Basic structure and function of cells

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CHAPTER 1 Basic structure and function of cells

CELL STRUCTURE

GENERAL CHARACTERISTICS OF CELLS

The shapes of mammalian cells vary widely depending on their interactions with each other, their extracellular environment and internal structures. Their surfaces are often highly folded when absorptive or transport functions take place across their boundaries. Cell size is limited by rates of diffusion, either that of material entering or leaving cells, or of diffusion within them. Movement of macromolecules can be much accelerated and also directed by processes of active transport across membranes and by transport mechanisms within the cell. According to the location of absorptive or transport functions, apical microvilli (Fig. 1.1) or basolateral infoldings create a large surface area for transport or diffusion.

Motility is a characteristic of most cells, in the form of movements of cytoplasm or specific organelles from one part of the cell to another. It also includes: the extension of parts of the cell surface such as pseudopodia, lamellipodia, filopodia and microvilli; locomotion of entire cells as in the amoeboid migration of tissue macrophages; the beating of flagella or cilia to move the cell (e.g. in spermatozoa) or fluids overlying it (e.g. in respiratory epithelium); cell division and muscle contraction. Cell movements are also involved in the uptake of materials from their environment (endocytosis, phagocytosis) and the passage of large molecular complexes out of cells (exocytosis, secretion).

Cells rarely operate independently of each other and commonly form aggregates by adhesion, often assisted by specialized intercellular junctions. They may also communicate with each other either by generating and detecting molecular signals that diffuse across intercellular spaces, or more rapidly by membrane contact, which may involve small, transient, transmembrane channels or interactions between membrane-bound signalling molecules. Cohesive groups of cells constitute tissues and more complex assemblies of tissues form functional systems or organs.

Most cells are between 5–50 μm in diameter: e.g. resting lymphocytes are 6 μm across, red blood cells 7.5 μm and columnar epithelial cells are 20 μm tall and 10 μm wide (all measurements are approximate). Some cells are much larger than this: e.g. megakaryocytes of the bone marrow are more than 200 μm in diameter. Large neurones and skeletal muscle cells have relatively enormous volumes because of their extended shapes, some of the former being over 1 metre in length.

CELLULAR ORGANIZATION

Each cell is contained within its limiting plasma, or surface, membrane which encloses the cytoplasm. All cells except mature red blood cells also contain a nucleus that is surrounded by a nuclear membrane or envelope (Fig. 1.1, Fig. 1.2). The nucleus includes the genome of the cell contained within the chromosomes, the nucleolus, and other sub-nuclear organelles. The cytoplasm contains several systems of organelles. These include a series of membrane-bound structures that form separate compartments within the cytoplasm, such as rough and smooth endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, mitochondria and vesicles for transport, secretion and storage of cellular components. There are also structures that lie free in the non-membranous, cytosolic compartment. They include ribosomes and several filamentous protein networks known collectively as the cytoskeleton. The cytoskeleton determines general cell shape and supports specialized extensions of the cell surface (microvilli, cilia, flagella). It is involved in the assembly of new filamentous organelles (e.g. centrioles) and controls internal movements of the cytoplasm and cytoplasmic vesicles. The cytosol contains many soluble proteins, ions and metabolites.

Cell polarity and domains

Epithelia (including endothelia and mesothelia) are organized into sheets or more complex structures (see Ch. 2) with very different environments on either side. These cells actively transfer macromolecules and ions between the two surfaces and are thus polarized in structure and function (Fig. 1.3). In polarized cells, particularly in epithelia, the cell is generally subdivided into domains that reflect the polarization of activities within it. The free surface, e.g. that facing the intestinal lumen or airway, is the apical surface, and its adjacent cytoplasm is the apical cell domain. This is where the cell interfaces with a specific body compartment (or, in the case of the epidermis, with the outside world). The apical surface is specialized to act as a barrier, restricting access of substances from this compartment to the rest of the body. Specific components are selectively absorbed from, or added to, the external compartment by the active processes, respectively, of active transport and endocytosis inwardly or exocytosis and secretion outwardly. The apical surface is often covered with small protrusions of the cell surface, microvilli, which increase the surface area, particularly for absorption.

