is defined as an abnormal number of chromosomes and can be detected by karyotyping. Examples include trisomy (the presence of a third chromosome of one type) and monosomy (the absence of one member of a chromosome pair). 1. Down syndrome (trisomy 21) is characterized by mental retardation, short stature, stubby appendages, congenital heart malformations, and other defects. Klinefelter syndrome (XXY) is aneuploidy of the sex chromosomes, characterized by infertility, variable degrees of masculinization, and small testes. 3. Turner syndrome (XO) is monosomy of the sex chromosomes charac- terized by short stature, sterility, and various other abnormalities. B. Transformed cells 1. Transformed cells have lost their ability to respond to regulatory signals controlling the cell cycle. They may undergo cell division indefinitely, thus becoming cancerous. Cell Biology and Histology Co 2. Vinca alkaloids may arrest these cells in mitosis; drugs that block purine and pyrimidine synthesis may arrest cells in the S phase of the cell cycle. Oncogenes represent mutations of certain regulatory genes, called proto-oncogenes, which normally stimulate or inhibit cell proliferation and development. L Genetic accidents or viruses may lead to the formation of oncogenes. 2. Oncogenes dominate the normal alleles (proto-oncogenes), causing a dereg- ulation of cell division, which leads to a cancerous state. 3. Bladder cancer and acute myelogenous leukemia are caused by oncogenes. Review Test Directions-- Each of the numbered items or incomplete statements in this section is followed by answers or by completions of the statement. Select the ONE lettered answer or completion that is BEST in each case. 1. The nuclear pore complex (A) permits free communication between the nu- cleus and the cytoplasm (B) is bridged by a unit membrane (C) is located only at specific nuclear pore sites (D) permits passage of proteins via receptor- mediated transport 2. Which one of the following nucleotides is pres- ent only in RNA? (A) Thymine (B) Adenine (C) Uracil (D) Cytosine (E) Guanine 3. Anticodons are located in (A) mRNA (B) rRNA (C) tRNA (D) snRNP (E) hnRNP 4. DNA is duplicated in the cell cycle during the (A) G phase (B) S.phase (C) M phase (D) G1 phase (E) Go phase 5. During mitosis, sister chromatids begin their migration to opposite poles during (A) telophase (B) metaphase (C) anaphase (D) prophase 6. An individual who exhibits the characteristics of Down syndrome is (A) aneuploid for chromosome 21 (B) aneuploid for the X chromosome (C) monoploid for chromosome 21 (D) monoploid for the Y chromosome 7. Transformed cells become malignant be- cause they (A) have a reduced amount of RNA polymerase (B) are unable to respond to regulatory signals in the cell cycle (C) cease mitotic activity (D) contain extra chromosomes (E) contain transcriptionally inactive hetero- chromatin 8. Microscopic observations of the proportions of the two types of chromatin in the nuclei of tissue sections can often be used to (A) determine the karyotype (B) identify the genome (C) diagnose Turner syndrome (D) identify DNA viruses (E) distinguish benign from malignant cells Cell Biology and Histology Directions: Each of the numbered items or incomplete statements in this section is negatively phrased, as indicated by a capitalized word such as NOT, LEAST, or EXCEPT. Select the ONE lettered answer or completion that is BEST in each case. 9. Which one of the following statements con- 10. Which one of the following statements con- cerning meiosis I is NOT true? cerning the human karyotype is NOT true? (A) Prophase I is divided into five stages: lepto- tene, zygotene, pachytene, diplotene, and diakinesis (B) During metaphase I, homologous chromo- somes line up at the equator in random fashion (C) The chromosome number is reduced from the diploid to the haploid number (D) During telophase I, each chromosome con- sists of one chromatid and a centromere (E) Diakinesis occurs at the end of prophase I (A) A normal human karyotype consists of 46 chromosomes (B) There are 22 pairs of autosomes in humans (C) There is one pair of sex chromosomes in women (D) Aneuploidy is considered to be a normal karyotype Directions: Each group of items in this section consists of lettered options followed by a set of numbered items. For each item, select the ONE lettered option that is most closely associated with it. Each lettered option may be selected once, more than once, or not at all. Questions 11-15 Questions 16-20 (A) Nuclear pore complex (B) Nucleolus (C) Heterochromatin (D) Outer nuclear membrane (E) Euchromatin Match the numbered items with the following structures. 11. An inclusion not bounded by a membrane that is observable only during interphase 12. Continuous with rough endoplasmic re- ticulum 13. Controls movement of proteins in and out of the nucleus 14. Site of transcriptional activity Match each description with the corresponding lettered structure in the micrograph of a nucleus. 15. Clumps of nucleoprotein concentrated near the periphery of the nucleus 16. Transcriptionally inactive chromatin 17. A fibrous layer containing lamins A, B, and C 18. Site of rRNA synthesis 19. Transcriptionally active chromatin 20. Passageway for transfer of materials Answers and Explanations 1-D. The nuclear pore complex contains a central aqueous channel, which permits passage of small, water-soluble molecules. However, movement of proteins in and out of the nuclens is selectively controlled by the nuclear pore complex via recepter-mediated transport. 2-C. DNA contains the purines, adenine and guanine, and the pyrimidines, cytosine and thymine. In RNA, uracil, a pyrimidine, replaces thymine. 8-C. Each tRNA possesses a triplet of nucleotides, called an anticodon, which recognizes the complementary codon in mRNA. 4-B. The S (synthesis) phase of the cell cycle is the period during which DNA replication and histone synthesis occur, resulting in duplication of the chromosomes. At the end of the S phase, each chromosome consists of two identical chromatids attached to one another at the centromere. 5-C. Anaphase begins when the sister chromatids separate and begin their migration to opposite poles of the cell. 6-A. Aneuploid is the term applied to an abnormal number of chromosomes in the genome. In Down syndrome, the individual has three number 21 chromosomes (trisomy 21), rather than the normal two. 7-B. Cells that lose their ability to respond to regulatory signals operating in the cell cycle are said to be transformed and ultimately become malignant. 