GENERAL MORPHOLOGY OF THE CELL

Department Of General Histology

CELL DIFFERENTIATION

The human organism presents about 200 different cell types, all derived from the zygote, the single cell formed by fertilization of an oocyte with a spermatozoon. The first cellular divisions of the zygote produce cells called blastomeres and as part of the inner cell mass blastomeres give rise to all tissue types of the adult. Explanted to tissue culture such cells have been termed embryonic stem cells. During their specialization process, called cell differentiation, the cells synthesize specific proteins, change their shape, and become very efficient in specialized functions. For example, muscle cell precursors elongate into fiber-like cells that synthesize and accumulate large arrays of actin and myosin. The resulting cell is specialized to efficiently convert chemical energy into contractile force.

The main cellular functions performed by specialized cells in the body are listed in Table 2–1. It is important to understand that the functions listed there can be performed by most cells of the body; specialized cells have greatly expanded their capacity for one or more functions during differentiation.

CELLULAR FUNCTIONS IN SOME SPECIALIZED CELLS

MEMBRANE STRUCTURE.(a): A TEM of a sectioned cell surface shows the trilaminar unit membrane with two dark (electron-dense) lines enclosing a clear (electron-lucent) band. These three layers of the unit membrane correspond to reduced osmium deposited on the hydrophilic phosphate groups present on each side of the internal bilayer of fatty acids where osmium is not deposited. The "fuzzy" material on the outer surface of the membrane represents the glycocalyx of oligosaccharides attached to phospholipids and proteins. Components of the glycocalyx are important for cell-cell recognition in many biological processes and for adsorption and uptake of many molecules by cells. X100,000.

(b): Schematic drawing depicts the trilaminar ultrastructure (left) and molecular organization (right) of the lipid bilayer in a cell membrane.

Membrane phospholipids, such as phosphatidylcholine (lecithin), consist of two non-polar (hydrophobic or water-repelling) long-chain fatty acids linked to a charged polar (hydrophilic or water-attracting) head group. Cholesterol is also present, often at nearly a 1:1 ratio with the phospholipids in plasma membranes. Membrane phospholipids are most stable when organized into a double layer (bilayer) with their hydrophobic fatty acid chains directed toward the middle away from water and their hydrophilic polar heads directed outward to contact water on both sides.

Cholesterol molecules insert among the close packed the phospholipid fatty acids, restricting their movement, and thus modulate the fluidity and movement of all membrane components. The lipid composition of each half of the bilayer is different. For example, in red blood cells phosphatidylcholine and sphingomyelin are more abundant in the outer half of the membrane, whereas phosphatidylserine and phosphatidylethanolamine are more concentrated in the inner half. Some of the lipids, known as glycolipids, possess oligosaccharide chains that extend outward from the surface of the cell membrane and thus contribute to the lipid asymmetry

THE FLUID MOSAIC MODEL OF MEMBRANE STRUCTURE

(a): The fluid mosaic model emphasizes that a membrane consisting of a phospholipid bilayer also contains proteins inserted in it or bound to the cytoplasmic surface (peripheral proteins) and that many of these proteins move within the fluid lipid phase. Integral proteins are firmly embedded in the lipid layers. Other proteins completely span the bilayer and are called transmembrane proteins. Hydrophobic amino acids of the integral membrane protein interact with the hydrophobic fatty acid portions of the membrane. Both the proteins and lipids may have externally exposed oligosaccharide chains. When cells are frozen and fractured (cryofracture), the lipid bilayer of membranes is often cleaved along the hydrophobic center.

(b): Membrane splitting occurs along the line of weakness formed by the fatty acid tails of membrane phospholipids, since only weak hydrophobic interactions bind the halves of the membrane along this line. Electron microscopy of cryofracture preparation replicas is a useful method of studying membranous structures. Most of the protruding membrane particles seen (1) are proteins or aggregates of proteins that remain attached to the half of the membrane adjacent to the cytoplasm (the P or protoplasmic face). Fewer particles are found attached to the outer half of the membrane (E or extracellular face). For every protein particle that bulges on one surface, a corresponding depression (2) appears in the opposite surface.