The surface of the cell opposite to the apical surface is the basal surface, with its associated basal cell domain. In a single-layered epithelium, this surface is apposed to the basal lamina. The remaining surfaces are known as the lateral cell surfaces. In many instances the lateral and basal surfaces perform similar functions and the cellular domain is termed the basolateral domain. Cells actively transport substances, such as digested nutrients from the intestinal lumen or endocrine secretions, across their basal (or basolateral) surfaces into the subjacent connective tissue matrix and the blood capillaries within it. Dissolved non-polar gases (oxygen and carbon dioxide) diffuse freely between the cell and the bloodstream across the basolateral surface. Apical and basolateral surfaces are separated by a tight intercellular seal, the tight junction, which prevents the passage of even small ions through the space between adjacent cells and thus maintains the difference between environments either side of the epithelium.

PLASMA MEMBRANE

Cells are bounded by a distinct plasma membrane, which shares features with the system of internal membranes that compartmentalize the cytoplasm and surround the nucleus. They are all composed of lipids (mainly phospholipids, cholesterol and glycolipids) and proteins, in approximately equal ratios. Plasma membrane lipids form a layer two molecules thick, the lipid bilayer. The hydrophobic ends of each lipid molecule face the interior of the membrane and the hydrophilic ends face outwards. Most proteins are embedded within, or float in, the lipid bilayer as a fluid mosaic. Some proteins, because of extensive hydrophobic regions of their polypeptide chains, span the entire width of the membrane (transmembrane proteins), whereas others are only superficially attached to the bilayer by lipid groups. Both are integral (intrinsic) membrane proteins, as distinct from peripheral (extrinsic) membrane proteins, which are membrane-bound only through their association with other proteins. Carbohydrates in the form of oligosaccharides and polysaccharides are bound either to proteins (glycoproteins) or to lipids (glycolipids), and project mainly into the extracellular domain.

Combinations of biochemical, biophysical and biological techniques have revealed that lipids are not homogenously distributed in membranes, but that some are organized into microdomains in the bilayer, called ‘detergent-resistant membranes’ or lipid ‘rafts’, rich in sphingomyelin and cholesterol (Morris et al 2004). The ability of select subsets of proteins to partition into different lipid microdomains has profound effects on their function, e.g. in T-cell receptor and neurotrophin signalling. The highly organized environment of the domains provides a signalling, trafficking and membrane fusion environment very different from that found in the disorganized fluid mosaic membrane.

In the electron microscope, membranes fixed and contrasted by heavy metals such as osmium appear in section as two densely stained layers separated by an electron-translucent zone – the classic unit membrane (Fig. 1.4). The total thickness is about 5 nm. Freeze-fracture cleavage planes usually pass along the midline of each membrane, where the hydrophobic tails of phospholipids meet. This technique has also demonstrated intramembranous particles embedded in the lipid bilayer; these are in the 5–15 nm range and in most cases represent large transmembrane protein molecules or complexes of molecules. Intramembranous particles are distributed asymmetrically between the two half-membranes, usually adhering more to one face than to the other. In plasma membranes, the inner or protoplasmic (cytoplasmic) half-membrane carries most particles, seen on its surface facing the exterior (the P face). Where they have been identified, particles usually represent channels for the transmembrane passage of ions or molecules.

Biophysical measurements show the lipid bilayer to be highly fluid, allowing diffusion in the plane of the membrane. Thus proteins are able to move freely in such planes unless anchored from within the cell. Membranes in general, and the plasma membrane in particular, form boundaries selectively limiting diffusion and creating physiologically distinct compartments. Lipid bilayers are impermeable to hydrophilic solutes and ions and so membranes actively control the passage of ions and small organic molecules such as nutrients, through the activity of membrane transport proteins. However, lipid-soluble substances can pass directly through the membrane so that, for example, steroid hormones enter the cytoplasm freely. Their receptor proteins are either cytosolic or nuclear, rather than being located on the cell surface.