8-E. The ratio of euchromatin to heterochromatin may be helpful in distinguishing benign from malignant cells, which are characterized by rapid, uncontrolled proliferation. Since euchromatin is transcriptionally active, whereas heterochromatin is not, cells with a high euchromatin-heterochro- matin ratio may be malignant. 9-D. During metaphase I of meiosis, homologous chromosomes line up at the equator of the spindle. The homologous chromosomes dissociate at anaphase I. At telophase I, the chromosomes, each consisting of two chromatids, arrive at the pole. Although the chromosomes have been reduced to the haploid number, the DNA content is 2CDNA (diploid amount). 10-D. The human karyotype contains 46 chromosomes, represented by 22 pairs of autosomes and 1 pair of sex chromosomes. Aneuploidy represents an abnormal number of chromosomes in the genome--either of the autosomes (e.g., in Down syndrome) or of the sex chromosomes (e.g., in Klinefelter and Turner syndromes). ll-B. The nucleolus is an inclusion not bounded by a membrane located within the nucleus. It is observable during interphase, but disappears during mitosis. 12-D. The outer nuclear membrane is continuous with the rough endoplasmic reticulum. 13-A. The nuclear pore complex selectively controls movements of water-soluble molecules and proteins in and out of the nucleus. 14-E. The pale-staining euchromatin is the transcriptionally active chromatin in the nucleus. 15-C. Heterochromatin is the dark-staining nucleoprotein located near the periphery of the nucleus. It is transcriptionally inactive but may be responsible for proper chromosome segregation during meiosis. 31 Cell Biology and Histology 16-E. Inactive chromatin, called heterochromatin, is dense and concentrated at the periphery of the nucleus. 17-A. The nuclear lamina is the cytoskeletal component located just inside the inner nuclear mem- brane. 18-D. Synthesis of rRNA and assembly of the ribosomal subunits occur in the nucleolus. 19-C. Euchromatin, the transcriptionally active form of chromatin, appears less dense than hetero- chromatin in electron micrographs. 20-Bo The nuclear envelope is interrupted by nuclear pores through which materials can be trans- ported in and out of the nucleus. 3 Cytoplasm II. Overview--The Cytoplasm. The cytoplasm contains three main struc- tural components: organelles, inclusions, and the cytoskeleton. The fluid component is called the cytosol. The functional interactions among certain organ- elles result in the uptake and release of material by the cell, protein synthesis, and intracellular digestion. Structural Components A. Organelles (Figure 3.1) are metabolically active units of living matter; they are usually bound by a membrane. 1. The plasma membrane is discussed in Chapter 1. 2: Ribosomes a. Structure. Ribosomes are 12 nanometers (nm) wide and 25 nm long and consist of a small and large subunit. The subunits are composed of several types ofrRqA and numerous proteins 1; see Figure 2.5). b..Ribosomes may exist free in the c or bound to membranes of the .oplasmic reticulum or outer nuclear mere rane. e er free or bound, the ribosomes constitute a single interchangeable population. c. /Lpolyribosome (polysome) is a cluster of ribosomes along a single strand of mRNA; pdlyribosomes have a spal confi d. Function. Ribosomes are the sites where mRNA is translated into (1) The small ribosomal subunit binds mRNA and activated tRN__R_NAs" the codons of the mRNA then bar with spondlng antlcodons of the___tRNA.. (2) The hrge ribosonml subunit then_ binds_to_the complex. Pe dyl transferase in the large subtmit catalyzes ]eptide-bond for- mat]on resuit]ng in addition o'" :--" amino acids to th-- growing polypep- tide chain. (3) A chain-termin __AA, UAG, or UGA) causes re- _ .la_se of the polypeptide from_ .h_e ri_bosSme, and thfi-osom units dissociate from the mRA_-- 33 34 ] Cell Biology and Histology GOLGI COMPLEX Vacuoles Concentrating Secretory Product Agronulor Membranes Vesicles ,=,,.__.=:. M e m b r o n e SECRETION Poducl GRANULES 0 Cross Section " % CENTRIOLES ERGASTOPLASM iboso esi I Perinu star NUCLEOLUS Ribosomes Gronules HONDRION ImiflOr Unil Membtone ! / PLASMA Glycogen MEMBRANE Filomenls ( 450 ) LIPID PARTICULATES DROPLETS of CYTOPLASMIC MATRIX Figure 3.1. Diagram of a eukaryotic cell and its major organelles and inclusions. (Adapted with permission from Fawcett DW: Bloom and Fawcett's Textbook of Histology, 12th ed. New York, Chapman & Hall, 1994, p 2.) Table 3.1. Ribosome Composition Subunit Large (60S) Small (40S) Number of rRNA types proteins 5S 49 5.8S 28S 18S 33 Cytoplasm [ 35 3. Rough eneloplasmic reticulum (RER) [see Figure 3.1]• a. Structure. RER is a system of sacs, or cavities, bounded by mem- branes. The outer surface of RER is studded with ribosomes (thus appearing rough). The interior rRER is calLe, d_the cisterna. The outer nuclear membrane is continuous with the RER m thubg]fig h-iucla2 cisrn-h-dbntinuity with the c]st-rne of the RER. The REaR _menbrane also___has rc_eptors ibophorin. si in its membrane to which the large ribosomal subunit binds. b. RER is abundant in cells synthesizing secretory protei__; in such cells, the RER is organized into many parallel arrays_. c. Function. The RER is the site where membrane-packageel pro- te_e_ns are synthesized, including secretory, plasma-membrane, and lyso- 4. Smooth endoplasmic reticulum (SER) [see Figure 3.1] a. StructureR is an_irregular netw0rkf__m_membrane-bounded_aha- a lkfi)ribosomes on its surface (thus.aplaring smooth). b. It usually appears as branching anastomosing tubules, or vesicles, whose membranes do not contain ribophorins____ rr_xe/ c. SER is 1.ess common th___an RER but is prominent in cells synthesizing seroi___._ds, triglycerides, and co--lesterol. el. Function. SER has different functions in different cell types. (1) Steroiel hormone synthes__is occurs in SER-rich cells such as the Leyd[-delIs of te testis, which make testosterone__. (2) Drug de_toxification occurs ine)atocy__tes followin, g proliferation of the E n response to phenobarbital; oxidases that metabolize "his drug are present in_the_SER. (3) Muscle contraction and relaxation involves the release and r__ecapture of calcium ions by the sarcoplasmic reticulum--(fh_term for SER in skeletal muscle cells). 