EXPERIMENT DEMONSTRATING THE FLUIDITY OF MEMBRANE PROTEINS. (a, b): Two types of cells were grown in

tissue cultures, one with fluorescently labeled transmembrane proteins in the plasmalemma (right) and one without. Cells of each type were fused together into hybrid cells through the action of Sendai virus.

(c): Minutes after the fusion of the cell membranes, the fluorescent proteins of the labeled cell spreads to the entire surface of the hybrid cells. Such experiments provide important data in support of the fluid mosaic model. However, in many cells most transmembrane proteins show very restricted lateral movements along the cell membrane and are anchored in place by other proteins linking them to the cytoskeleton.

MEMBRANE PROTEINS

FORMATION AND MATURATION OF CELL MEMBRANE PROTEINS

Membrane proteins of the plasmalemma are synthesized in the rough endoplasmic reticulum and then move in transport vesicles to a Golgi apparatus, another cytoplasmic structure with several flattened membrane saccules or cisternae. While in the Golgi apparatus, the oligosaccharide chains are added (glycosylation) to many membrane proteins by enzymes in the Golgi saccules. When glycosylation and other posttranslational modifications are complete, the mature membrane proteins are isolated within vesicles that leave the Golgi apparatus. These vesicles move to the cell membrane and fuse with it, thus incorporating the new membrane proteins (along with the lipid bilayer of the vesicle) into the cell membrane.

EXPERIMENT DEMONSTRATING THE FLUIDITY OF MEMBRANE PROTEINS

(a, b): Two types of cells were grown in tissue cultures, one with fluorescently labeled transmembrane proteins in the plasmalemma (right) and one without. Cells of each type were fused together into hybrid cells through the action of Sendai virus. (c): Minutes after the fusion of the cell membranes, the fluorescent proteins of the labeled cell spreads to the entire surface of the hybrid cells. Such experiments provide important data in support of the fluid mosaic model. However, in many cells most transmembrane proteins show very restricted lateral movements along the cell membrane and are anchored in place by other proteins linking them to the cytoskeleton.

FORMATION AND MATURATION OF CELL MEMBRANE PROTEINS

THREE MAJOR FORMS OF ENDOCYTOSIS

Endocytosis is a process in which a cell takes in material from the extracellular fluid using dynamic movements and fusion of the cell membrane to form cytoplasmic, membrane-enclosed structures containing the material. Such cytoplasmic structures formed during endocytosis fall into the general category of vesicles or vacuoles. (a): Phagocytosis involves the extension from the cell of large folds called pseudopodia which engulf particles, for example bacteria, and then internalize this material into a cytoplasmic vacuole or phagosome. (b): In pinocytosis the cell membrane invaginates (dimples inward) to form a pit containing a drop of extracellular fluid. The pit pinches off inside the cell when the cell membrane fuses and forms a pinocytotic vesicle containing the fluid. (c): Receptor-mediated endocytosis includes membrane proteins called receptors which bind specific molecules (ligands). When many such receptors are bound by their ligands, they aggregate in one membrane region which then invaginates and pinches off to create vesicle or endosome containing both the receptors and the bound ligands.

ENDOCYTOSIS AND MEMBRANE TRAFFICKING.

Ligands, such as hormones and growth factors, are internalized by receptor-mediated endocytosis, which is mediated by the cytoplasmic peripheral membrane protein clathrin or other proteins which promote invagination and temporarily coat the newly formed vesicles. Such coated vesicles can be identified by TEM. After detachment of the coating molecules, the vesicle fuses with one or more vesicles of the endosomal compartment, where the ligands detach from their receptors and are sorted into other vesicles. Vesicles of membrane with empty receptors return to the cell surface and after fusion the receptors are ready for reuse. Vesicles containing the free ligands typically fuse with lysosomes, as discussed below. The cytoskeleton with associated motor proteins is responsible for all such directional movements of vesicles.