Plasma membranes are able to generate electrochemical gradients and potential differences by selective ion transport, and actively take up or export small molecules by energy dependent processes. They also provide surfaces for the attachment of enzymes, sites for the receptors of external signals, including hormones and other ligands, and sites for the recognition and attachment of other cells. Internally, plasma membranes can act as points of attachment for intracellular structures, in particular those concerned with motility and other cytoskeletal functions. Cell membranes are synthesized by the rough endoplasmic reticulum in conjunction with the Golgi apparatus.

The cell coat (glycocalyx)

The plasma membrane differs structurally from internal membranes in that it possesses an external, diffuse, carbohydrate-rich coat, the cell coat or glycocalyx. The cell coat forms an integral part of the plasma membrane, projecting as a diffusely filamentous layer 2–20 nm or more from the lipoprotein surface (see Fig. 1.5). The overall thickness of the plasma membrane is therefore variable, but is typically 8–10 nm. The cell coat is composed of the carbohydrate portions of glycoproteins and glycolipids embedded in the plasma membrane (Fig. 1.4).

The precise composition of the glycocalyx varies with cell type: many tissue and cell type-specific antigens are located in the coat, including the major histocompatibility antigen systems and, in the case of erythrocytes, blood group antigens. It also contains adhesion molecules, which enable cells to adhere selectively to other cells or to the extracellular matrix. They have important roles in maintaining the integrity of tissues and in a wide range of dynamic cellular processes, e.g. the formation of intercommunicating neural networks in the developing nervous system and the extravasation of leukocytes. Cells tend to repel each other because of the predominance of negatively charged carbohydrates at cell surfaces. There is consequently a distance of at least 20 nm between the plasma membranes of adjacent cells, other than at specialized junctions.

Cell surface contacts

The plasma membrane is the surface which establishes contact with other cells and with structural components of extracellular matrices. These contacts may have a predominantly adhesive role, or initiate instructive signals within and between cells, or both; they frequently affect the behaviour of cells. Structurally, there are two main classes of contact, both associated with cell adhesion molecules. One class is associated with specializations at discrete regions of the cell surface that are ultrastructurally distinct. These are described on page 6. The second, general, class of adhesive contact has no obvious associated ultrastructural features.

General adhesive contacts

One class of transmembrane or membrane-anchored glycoproteins that project externally from the plasma membrane, and which form adhesive contacts, are the cell adhesion molecules. There are a number of molecular subgroups, which are broadly divisible on the basis of their calcium dependence. Their numerous functions include tissue formation and morphogenesis and there is evidence for coordinated interaction between the subgroups.

Calcium-dependent adhesion molecules

Cadherins, selectins and integrins are calcium-dependent adhesion molecules. Cadherins are transmembrane proteins, with five heavily glycosylated external domains. They are responsible for strong general intercellular adhesion, as well as being components of some specialized adhesive contacts, and are attached by linker proteins (catenins) at their cytoplasmic ends to underlying cytoskeletal fibres (either actin or intermediate filaments). Different cell types possess different members of the cadherin family, e.g. N-cadherins in nervous tissue, E-cadherins in epithelia, and P-cadherins in the placenta. These molecules bind to those of the same type in other cells (homophilic binding), so that cells of the same class adhere to each other preferentially, forming tissue aggregates or layers, as in epithelia. For a review of cadherin-mediated adhesion in morphogenesis, see Gumbiner (2005).

Selectins are found on leukocytes, platelets and vascular endothelial cells. They are transmembrane lectin glycoproteins that can bind with low affinity to the carbohydrate groups on other cell surfaces to permit movement between the two, e.g. the rolling adhesion of leukocytes on the walls of blood vessels (p. 136). They function cooperatively in sequence with integrins, which strengthen the selectin adhesion.

Integrins are glycoproteins that typically mediate adhesion between cells and extracellular matrix components such as fibronectin, collagen, laminin. They integrate interactions between the matrix and the cell cytoskeleton to which they are linked, and so facilitate cell migration within the matrix. An integrin molecule is formed of two subunits (α and β), each of which has several subtypes. Combinations of alternative subunits provide some 24 known integrin heterodimers, each one directed to a particular extracellular molecule, although there is considerable overlap in specificity. Some integrins depend for their binding on magnesium, rather than calcium.