5. Annulate lamellae a. Structure. Annulate lamella are parallel stacks of membranes (usually 6TWhich resemble the nuclea envelope, including its or_p_m 5 • hey are often arrag_ed with their annuli (pore____s) in registe-- and are frequently continuous with the RER.- b. Function. Annulate lamellae are found in rapidly growing cells (e.g., .germ cells, .e_m_b_rqp_ic _ce.s, and tumor cells), but their function and significance rema!n unknown. 6. Mitochondria (see Figure 3.1) a. Structure. Mitochondria are rod-shaped organelles [0.2 micrometers (Lm) wide and up to 7 p;m long]. They possess an_o2ater membrane, which su__rrounds the organelle, and an inner__membrane_, which invaginates---o _form cristae. They are subdivided into an intermembrn-mpart- ment, which is located between the two membranes, and r m-ta fix corn arp__a_nt. Granules within the matrix bind the divalent • cativns Mg u and Ca "ч. .... "------ 36 [ Cell Biology and Histology Cytoplasm Outer mitochondrial membrane H + H ч . +H + H + .+ H + + H + H + H + . + H + Intermembranespace H + H +H + + H + . H. H ч H +H H+H H+, H+1-t . H + • H + H +'' H + 1 " H + H + . H + =H + H* r-- -- \o, ...... / I, ,. \-" ..... / H+ I H+ ' ..... -/ H+lH+ ADP +" Pi ATP + H20 ADP + Pi ATP + H20 ADP + Pi ATP + H20 Matrix space Figure 3.2. Chemiosmotic coupling mechanism for generating ATP in mitochondria. During electron transport, H* ions (protons) are pumped from the inner matrix compartment across the inner mitochondrial membrane into the intermembrane compartment. The electrochemical proton gradient thus created drives ATP synthase to catalyze the conversion of ADP and P to ATP. b. Enzymes and genetic apparatus. Mitochondria contain: (1) All the enzymes of the Krebs (TCA) cycle in the matrix, except for succinate dehydrogge, which is located on the "tochon- drial memJrae. (2) Elementary particles (visiblebn ne_Ka_tively sta) that -cShain ATP synthase, a special enzyme involved in coupling oxidatorylation of ADP to form ATP.-------- (3) A ge.n_eti_c_app__ar_atu_ i_nth__matT]_:composed of circular DNA, mRNA, tRNA____L, and._r_RN___A (with___a limited coding capacity). How- ever, most mitochondrial protein_____s are encoded by nuclear DNA.. c. Origin and proliferation (1) Mitochondria may have originated as symbionts (intracellular parasites). According to this theory, anaerobic eukaryotic cells endo- cytosized aerobic microorganism that evolved into mitochondria, which function in oxidative proce'sses. (2) Mitocte by n (fission) of preexisting mito- chondria and typically ]ave lO-day life sp-----an. d. Mitochondrial ATP synthesis (1) Mitochondria svnth ATP via tcle, which he----l energ ad produ-ces -I; btion of fatty cids, (2) ATP is also synthesized via a chemiosmotic coupling mecha- -n_i_sm_inv--lffig enzy-ine complexes of the electroK tansport chain_and elementary particles present in the cristae (Fig- ure 3.2). Cytoplasm e. Condensed mitochondria result from a_cpnformational change in the orthodox form (ypcal morphology). The change occurs in re= ' nse an uncoup]ing of (1) In condensed mitochondria, the size of the inner__compartment is decreased and the matr-density is increased. The intermnb-rane compartment is enlarged. (2) Condensed mitochondria are present in brown fat cells, which produce heat rather than ATP due to transport protein in their inner menbrane that uncouplration from ATP .synthesis (see Chapter 6 IV B 5 b). (3) They.swell in responseto calciu_m_, pho_sp_h__te , and thyroxine__, which inducean increas-n water uptake and an uncoupling of phosphory- lation; ATP reverses the swelling. 7. Golgi complex (apparatus) [see Figure 3.1] a. Structure. The Golgi complex consists of several disk-shaped cister- nae (saccules) arranged in a stack. Cisternae are slightly curved, with flat centers and dilated rims, but their size and shape vary. A distinct polarity exists across the cisternal stack. b. Regions (1) The cislgi network (CGN) of the Go_mp_lex consists of tubu____lar and cisternal elements that are located at the side of t_he Golgi stack facing the RER and small transfer vesicles; this region is involved in protein sorting and retrieval (see III B 1 c). (2} The cis (entry) face of the Golgi complex comprises the forming cisternae--nd islS-cated next to the CGN. (3) The medial compartment of the Golgi complex comprises a few cist_ern-lyl, bt--Khe___cisand trans faces. (4) The_tran_sxi_t) face of the Golgi complex comprises the maturing cisternae , which are located at the side of the stack facing vacuoles and secretory granules. (5) The trans Golgi network (TGN) lies.apart from the last cisterna attle-'rans a and s seeparated from the Golgi stack. It proteins Ior their final destinations: c. Functions. The Golgi complex processes membrane-packaged proteins synthesized in the RER and recycles and redistributes membranes. Coated vesicles are characterized by a visible cytoplasmic surface coat. At let tw'types of coated vesicles exist. no Clathrinocoated vesicles (1) Structure. These vesicles are coated with clathrin, which consists of three large and three small polypeptide chains that form a tri- skelion (three-legged structure). Thirty-six clathrin triskelions as- sociate to form a polyhedral cage-like lattice around the vesicle. Cell Biology and Histology (2) Function (a) These vesicles are formed during receptor-mediated up- take (endocytosis) of specific molecules by the cell. After uptake, the vesicles quickly lose their coats, and clathrin re- turns to the plasma membrane for recycling (Figure 3.3). (b) They also function in the signal-directed (regulated) transport of proteins from the trans Golgi network to lyso- somes or secretory granules. b. Coatomer-coated vesicles (1) Structure. These vesicles have coats consisting of coatomer, which does not form a cage-like lattice around vesicles. Coatomer is a large protein complex formed by individual coat protein subunits called COPs. (2) Function (a) Coatomer-coated vesicles mediate the continuous constitu- tive protein transport (default pathway; bulk flow) within the cell. (b) They also transport proteins from the RER to the Golgi com- plex, from one Golgi cisterna to another, and from the TGN to the plasma membrane. (i) COPII transports molecules from the RER to the Golgi (anterograde transport). (ii) COPI transports anterograde but also facilitates retro- grade transport (from the CGN, Golgi, and TGN back to the RER). 