INTERNALIZATION OF LOW-DENSITY LIPOPROTEINS

Endocytosis of low-density lipoproteins (LDL) is an important mechanism that keeps the concentration of LDL in extracellular body fluids low and is a well-studied example of endocytosis and membrane trafficking. LDL, which is often rich in cholesterol, binds with high affinity to its specific receptors in the cell membranes. This binding activates the formation of clathrin-coated endocytotic pits that form coated vesicles. The vesicles soon lose their coat proteins, which return to the inner surface of the plasmalemma. The uncoated vesicles fuse with endosomes and the free LDL and the receptors are sorted into separate vesicles. Receptors are returned to the cell surface and the LDL is transferred to lysosomes for digestion and separation of their components to be utilized by the cell.

EXOCYTOSIS In exocytosis a membrane-limited cytoplasmic

vesicle fuses with the plasma membrane, resulting in the release of its contents into the extracellular space without compromising the integrity of the plasma membrane (Figure 2–6). Often exocytosis of stored products from epithelial cells occurs specifically at the apical domains of cells, such as in the exocrine pancreas and the salivary glands (see Chapter 4). The fusion of membranes during exocytosis is a highly regulated process involving interactions between several specific membrane proteins. Exocytosis is triggered in many cells by transient increase in cytosolic Ca2+.

SIGNAL RECEPTION AND TRANSDUCTION In endocrine signaling, the signal molecules (called

hormones) are carried in the blood to target cells throughout the body.

In paracrine signaling, the chemical mediators are rapidly metabolized so that they act only on local cells very close to the source.

In synaptic signaling, a special kind of paracrine interaction, neurotransmitters act only on adjacent cells through special contact areas called synapses.

In autocrine signaling, signals bind receptors on the same cell type that produced the messenger molecule.

In juxtacrine signaling, important in early embryonic tissue interactions, signaling molecules remain part of a cell's surface and bind surface receptors of the target cell when the two cells make direct physical contact.

RECEPTORS AND THEIR LIGANDSCells respond to certain external chemical signals that act as ligands, such as hormones and lipoproteins, according to the library of receptors they have. Such receptors are always proteins, typically transmembrane proteins. In this schematic representation, three cells appear with different receptors. The extracellular environment is shown to contain several ligands, which can interact only with the appropriate specific receptors. Considering that the extracellular environment contains a multitude of molecules, it is important that ligands and the respective receptors exhibit complementary morphology and great binding affinity.

Hydrophilic signaling molecules, including most hormones, local chemical mediators (paracrine signals), and neurotransmitters activate receptor proteins on the surface of target cells. These receptors, often transmembrane proteins, relay information to a series of intracellular intermediaries that ultimately pass the signal (first messenger) to its final destination in either the cytoplasm or the nucleus in a process called signal transduction. One of the best-studied classes of such intermediary proteins, the G proteins, binds guanine nucleotides and acts on other membrane-bound intermediaries called effectors which propagate the signal further into the cell.

Effector proteins are usually ion channels or enzymes that generate large quantities of small second messenger molecules, such as 1,2-diacyglycerol (DAG), cyclic adenosine monophosphate (cAMP), and inositol 1,4,5-triphosphate (IP3). The ions or second messengers diffuse through the cytoplasm, amplifying the first signal and triggering a cascade of molecular reactions that lead to changes in gene expression or cell behavior.

G PROTEINS AND INITIATION OF SIGNAL TRANSDUCTION

When a hormone or other signal binds to a membrane receptor, the hormone can begin to cause changes in the cell's activities after a signal transduction process initiated by the bound receptor. The first step in receptor signaling often involves G proteins which bind guanosine diphosphate (GDP) when inactive and are activated when GDP is exchanged for GTP. A simplified version of G protein activity is shown here. Conformational changes occur in the receptor when it binds its ligand and the changed receptor activates the G protein–GDP complex. A GDP-GTP exchange releases the subunit of the G protein, which then moves laterally to bind with a transmembrane effector protein, activating it to propagate the signal further by various mechanisms. The subunit GTP is rapidly converted back to GDP, allowing the polypeptide to reassociate with the rest of the G protein complex, ready to be activated again when the receptor is again bound by hormone.