Specialized adhesive contacts

Specialized adhesive contacts, some of which mediate activities other than simple mechanical cohesion, are localized regions of the cell surface with particular ultrastructural characteristics. Three major classes exist: occluding, adhesive and communicating junctions (Fig. 1.5).

Occluding junctions (tight junctions, zonula occludens)

Occluding junctions create diffusion barriers in continuous layers of cells, including epithelia, mesothelia and endothelia, and prevent the passage of materials across the cellular layer through intercellular spaces. They form a continuous belt (zonula) around the cell perimeter, near the apical surface in cuboidal or columnar epithelial cells. At a tight junction, the membranes of the adjacent cells come into contact, so that the gap between them is obliterated. Freeze-fracture electron microscopy shows that the contacts between the membranes lie along branching and anastomosing ridges formed by the incorporation of chains of intramembranous protein particles on the P face of the lipid bilayer (Fig. 1.5C).

This arrangement ensures that substances can only pass through the layer of cells by diffusion or transport through their apical membranes and cytoplasm. The cells thus selectively modify the environment on either side of the layer. Occluding junctions also create regional differences in the plasma membranes of the cells in which they are found. For example, in epithelia, the composition of the apical plasma membrane differs from that of the basolateral regions (see Fig. 1.3), and this allows these regions to engage in functions such as directional transport of ions and uptake of macromolecules. Because tight junctions have high concentrations of fixed transmembrane proteins, they act as barriers to lateral diffusion of lipid and protein within membranes. The integrity of tight junctions is calcium-dependent. Cells can transiently alter the permeability of their tight junctions to increase passive paracellular transport in some circumstances.

Adhesive junctions

Adhesive junctions include intercellular and cell–extracellular matrix contacts, where cells adhere strongly to each other or to adjacent matrix components. Intercellular contacts can be subdivided according to the extent and location of the contact. They all display a high concentration of cell adhesion molecules, which externally bind adjacent cells, and internally link to the cytoskeleton via intermediary proteins.

Desmosomes (maculae adherentes)

Desmosomes are limited, plaque-like areas of particularly strong intercellular contact. They can be located anywhere on the cell surface. In epithelial cells, there may be a circumferential row of desmosomes parallel to the tight and intermediate junctional zones, an arrangement that forms the third, most basally situated, component of the epithelial apical junctional complex (Fig. 1.5). The intercellular gap is approximately 25 nm, is filled with electron-dense filamentous material running transversely across it and is also marked by a series of densely staining bands running parallel to the cell surfaces. Adhesion is mediated by calcium-dependent cadherins, desmoglein and desmocollin. Within the cells on either side, a cytoplasmic density underlies the plasma membrane and includes the anchor proteins desmoplakin and plakoglobin, into which the ends of intermediate filaments are inserted. The type of intermediate filament depends on cell type, e.g. keratins are found in epithelia and desmin filaments in cardiac muscle cells. Desmosomes form strong anchorage points, likened to spot-welds, between cells subject to mechanical stress, e.g. in the prickle cell layer of the epidermis, where they are extremely numerous and large.

Gap junctions (communicating junctions)

Gap junctions resemble tight junctions in transverse section, but the two apposed lipid bilayers are separated by an apparent gap of 3 nm which is bridged by numerous transmembrane channels (connexons). Connexons are formed by a ring of six connexin proteins in each membrane. Their external surfaces meet those of the adjacent cell in the middle. A minute central pore links one cell to the next (Fig. 1.5). These channels may exist in small numbers, and this makes them difficult to detect structurally. However, they lower the transcellular electrical resistance and so can be detected by microelectrodes. Larger assemblies of many thousands of channels are often packed in hexagonal arrays (Fig. 1.5B). Such junctions form limited attachment plaques rather than continuous zones, and so allow free passage of substances within the adjacent intercellular space, unlike tight junctions. They occur in numerous tissues including the liver, epidermis, pancreatic islet cells, connective tissues, cardiac muscle and smooth muscle, and are also common in embryonic tissues. In the central nervous system, they are found in the ependyma and between neuroglial cells, and they form electrical synapses between some types of neurone, although this is rare in humans. Very recently, a second family of gap junctional proteins has been discovered, the pannexins. In humans, expression of pannexins has been most extensively studied in the nervous system (reviewed in Litvin et al 2006).