9. Lysosomes a. Structure. Lysosomes are _d2e, membrane-bound organelles of d_:_ verse shape and size. They may be identified in cell sections bztchem- ical staining for acid phosphatase. Lysosomes possess special mem- brane p_r_o_teins a-more than 4.0 hyffrolases, which are synthesized in _he: AWP-powered proton u- in their men-ra- ritin an acid pH (__. b. Formation. L.:sosomes are formed when seqtered material fuses - _with a late endosome [ enzymatic degradation begins. Formation of lys6SSngiath-lysosomal pathwa-(e Figffre 33) involves the following intermediates. (1) Early (a) Theser, eripherally located vesicles contain- ligand complexes. (b) They are also known as the compartme_nt for uncnpling of ._receltor and li__!mad (CURL)... (c) Their__acidic__terior (pHi6) are maintained byATP-__driven p__rot_o_n_umts. T_hhe acidity aids in the un_coupling of receptors a; receptors__return to the plasma membrane and ligands move o a late endosome. Cytoplasm [ 39 Receptors --- Ligand in solution for ligand. 1 Clathrin Clathrin triskelions meeCYmCblatn°ePlasma . ), pit Clathrin-coated endocytic vesicle /Plasma membrane ( Residual body Uncoated endocytic vesicle Multivesicular j--.T_.,-'.._ 0 L() body (type of / Early endosome ,) y lysosome) (CURL) pH 6.0 pH 5.0 " Recycling of Hydrolases q receptors to Lysosomal membrane plasma ( membrane Late endosome pH 5.5 Clathrin-coated vesicles containing lysosomal hydrolases or lysosomal membrane proteins Clathrin Tnsgolginetwork gculum Figure 3.3. Diagram illustrating receptor-mediated endocytosis of a ligand (e.g., low-density lipoproteins) and the lysosomal degradative pathway Clathrin triskelions quickly recycle back to the plasma membrane. The receptors and ligands then uncouple in the early endosome (CURL), which is followed by recycling of receptors back to the plasma membrane. The late endosome is the primary intermediate in the formation of lysosomes (e.g., multivesicular bodies.). Material that is phagocytosod or organelles that undergo autophagy do not use the eady endosomal pathway. Cell Biology and Histology (2) Late endosomes (a) They are also known as endolysosomethe intermediate co_mpart__nt. (b) Theseir.regular versicles (pH 5.5) can receive ligands from .early en_dosomes (either b vesiclransport or-by ar[v endm- somes "maturing" and becoming late endosomes). (c) Late endosomes contain both lysosomal hydrolases and lysosomal membrane proteins; these are fo the RER, transported to the Golgi complex for processing, and delivered in separate vesicl to late endosome--6-. c. Types of lysosomes (gee Figure 3.7). Lysosomes are named after the content of recognizable material; otherwise, the general term lysosome is used. (1) Multivesicular bodies are formed by_fusion of an ear_ endosome containing endocytic vesicles with a late endosome. (2) l-alysosomes are formed ly fusion o-a phago-ytic vacuole with a late endosome or a lysosome. (3) Autophagolysosomes are formed by fusion of an autophagic vacuole with a late endosome or lysosome. Autophagic vacuoles are formed when cell components targeted for destruction become enveloped by membranes from the RER. (4) Residual bodies are lysosomes of any type that have expended their capacity to degrade material. They contain undegraded ma- terial (e.g., lipofuscin and hemosiderin) and eventually m_maY be excreted from the cell. I.Peroxisomes (see Figure 3.1) a. Structure. Peroxisomes are also known as microbodies and are membrane-bound, spherical or ovoid organelles that may be identified in cells by a cytochemical reaction for catalase. In stained prepara- tions, they appear as small organelles (0.15-0.25 m in diameter); they may be larger in hepatocytes. Peroxisomes may contain a nucleoid, a crystalline core consisting of urate oxidase; the human peroxisome lacks a nucleoid. b. They originate from preexisting peroxisomes, which grow by im- porting specific cytosolic proteins and then divide by fission. The life span of a peroxisome is approximately 5-6 days. c. Function. Peroxisomes always contain the oxidative enzymes D- amino acid oxidase, urate oxidase, and catala of ohr enzymes whose functions vary fation of fatty acids to the detoxification of substances such as ethan---. 11. Centrioles (see Figures 3.1 and 2.7) a. Structure. Centrioles are a pair of cylindrical rods (each 0.2 m wide and 0.5 m long) oriented at right angles to one another. Each number of the pair is composed of nine triplets of microtubules (9 + 0 axoneme pattern). 1. The terms primary and virgin lysosomes, formerly used for tiny vecles believed to be lysosomes that have not yet engaged in digestive activity, are no longer used. C ytoptasm [ 1 b. Centrioles are located in the centrosome (cell center) where a number of small dense bodies, centriolar satellites, are also present. c. Centrioles self-duplicate in the S phase of the cell cycle, as each parent centriole forms a procentriole at right angles to itself. d. Function (1) Centrioles are associated with. microtubule-organizing cen- ter__s (.MTOCs), which are dense r]cen2i61ar-ae-l]ite-t-hat gve rise to microtubules. (2) They are present at the poles of the mitotic spindle where microtu- bules originate or converge. (3) They also form basal bodies, which give rise to the axonemes of cilia and flagella. B. Inclusions. Inclusions are "lifeless" accumulations of material that are not metabolically active and usually are present in the cytosol only tempo- rarily. 1. Glycogen appears as small clusters (or in hepatocytes as larger aggre- gates, known as rosettes) of electron-dense, 20- to 30-nm -particles, which are similar in appearance to, but larger than, ribosomes. Glycogen is not bound by a membrane but frequently lies close to the SER. Glycogen serves as a stored energy source that can be degraded to glucose, which enters the bloodstream to elevate blood sugar levels. 2. Lipid droplets vary markedly in size and appearance depending on the method of fixation and are not bound by a membrane. Lipid droplets are storage forms of triglycerides (an energy source) and cholesterol (used in the synthesis of steroids). 