THE CHEMIOSMOTIC PROCESS OF ENERGY TRANSDUCTION.

The movement of electrons along the units of the inner mitochondrial membrane electron transport system (middle) is accompanied by the directed movement of protons (H+) from the matrix into the intermembranous space. The inner membrane is highly impermeable to protons and the result is an electrochemical gradient across the membrane. The other membrane-associated proteins (left) make up the ATP synthase systems, each of which forms a 10 nm, multisubunit globular complex on a stalk-like structure projecting from the matrix side of the inner membrane.

A channel runs through this enzyme complex and specifically allows protons to flow through it, down the electrochemical gradient and across the membrane back into the matrix. Passage of protons through this narrow path causes rapid spinning of specific polypeptides in the globular ATP synthase complex. In this manner the energy of proton flow is converted into the mechanical energy of protein movement.

Other subunit proteins of the complex store this energy in the new phosphate bond of ATP which leaves the mitochondrion for use throughout the cell. It is estimated that each ATP synthase complex produces more than 100 molecules of ATP per second. In some mitochondria, particularly those in cells of multilocular adipose tissue, another inner membrane protein called thermogenin forms a shunt for the return of protons into the matrix (right).

This reflux of protons does not produce ATP, but instead dissipates energy as heat which warms blood flowing through the tissue

CELL CYTOPLASM

MITOCHONDRIAThe two mitochondrial membranes and central matrix can be seen here in the diagram and the TEM. The outer membrane is smooth and the inner membrane, shown at left, has many sharp folds called cristae which increase its surface area greatly. Cristae are most numerous in mitochondria of highly active cells. The matrix is a gel containing numerous enzymes. The inner membrane surface in contact with the matrix is studded with many multimeric protein complexes resembling globular units on short stalks. These contain the ATP synthase complexes that generate most of the cell's ATP.

MITOCHONDRIA IN THE LIGHT MICROSCOPE

In sectioned cells stained with H&E, such as certain cells of the stomach inner lining, mitochondria typically appear as numerous eosinophilic structures throughout the cytoplasm. The mitochondria usually appear round or slightly elongated and are more numerous in cytoplasmic regions with higher energy demands, such as near the cell membrane in cells undergoing much active transport. The central nuclei are also clearly seen in these cells.

Entire mitochondria can be shown in cultured cells, such as the endothelial cells shown here and often appear as the elongated structures (shown in yellow or orange here), usually arrayed in parallel along microtubules. These preparations along with TEM studies indicate that the elongated shape is typical of mitochondria and that their shape can be quite plastic and variable. Specific mitochondrial staining such as that shown here involves incubating living cells with specific fluorescent compounds that are specifically sequestered into these organelles, followed by fixation and immunocytochemical staining of the microtubules. In this preparation, microtubules are stained green and mitochondria appear yellow or orange, depending on their association with the green microtubules. The cell nucleus was stained with DAPI. (Figure 2–11b, with permission, from Invitrogen.)

POLYRIBOSOMES

(a): Many ribosomes attach to the same mRNA and move along it during translation, with each ribosome producing and at the end of the mRNA releasing one copy of the protein encoded by that message.

(b): Proteins that are to be incorporated into membranes, or eventually to be extruded from the cytoplasm (secreted proteins) or sequestered into lysosomes, are made on polysomes that adhere to the membranes of endoplasmic reticulum. The proteins produced by these ribosomes are segregated during translation into the interior of the endoplasmic reticulum's membrane cisternae.

ENDOPLASMIC RETICULUM(a): Electron microscopy shows that some regions of endoplasmic reticulum, called smooth ER (foreground), are devoid of ribosomes, the small granules that are present in the rough ER (background). Both types of ER are continuous with one another. The interconnected membranous cisternae of the smooth ER are frequently tubular, whereas those in the rough ER are flattened sacs.