3. Lipofuscin appears as membrane-bound, electron-dense granular mate- rial varying greatly in size and often containing lipid droplets. Lipofuscin represents a residue of undigested material present in residual bodies. Because the amount of this material increases with age, it is called age pigment. It is most common in nondividing cells (e.g., cardiac muscle cells; neurons ,and hepatocytes). C. Cytoskeleton. The cytoskeleton is the structural framework within the cyto- sol. It functions in maintaining cell shape, stabilizing cell attachments, facilitat- ing endocytosis and exocytosis, and promoting cell motility. It includes the following components: 1. Microtubules a. Structure. Microtubules are straight, hollow tubules 25 nm in diameter and several micrometers long. They have a rigid wall composed of 13 protofllaments, each of which consists of a h-fiar arrangement o - ubulin dimrs. b. They are polar, with polymerization (assembly) and depolymerization (disasseml occurringpreferentially at the Cell Biology and Histology III. c. Microtubul___es contain microtubule-associated proteins (MAPs), which stabilize them and bind them to other cytoskeletl components and organelles'----they--e alo are associated with kinesin--nd two forc--geroteins which serve as or ve organelle movement. d. Function. Microtubulese; aid in the transport of macromolecules within the cosql; and promote the move--nt of cilia, -Iagella, and es. 2. Microfilaments a. Structure. Microfilaments are also known as F actin or actin fila- ments. They are 6 nm in diameter and are_o__m._posed of globular actin monomers (G actin) linked into a double helix. b. ay polarity like microtubules; that is, their polymerization and depolymerization occur preferentially at the plus end. c. Microfilaments are abundant at the periphery of the cell where they are anchored to the plasma membra--cih-bh-g-6Vmre intermediary prot'mg-(:g., -actinin, vinculin, talin). d. Function. Microfilaments are involved in many cellular processes, such as establishing focal contacts between the cell and the extracellular matrix, locomotion of nonmuscie cells, formation of the contractile ring (in dividiils- f0]difi-bf 6iithelia into tubes during development. 3. Intermediate filaments are 8-11 nm in diameter. They constitute a population of heterogeneous filaments that includes keratin., vimentin, des- min__, glial fibriry acid protein (GFAP), lamins, 3.2). I general, they provide mechanl"-c-a-l-Ten- Interactions Among Organelles. Organelles are involved in important cellular processes, such as the uptake and release of material by cells, protein synthesis, and intracellular digestion. These interactions provide the basis for a functional approach to the dynamics of cell biology. A. Uptake and release of material by cells 1. Endocytosis is the uptake (internalization) of material by cells. Endocytosis includes pinocytosis, rec___eptor-mediated endocytosis, and hagocytosis. a. Pinoeytosis ("cell drinking") is the nonspecific (random) take of extracellular fluid and material in blution into pinocytic vesicles. b. Receptor-mediated endocytosis is the specific uptake of a sub- tan6 (e.--7., ]ow-de-nS ..lipproteins and lrotein hormones) by a cell that has a llasma-membrane receptor for that subta-ffce (which is termed a ligand). It involves the following sequence of events (see Figure 3.3." (1) A ligand binds specifically to its receptors o_n the cell surface. (2) Ligand-recepto-_c-p|_s custer into a clathrin--0-oated pit, which invaginates and gives rise to a clathrin-coated veic elon- .ti_n'_mg the gand. Cytoplasm Table 3.2. Intermediate Filaments Protein Location Function Keratin 19 distinct forms (acidic, neutral, and basic Tonofilaments Epithelial cells Desmin Vimentin Provides struct tension-bearing role; _markers _ "fortumors o_f e_pt_helial origin _ Desmosome--hemidesmosome association Forms a framework linking myofibrils/myofilaments; marker for tumors of muscle origin Is associated with outer nuclear envelope and pores; marker for connective tissue tumors Glial fibrillary acidic protein (GFAP) Neurofilaments Skeletal muscle Cardiac muscle Smooth muscle Fibroblasts Endothelial cells Vascular smooth muscle Chondroblasts Macrophages Mesenchymal cells Astrocytes Oligodendrocytes Schwann cells Neurons Lamins A, B, and C Nuclear lamina of all cells Provides structural support; marker for glial tumors Provide usgp_9_rt for axons and .dendrites; facilitate gel state of the cytosol "' O:ganize peripheral chromatin; control assembly/disassembly of nuclear envelope (3) The _cy. pplas _mic clathrin coat is rapidly lost, leaving an uncoated endocytic vesicle containing the lig--d.- c. Phags ("c') is the uptake of microorganisms, other cells, and particulate matter (frequently by a cell. P-ocyt0sis usually involves cell:surface recep__tors. It is characteristic of cells--particularly macrophagesthat degrade prote'_ms and cellu- lar debris and involve-ie fSllowing sequence of events: (1) A macrophage binds, via its Fc receptors, to an antibody-coated (lg-coated) bacterium, or via its C3b receptors to a co_mplement- coated bacterium. (2) Binding progresses lasma membrane completely envelops the bacterium, forming a phagocytic vacuole. 2. xocytosis is the release of_material from the cell via fusion of a secretory granule nembrane with the plasma membrane. It requires interaction of receptors in both the granule and plasma mem- br.xane, as well as the coalescence (adhe__rence ad joining) of the two_ holiDid membrane Exocytosis takes place in_____both regulated and constitutive secretion. Regulated cretion (d) is the release, in response to __an extracellular signal, of proteins and other material stored in the cell. ----- Cell Biology and Histology b. _C__o_.n_s_t_i.tutive secretion (default pathway) is the more-or-less con__Q_O_- __Ug_us_release of material (lagen and serum proteins) without a, ny intermediate storagestep. An extracellulr signal is not rd for constitutive secretion. _ 3. Membrane recycling maintains a relatively constant plasma-membrane surface area following exocytosis. In this process, the secretory granule • fnembrane added to the plasma-membrane surfaf dring exoc:9is is retrieved during enc_ytosis via clathrin-coated vesicles. This -nembrane is returned toLe_TG. N- iia early en-somes) forrecycling=. Figure 3.4 illustrates endocytotic pathways used by cells. Protein synthesis 1. Synthesis of membrane-packaged proteins involves translation of mRNAs encoding secretory, membrane, and lysosomal proteins on ribosomes at the surface of the RER; transport of the growing polypeptide chain across the RER membrane and into the cisterna; and its processing within the RER. a. Transport of newly formed peptide into the RER cisterna is thought to occur by a mechanism described by the signal hypothesis as follows (Figure 3.5): (1) mRNAs for secretory, membrane, and lysosomal proteins contain codons that encode a signal sequence. (2) When the signal sequence is formed on the ribosome, a signal recognition particle (SRP) present in the cytosol binds to it. (3) Synthesis of the growing chain stops until the SRP facilitates the relocation of the polysome to SRP receptors in the RER mem- brane. (4) The large subunits of the ribosomes interact with ribosome receptor proteins, which "bind" them to the RER membrane. The SRP de- taches, and multisubunit protein translocators form a pore across the RER membrane. Synthesis resumes and the newly formed polypeptide is threaded through the pore and into the RER cisterna. b. Posttranslational modification in the RER (1) After the newly formed polypeptide enters the cisterna, a signal peptidase cleaves the signal sequence from it. (2) The polypeptide is glycosylated. (3) Disulfide bonds form, converting the linear polypeptide into a globu- lar form. c. Protein transport from the RER to the CGN (Figure 3.6) (1) Transitional elements of the RER give rise to transfer vesicles (coatomer-coated) containing newly synthesized protein. (2) Transfer vesicles move to the cis Golgi Network (CGN) where - they deliver the protein. (3) From the CGN, proteins either move forward toward the cis Golgi face, or (if they are RER-resident proteins) they are captured by a specific membrane receptor protein and returned in vesicles to the RER along a microtubule-guided pathway. Cytoplasm ( TRANSCYTOSIS I lBasal_lateral eady endosome ,. Clathrin - Apical early endosome Late endosome Lysosome Clathrin triskelions Figure 3.4. Pathways used by receptors and ligands following endocytosis. (1) Recycling of receptors to the same plasma membrane surface. (2) Transcytosis from one surface (e.g., basal-lateral) to another (e.g., apical). Transcytosis can occur in either direction, but separate eady endosome compartments exist near the domain of vesicle entry. (3) Degradation. If not retrieved from either eady endosome compartment, the ligands move to a common late endosome, which subsequently becomes a lysosome where degradation Is completed. 46 / Cell Biology and Histology subuns Signal peptidase SRP receptor Ribosome receptor Figure 3.5. Diagram illustrating the signal hypothesis, which proposes a mechanism whereby newly formed secretory polypeptides become segregated within the cistema of the RER. SRP = signal recognition particle. (Reprinted with permission from Widnell CC and Pfenninger KH: Essential Cell Biology. Baltimore, Williams & Wilkins, 1990, p 108.) Protein processing in the Golgi complex (see Figure 3.6) occurs as proteins move from the cis to the trans face of the Golgi complex through distinct cisternal subcompartments. This movement oc- curs by the budding-off of coatomer-coated vesicles from one cisterna and their fusion with the dilated rim of another cisterna. Protein pro- cessing may include the following events, each of which occurs in a different cisternal subcompartment: (1) Phosphorylation of mannose residues on lysosomal hydrolases (2) Removal of mannose (3) Terminal glycosylation of some proteins with sialic acid residues and galactose (4) Sulfation and phosphorylation of amino acid residues (5) Acquisition of a membrane similar in composition and thickness to the plasma membrane (6) Capture and return of RER-resident proteins from any of the Golgi cisternae e. Sorting of proteins in TGN (see Figure 3.6) (1) Regulated secretory proteins are sorted from membrane and lysosomal proteins and delivered via clathrin-coated vesicles to con- densing vacuoles, where removal of water, via ionic exchanges, yields secretory granules. Cytoplasm Plasma membrane proteins Secrstory ,.osome granule Clathrin- Coatomer- Late endosome '--vesicle v vesicle Clathrin 1' t.skelions Cl=hin Cl=hn / ';'"opate Soing o[ preins coated / ( Removal of mannose traner / vesicles Phospholation of mannose CIS G Plasma membrane O L G I Secretory Lysosomal Plasma membrane proteins proteins proteins Figure 3.6. Diagram illustrating the modification of proteins in separate compartments of the Golgi complex and their sorting for movement to other cellular sites. Proteins synthesized In the rough endoplasmic reticulum (RER) Include secretory (A), membrane (C), and lysosomal (&) proteins. These proteins pass through the Golgl complex, whereas cytosollc proteins do not. Movement of a secretory protein Is illustrated In the diagram. Note that coatomer-coated vesicles have formed by pinching off the RER, CGN, and Golgi clstemae to shuttle protein molecules from one site to another. The coatomer protein COPI retrieves RER resident proteins from the Golgl or TGN (retrograde transport). All proteins do not undergo all of the chemical modifications (e.g., only lysosomal proteins undergo mannose phosphorylation). 48 ] Cell Biology and Histology Receptor-mediated endocytoss Phagocytosis hydrolases and ' 0,_ ".A V lysosomal membrane in separate vesicles { .-.0 1, /'/ \ I,J Eaarly endosome (CURL) _ . I /I l/3 k.,_..../" i I i- -\\ 'nagocy[c I i" 1/120 \ k,J Lain eneosome Mitochondrion Residual AutophagiCvacuole Autophagolysosome body B AUTOPHAGY Figure 3.7. Diagram illustrating the different pathways of intracellular lysosomal digestion and the types of lysosomes involved in each. (2) Lysosomal proteins are sorted into clathrin-coated regions of the TGN that have receptors for mannose 6-phosphate and are delivered to late endosomes via clathrin-coated vesicles. (3) Plasma-membrane proteins are sorted into coatomer-coated regions of the TGN and delivered to the plasma membrane in coa- tomer-coated vesicles. Synthesis of cytosoHc proteins takes place on polyribosomes free in the cytosol and is directed by mRNAs that lack signal codons. The proteins (e.g., protein kinase and hemoglobin) are released directly into the cytosol. 