ENDOPLASMIC RETICULUM

In a very thin cultured endothelial cell, both ER (green) and mitochondria (orange) can be visualized with vital fluorescent dyes that are sequestered specifically into those organelles. This staining method with intact cells clearly reveals the continuous, lacelike ER present in all regions of the cytoplasm. (Figure 2-15b, with permission, from Invitrogen.)

ROUGH AND SMOOTH ERAs seen with the TEM the cisternae of rough ER are flattened, with polyribosomes on their outer surfaces and concentrated material in their lumens. Such cisternae appear separated in sections made for electron microscopy, but they actually form a continuous channel or compartment in the cytoplasm. Smooth ER is continuous with rough ER but is involved with a much more diverse range of functions. Three major activities associated with smooth ER are (1) lipid biosynthesis, (2) detoxification of potentially harmful compounds, and (3) sequestration of Ca++ ions. Specific cell types with well-developed smooth ER are usually specialized for one of these functions.

MOVEMENT OF POLYPEPTIDES INTO THE RER.

PROTEIN SYNTHESIS AND CELL MORPHOLOGY.

(a): Cells that make few or no proteins for secretion have very little rough ER, with essentially all polyribosomes free in the cytoplasm.

(b): Cells that synthesize, segregate, and store various proteins in specific secretory granules or vesicles always have rough ER, a Golgi apparatus, and a supply of granules containing the proteins ready to be secreted.

(c): Cells with extensive RER and a well-developed Golgi apparatus show few secretory granules because the proteins undergo exocytosis immediately after Golgi processing is complete. Many cells, especially those of epithelia, are polarized, meaning that the distribution of RER and secretory vesicles is different in various regions or poles of the cell.

(d): Epithelial cells specialized for secretion have distinct polarity, with RER abundant at their basal ends and mature secretory granules at the apical poles undergoing exocytosis into an enclosed extracellular compartment, the lumen of a gland.

PHOSPHOLIPID TRANSPORTPhospholipids or more complex lipids such as cholesterol are generally synthesized by enzymes located in smooth ER. The products are inserted into the lipid bilayers of that organelle and are distributed in membranes throughout the cell by movement through the ER, Golgi apparatus, secretory vesicles, and other organelles. As shown here however, individual phospholipids can also be transported from smooth ER directly to membranes elsewhere in the cell but only after binding a water-soluble transporting protein. There is a specific transfer protein (also called exchange proteins) for each specific type of phospholipid and each can be reused many times. Phospholipid transfer proteins are an important mechanism for redistributing lipids between different membrane-enclosed compartments or organelles, such as the ER and mitochondria.

GOLGI APPARATUS

GOLGI APPARATUS

Morphological aspects of the Golgi apparatus are revealed by the SEM, which shows a three-dimensional snapshot of the region between RER and the Golgi membrane compartments. Cells may have multiple Golgi apparatuses, each with stacks of cisternae and dynamic cis and trans faces, and these typically are situated near the cell nucleus. This has been shown in careful TEM studies but is also clearly seen in intact cultured cells.

(c): The fibroblast was processed by immunocytochemistry using an antibody against golgin-97 to show many complexes of Golgi vesicles (green), all near the nucleus, against a background of microfilaments organized as stress fibers and stained with fluorescent phalloidin (violet). Because of the abundance of lipids in its many membranes, the Golgi apparatus is difficult to visualize by light microscopy in typical paraffin-embedded, H&E stained sections. In cells with very active Golgi complexes however, such as developing white blood cells, the organelle can sometimes be seen as a faint unstained juxtanuclear region (sometimes called a "Golgi ghost") surrounded by basophilic cytoplasm.