8 C. Intracellular digestion 1.._Nonlysosomal_ digestion is the degradation of c__osolic constituents by mechanisms outside of the vacuolar lysosomal pathway. In these mecha- ni-m-, Sh-Ii-d-rotire degraded by different classes of nonlyso- somal proteases. 2.Lgestion (Figure 3.7) is the degradation of material within _various types of lysosomes by lysosomal s. It occurs bv different pathway#_depending on te orig-n of the material to be degraded. a. Heterophagy is thegn and degradation of foreign material taken into tlcell by receptor-mediated endoytosih@bytsis. (1) Digestion of endocytosed hgands occurs m multlvescular bod- ies (see Figure 3.3). Cytoplasm (2) Digestion of. p_hagocytosed microorganisms and foreign particles begins and may be completed in phagolysosomes. Autophagy is the segregation of an organelle or other cell constituents within-membranes from the RER to form an autophagic vacuole, s subsequently digestan asosome. Crinophagy is the fusion of hormone secretory granules with lysosomes and their subsequent digestio n. CriKSphagy is used to remove excess numbers of secretory granules from the ceil. IV. Clinical Considerations A. _Lyso.soal storage dis_e_ases are hereditary conditions in which the synthe- s.isof specific lysosomal acid hydrolases is impaired. They are characterized by the inability of lysosomes to degrade certain compounds, which accumulate and iterfere with cell functioning. 1. In Tay-Sachs disease, glycolipids accumulate in neurons. 2. In glycogen storage di_se_ase, glycogen is abundant in the liver and musl. 3. In H__urler'_s syndrome, glycosaminoglycans accumul_ate in many tissues and organs. B. Familial hypercholesterolemia is associated with a decreased ability of cells to take in cholesterol, which normally is ingeste-sd-b receptor- mediated endocytosis of low-density lipoproteins (LDLs). 1. This disease is caused by an inheritedgenetic defect, that results in an inability to synthesize LDL re6pto_r]n t-sis of defective recep- or to clath-n-coated pits. 2. It is characterized by an elevated level of cholesterol in the bloodstream, which facilitates early development of atlerosclerosis, which may fatal. C. Tumor diagnosis is often based2_n immunocytochemical identification of the "ntemediate filamen_ in tumor cells, since the type bf intermediate filamen present identifies thee tissu_e from which the metastatic cancer cells originat-d. Review Test Directions: Each of the numbered items or incomplete statements in this section is followed by answers or by completions of the statement. Select the ONE lettered answer or completion that is BEST in each case. 1. Which of the following organelles divides by fission? (A) Golgi complex (B) Rough endoplasmic reticulum (C) Peroxisome (D) Smooth endoplasmic reticulum (E) Centriole 2. A 30-year-old man with very high blood cho- lesterol levels (290 milligrams per deciliter) has been diagnosed with premature atherosclerosis. His father died of a heart attack at age 45, and his mother, age 44, has coronary artery disease. Which of the following is the most likely explana- tion of his condition? (A) He has a lysosomal storage disease and can- not digest cholesterol (B) He suffers from a peroxisomal disorder and produces low levels of hydrogen peroxide (C) The smooth endoplasmic reticulum (SER) in his hepatocytes has proliferated and pro- duced excessive amounts of cholesterol (D) He has a genetic disorder and synthesizes defective LDL receptors (E) He is unable to manufacture endosomes Directions: Each of the numbered items or incomplete statements in this section is negatively phrased, as indicated by a capitalized word such as NOT, LEAST, or EXCEPT. Select the ONE lettered answer or completion_that is BEST in each case. 3. Which one of the following statements con- cerning the cytoskeleton is NOT true? (A) Keratin filaments are associated with the plaques of desmosomes (B) Intermediate filaments provide mechanical strength to cells (C) Desmin filaments are present in all three types of muscle cells (D) Actin microfilaments are associated with hemidesmosome plaques (E) Microtubules are associated with a molecu- lar motor called kinesin 4. Which of the following vesicles does NOT con- tain a full comple__ment of acid hydrolases? (A) Multivesicular body (B) Early endosome (C) Autophagolysosome (D) Lysosome (E) Phagolysosome 5O Cytoplasm / 51 Directions: Each group of items in this section consists of lettered options followed by a set of numbered items. For each item, select the ONE lettered option that is most closely associated with it. Each lettered option may be selected once, more than once, or not at all. Questions 5- (A) Clathrin-coated vesicle (B) Coatomer-coated vesicle (C) Both (D) Neither Match each process below with the option that fits best. 5. Movement of protein from the RER to the cis Golgi network 6. Retrieval of secretion-granule membrane after exocytosis 7. Movement of glycoprotein from cis to medial Golgi cisternae 8. Uncoupling of endocytosed ligands from re- ceptors 9. Movement of acid hydrolases from the trans Golgi network to a late endosome Questions 14-17 Questions 10-13 (A) Microfilament (B) Intermediate filament (C) Microtubule Match each description below with the cell com- ponent that it best describes. 10. Is associated with kinesin 11. Consists of globular actin monomers linked into a double helix 12. Has a rigid wall composed of 13 protofila- ment strands 13. Provides structural support to astrocytes Match each description with the corresponding lettered structure in the electron micrograph. 14. Site where fmal sorting of proteins occurs 15. Site that receives transport vesicles from the RER 16. Site where terminal glycosylation of mem- brane-packaged proteins occurs 17. Site where SRP receptor proteins are lo- cated Cell Biology and Histology Questions 18-20 D Match each description with the corresponding lettered structure in the electron micrograph. 18. Site of the elementary particles associated with chemiosmotic coupling and ATP production 19. Site of enzymes associated with fatty acid oxidation 20. Site where divalent cations are bound Answers and Explanations l-C. A peroxisome originates from preexisting peroxisomes. It imports specific cytosolic proteins and then undergoes fission. The other organelle that divides by fission is the mitochondrion. 