Though only snapshots of this highly dynamic organelle, electron micrographs of the Golgi apparatus provided early evidence about how this organelle functions, evidence that has now been strengthened by biochemical and other studies. To the right is a cisterna (arrow) of the rough ER containing granular material. Close to it are small vesicles containing apparently similar material. These are very close to the cis face of the Golgi apparatus. In the center are the characteristic flattened, curved, and stacked medial cisternae of the complex. Dilatations (upper left arrow) are seen extending from the ends of the cisternae. Similar dilatations gradually detach themselves from the cisternae and fuse at the trans face, forming the secretory granules (1, 2, and 3). Near the plasma membranes of two neighboring cells is more rough ER and smooth ER. X30,000. Inset: a small region of a Golgi apparatus in a 1- m section impregnated with silver, which demonstrates the abundance of glycoproteins within some cisternae. X1200.

SUMMARY OF GOLGI APPARATUS STRUCTURE AND FUNCTION

SECRETORY GRANULESTEM of one area of a pancreatic acinar cell shows numerous mature, electron-dense secretory granules (S) in association with condensing vacuoles (C) of the Golgi apparatus (G). Such granules form as the contents of the Golgi vacuoles becomes more condensed. In H&E stained sections secretory granules are often shown as intensely eosinophilic structures, which in polarized epithelial cells are concentrated at the apical region prior to exocytosis.

LYSOSOMES

Cells in a kidney tubule show numerous purple lysosomes (L) in the cytoplasmic area between the basally located nuclei (N) and apical ends of the cells at the center of the tubule. Using endocytosis, these cells actively take up small proteins in the lumen of the tubule, degrade the proteins in lysosomes, and then release the resulting amino acids for reuse.

Lysosomes in cultured vascular endothelial cells can be specifically stained using fluorescent dyes sequestered into these organelles (green), which are abundant around the blue Hoechst-stained nucleus. Mitochondria (red) are scattered among the lysosomes.

In the TEM lysosomes (L) have a characteristic very electron-dense appearance and are shown here near groups of Golgi cisternae (G) and a centriole (C). Less electron-dense lysosomes represent heterolysosomes in which digestion of the contents is underway. The cell is a macrophage with numerous fine cytoplasmic extensions (arrows).

LYSOSOMAL FUNCTIONSSynthesis of the digestive enzymes occurs in the rough ER, and the enzymes are packaged in the Golgi apparatus. Heterophagosomes, in which bacteria are being destroyed, are formed by the fusion of the phagosomes and lysosomes. Autophagosomes, such as those depicted here with ER and mitochondria in the process of digestion, are formed after nonfunctional or surplus organelles become enclosed with membrane and the resulting structure fuses with a lysosome. The products of digestion can be excreted from the cell by exocytosis, but may remain in a membrane-enclosed residual body, containing remnants of indigestible molecules. Residual bodies can accumulate in long-lived cells and be visualized as lipofuscin granules. In some cells, such as osteoclasts, the lysosomal enzymes are secreted to a restricted extracellular compartment.

AUTOPHAGOSOMES

Autophagy is a process in which the cell uses lysosomes to dispose of obsolete or non-functioning organelles or membranes. Details of the process are highly regulated but not well-understood. Membrane of unknown origin encloses the organelles to be destroyed, forming an autophagosome which then fuses with a lysosome for digestion of the contents. In the TEM autophagosomes can sometimes be recognized by their contents, as shown here. Upper right: Two autophagosomes containing portions of the RER that are slightly more electron-dense than neighboring normal RER. Center: An autophagosome containing what may be mitochondrial membranes (arrow) plus RER. Left: A vesicle that may represent a residual body with indigestible material.

PEROXISOMES

(a): By TEM peroxisomes generally show a homogenous matrix of moderate electron-density, but may include darker crystalloid internal structures representing very dense concentrations of enzymes. The arrows indicate small aggregates of glycogen. (x30,000) (b): A cultured endothelial cell processed by immunocytochemistry shows many peroxisomes (green) distributed throughout the cytoplasm among the vitally stained elongate mitochondria (red) around the DAPI-stained nucleus (blue). Peroxisomes shown here were specifically stained using an antibody against the membrane protein PMP70.