2-D. Cells import cholesterol by the receptor-mediated uptake of low-density lipoproteins (LDLs) in coated vesicles. Certain individuals inherit defective genes and cannot make LDL receptors, or they make defective receptors that cannot bind to clathrin-coated pits. The result is an inability to internalize LDLs, which leads to high levels of LDLs in the bloodstream. High LDL levels predispose the individual to premature atherosclerosis and increase the risk of heart attack. 3-D. Actin filaments are involved in cellular locomotion, the formation of the contractile ring in dividing cells, and contraction. Keratin filaments are associated with plaques of desmosomes and hemidesmosomes. 4-B. The early endosome is a mildly acidic compartment near the periphery of the cell in which the uncoupling of ligands and receptors takes place. It does not contain acid hydrolases. 5-B. Transport of protein from the RER to the cis Golgi network occurs via coatomer-coated vesicles. 6-A. Membrane recycling after exocytosis of the contents of a secretion granule occurs via clathrin- coated vesicles. 7-B. Transfer of material among the cisternae of the Golgi complex takes place via coatomer- coated vesicles. Cytoplasm I 53 8-D. The uncoupling of ligands and receptors internalized by receptor-mediated endocytosis occurs in the early endosome. 9-A. Proteins targeted for lysosomes (via late endosomes) leave the trans Golgi network in clathrin- coated vesicles. 10-C. Dynein is a force-generating protein associated with microtubules. It serves as a molecular motor for the transport of organelles and vesicles. ll-A. Globular actin monomers (G actin) polymerize into a double helix of filamentous actin (F actin) or microfilament in response to the regulatory influence of a number of actin-binding proteins. 12-C. A microtubule consists of a[-tubulin dimers polymerized into a spiral around a hollow lumen to form a fairly rigid tubule. When cross-sectioned, the microtubule reveals 13 protofilament strands, which represent the tubulin dimers present in one complete turn of the spiral. 13-B. Glial filaments are a type of intermediate filament, composed of glial fibrillary acidic protein, present in fibrous astrocytes. These filaments are supportive, but they may also play additional roles in both normal and pathologic processes in the central nervous system. 14-B. The trans Golgi network is the site where the final sorting of protein occurs. 15-C. The cis Golgi network is the site receiving transfer vesicles from the RER. 16-A. The medial Golgi cisterna, which lies between the cis and trans face of the Golgi complex, is the site where terminal glycosylation of membrane-packaged proteins occurs. 17-D. The RER membrane is the site where SRP receptor proteins are located. During protein synthesis, the SRP-bound ribosome is delivered to the SRP receptor located in the RER membrane. 18-B. The inner mitochondrial membrane (which infolds to form cristae) is where the globular elementary particles associated with ATPsynthase are located. This enzyme converts ADP and P to ATP, a reaction driven by energy from the electrochemical proton gradient existing across the inner mitochondrial membrane. 19-D. The mitochondrial matrix is the site where enzymes associated with fatty acid oxidation are located. 20-C. Mitochondrial matrix granules bind divalent cations such as Ca 2ч and Mg u. this is for non-commercial use by Russian-speakers only ! if you are not a Russian-speaker delete this file immediately ! /I, aHHblH CKaH npep, HO3HaHeH HI.Ub /I,R : 1. pyccKHx nob3OBaTeeH- BpaHeH H yHeHblX B Oco6eHHOCTH 2. BECrl/1ATHOFO pacnpocTpaHeHHR cI4aHHpOBaHO rlOTOM H 14pOBblO lvlYCAH, T 4 Extracellular Matrix I. Overview--The Extracellular Matrix Structure. The extracellular matrix is an organized meshwork of macro- molecules surrounding and underlying cells. Although it varies in composi- tion, in general it consists of an amorphous ground substance (containing primarily glycosaminoglycans, proteoglycans, and glycoproteins) and fibers (Figure 4.1). Bo Functions. The extracellular matrix, along with water and other small mole- cules (e.g., nutrients, ions), constitutes the extracellular environment. By affecting the metabolic activities of cells in contact with it, the extracellular matrix may alter the cells as well as influence their shape, migration, division, and differentiation. Collagen fibrils k , /:"'/' I Hyalc°mnicecules Proteoglycan " "" ore protein "L'J I Glycosammoglycans I=llure 4.1. Illustration showing the major components of the extracellular matdx. (Adapted with permission from Fawcett DW: Bloom and Fawcett's A Textbook of Histology, 12th ed. New York, Chapman & Hall, 1994, p 134.) 56 / Cell Biology and Histology Table 4.1. Classification of Glycosaminoglycans (GAGs) Linked to Type Core Protein Sulfated Hyalur0nic acid No No Chondroitin sulfate Yes Yes Dermatan sttlfate Yes Yes Heparin Yes Yes Heparan sulfate Yes Yes Keratan sulfate Yes Yes Major Locations in Body Synovial fluid, vitreous humor, cartilage, skin, most connective tissues Cornea, cartilage, bone, adventitia of arteries Skin, blood vessels, heart valves Lung, skin, liver, mast cells Basal laminae, lung, arteries, cell surfaces Cornea, cartilage, nucleus pulposus of intervertebral disk II. Ground Substance A. Glycosaminoglycans (GAGs) are long, unbranched polysaccharides com- posed of repeating disaccharide units. 1. An amino sugar (either N-acetylglucosamine or N-acetylgalactosamine) is always one of the repeating disaccharides. o Because GAGs are commonly sulfated and usually possess a uronic acid sugar (which has a carboxyl group) in the repeating disaccharide unit, they have a strong negative charge. 3. GAGs are generally linked to a core protein. The attraction of osmotically active cations (e.g., Na ч) to GAGs results in a heavily hydrated matrix which strongly resists compression. Their extended random coils occupy large volumes of space because they are unable to fold compactly. o GAGs are classified into four main groups based on their chemical structure (Table 4.1). a. Hyaluronic acid is a very large, nonsulfated molecule up to 20 microm- eters (m) in length that is not attached to a core protein. b. The other three groups are chondroitin sulfate and dermatan sul- fate; heparin and heparan sulf