MOLECULAR ORGANIZATION OF A MICROTUBULE

Microtubules are rigid structures which assemble from heterodimers of and tubulin. Microtubules have an outer diameter of 24 nm and a hollow lumen 14 nm wide. Tubulin molecules are arranged to form 13 protofilaments, as seen in the cross section in the upper part of the drawing. The specific orientation of the tubulin dimers results in structural polarity of the microtubule. Microtubules elongate or rapidly shorten by the addition or removal of tubulin at the ends of individual protofilaments. The lengths and locations of cytoplasmic microtubules vary greatly during different phases of cell activity, with assembly dependent on shifting balances between polymerized and unpolymerized tubulin and other factors in "dynamic instability."

Actin micro filaments (MF) and microtubules (MT) can both be clearly distinguished in this TEM photo of fibroblast cytoplasm. The image also provides a good comparison of the relative diameters of these two cytoskeletal components.

The ultrastructural view can be compared to the appearance of microfilaments and microtubules in a cultured cell stained by immunocytochemistry. Actin filaments (red) are most concentrated at the cell periphery, forming prominent circumferential bundles from which finer filaments project into the transient cellular extensions at the edge of the cell and push against the cell membrane. Such an arrangement of actin filaments forms a dynamic network important for cell shape changes such as those during cell division, locomotion, and formation of cellular processes, folds, pseudopodia, lamellipodia, veils, microvilli, etc. which serve to change a cell's surface area or give direction to a cell's crawling movements. Microtubules (green/yellow) are present throughout the cytoplasm and are oriented in arrays which generally extend from the area around the nucleus into the most peripheral extensions.

CILIA

Cilia are motile structures projecting from a cell, typically the apical end of epithelial cells. Each cilium is covered by the cell membrane and contains cytoplasm dominated by a specialized assembly of unusually stable microtubules, the axoneme. Shifting movements between microtubules of an axoneme produce whip-like motions of the cilia. Most epithelial cells lining the respiratory tract, such as those shown in the three micrographs here, have numerous cilia which move to propel mucus along the tract toward the pharynx. Between the ciliated cells are mucus-producing, non-ciliated goblet cells (G) with basal nuclei and apical cytoplasm filled with mucus granules. The relative size and spacing of the ciliated cells and goblet cells is seen in micrographs. (a): Light micrograph. X400. Pararosaniline-toluidine blue,

CILIA

(b): SEM. X300.

(c): TEM shows the axonemes of cilia cut in different orientations and their basal bodies in the apical cytoplasm. X9200.

MICROTUBULES

(a): cross-section by TEM after fixation with tannic acid in glutaraldehyde, which leaves the unstained tubulin subunits delineated by the dense tannic acid. Cross sections of tubules reveal the ring of 13 subunits of dimeric tubulin which are arranged lengthwise as protofilaments. Changes in microtubule length are caused by the addition or loss of individual tubulin subunits from protofilaments.

CILIA(b): A diagrammatic cross-section through a cilium reveals a cytoplasmic core of microtubules called an axoneme. The axoneme consists of two central microtubules surrounded by nine peripheral microtubular doublets associated with several other proteins. In the doublets, microtubule A is complete, consisting of 13 protofilaments, whereas microtubule B shares some of A's protofilament heterodimers. A series of protein complexes containing ciliary dynein, the inner and outer dynein arms, are bound to microtubule A along its length. When activated by ATP, the dynein arms briefly link microtubule B of the adjacent doublet and provide for slight sliding of the doublets against each other, which is then immediately reversed. This rapid back-and-forth shift between adjacent doublets, produced by the ciliary dynein motors, causes the rhythmic changes of axonemal shape that bring about the flailing motion of the entire cilium. Each axoneme is continuous with a basal body located at the base of the cilium. Basal bodies are structurally very similar to centrioles, which nucleate and organize the growth of microtubules during formation of the mitotic spindle.

CENTRIOLES

(c): Each centriole consists of nine relatively short microtubular triplets linked together in a pinwheel-like arrangement. In the triplets, microtubule A is complete and consists of 13 protofilaments, whereas microtubules B and C share protofilaments. Under normal circumstances, these organelles are found in pairs and are oriented at right angles to one another. The pair of centrioles is called a centrosome.

CENTROSOME

ACTIN FILAMENT TREADMILLINGActin filaments or microfilaments are helical two-stranded polymers assembled from globular actin subunits. The filaments are flexible structures, with diameters in various cells of 5-9 nm, depending on associated proteins. Assembly of actin filaments (F-actin) results in their polarity, with actin subunits (G-actin) added to the plus (+) end and removed at the minus (–) end. Even actin filaments of a constant length are highly dynamic structures, balancing G-actin assembly and disassembly at the opposite ends, with a net movement or flow along the polymer known as treadmilling.

WITHIN CELLS, ACTIN MICROFILAMENTS (F-ACTIN) CAN BE ORGANIZED IN SEVERAL FORMS. 1. In skeletal muscle, they assume a stable array

integrated with thick (16-nm) myosin filaments. 2. In most cells, microfilaments form a thin sheath or

network just beneath the plasmalemma. These filaments are involved in all cell shape changes such as those during endocytosis, exocytosis, and cell locomotion.

3. Microfilaments are intimately associated with several cytoplasmic organelles, vesicles, and granules and play a role in moving or shifting cytoplasmic components (cytoplasmic streaming).

4. Microfilaments are associated with myosin and form a "purse-string" ring of filaments whose constriction results in the cleavage of mitotic cells.

5. In crawling cells actin filaments are organized into parallel contractile bundles called stress fibers

INTERMEDIATE FILAMENTS OF KERATIN

Intermediate filaments display an average diameter of 10-12 nm, between that of actin filaments and microtubules, and serve to provide mechanical strength or stability to cells. Unlike the other two cytoskeletal polymers, intermediate filaments are composed of various protein subunits in different types of cells. All such subunits appear to be rodlike rather than globular and undergo step-wise assembly into a structure resembling a cable with many strands. A large and important class of intermediate filaments is composed of keratin subunits, which are prominent in epithelial cells. Bundles of keratin filaments associate with certain classes of intercellular junctions common in epithelial cells and are easily seen with the TEM, as shown here in two extensions in an epidermal cell bound to a neighboring cell.

CELLULAR INCLUSIONS-1

Lipid droplets are abundant in cells of the adrenal cortex, and appear with the TEM as small spherical structures with homogenous matrices (L). Mitochondria are also seen here. As aggregates of hydrophobic lipid molecules these inclusions are enclosed by a single monolayer of phospholipids with various peripheral proteins, including enzymes for lipid metabolism. In routine processing of tissue for paraffin sections fat droplets are generally removed, leaving empty spaces in the cells. Common fat cells have cytoplasm essentially filled with one large lipid droplet. X19,000.

CELLULAR INCLUSIONS-2

TEM of a liver cell cytoplasm shows numerous individual or clustered electron-dense particles representing glycogen granules, although these granules lack membrane. Glycogen granules usually form characteristic aggregates such as those shown. Glycogen is a ready source of energy and such granules are often abundant in cells with high metabolic activity. X30,000.

CELLULAR INCLUSIONS-3

Pigment deposits (PD) occur in many cell types and may contain various complex substances, such as lipofuscin or melanin. Lipofuscin granules represent an accumulating by-product of lysosomal digestion in long-lived cells, but melanin granules serve to protect cell nuclei from damage to DNA caused by light. Many cells contain pigmented deposits of hemosiderin granules containing the protein ferritin, which forms a storage complex for iron. Hemosiderin granules are very electron-dense, but with the light microscope appear brownish and resemble lipofuscin. The liver cells shown have large cytoplasmic regions filled with pigment deposits which probably represent iron-containing hemosiderin. X400

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