Although gap junctions form diffusion channels between cells, the size of their apertures limits diffusion to small molecules and ions (up to a molecular weight of about 1000 kDa). Thus they admit sodium, potassium and calcium ions, various second messenger components, and a number of metabolites, but they exclude messenger RNA and other macromolecules. In some excitable tissues (e.g. cardiac and smooth muscle), one cell can activate another electrically by current flow through gap junctions. Communicating junctions probably permit metabolic cooperation between groups of adjacent cells; the significance of this activity in embryogenesis, normal tissue function, homeostasis and repair is only beginning to be understood.

Cell signalling

Cellular systems in the body communicate with each other to coordinate and integrate their functions. This occurs through a variety of processes known collectively as cell signalling, in which a signalling molecule produced by one cell is detected by another, almost always by means of a specific receptor protein molecule. The recipient cell transduces the signal, which it most usually detects at the plasma membrane, into intracellular chemical messages that change cell behaviour.

The signal may act over a long distance, e.g. endocrine signalling through the release of hormones into the bloodstream, or neuronal synaptic signalling via electrical impulse transmission along axons and subsequent release of chemical transmitters of the signal at synapses (p. 44) or neuromuscular junctions (p. 62). A specialized variation of endocrine signalling (neurocrine or neuroendocrine signalling) occurs when neurones or paraneurones (e.g. chromaffin cells of the suprarenal medulla) secrete a hormone into interstitial fluid and the bloodstream.

Alternatively, signalling may occur at short range through a paracrine mechanism, in which cells of one type release molecules into the interstitial fluid of the local environment, to be detected by nearby cells of a different type that express the specific receptor protein. Neurocrine cell signalling uses chemical messengers found also in the central nervous system which may act in a paracrine manner via interstitial fluid or reach more distant target tissues in the bloodstream. Cells may generate and respond to the same signal. This is autocrine signalling, a phenomenon that reinforces the coordinated activities of a group of like cells, which respond together to a high concentration of a local signalling molecule. The most extreme form of short-distance signalling is contact-dependent (juxtacrine) signalling, where one cell responds to transmembrane proteins of an adjacent cell that bind to surface receptors in the responding cell membrane. Contact-dependent signalling also includes cellular responses to integrins on its surface binding to elements of the extracellular matrix. Juxtacrine signalling is important during development and in immune responses.

These different types of intercellular signalling mechanism are illustrated in Figure 1.6. For further reading, see Alberts et al (2002) and Pollard & Earnshaw (2007).

Signalling molecules and their receptors

The majority of signalling molecules (ligands) are hydrophilic. They cannot cross the plasma membrane of a recipient cell to effect changes intracellularly unless they first bind to a plasma membrane receptor protein. Ligands are mainly proteins (usually glycoproteins), polypeptides or highly charged biogenic amines. They include: classic peptide hormones of the endocrine system; cytokines, which are mainly of haemopoietic cell origin and involved in inflammatory responses and tissue remodelling (e.g. the interferons, interleukins, tumour necrosis factor, leukaemia inhibitory factor); polypeptide growth factors (e.g. the epidermal growth factor superfamily, nerve growth factor, platelet-derived growth factor, the fibroblast growth factor family, transforming growth factor beta and the insulin-like growth factors). Polypeptide growth factors are multifunctional molecules with more widespread actions and cellular sources than their names suggest. They and their receptors are commonly mutated or aberrantly expressed in certain cancers. The cancer-causing gene variant is termed a transforming oncogene and the normal (wild-type) version of the gene is a cellular oncogene or proto-oncogene. The activated receptor acts as a transducer to generate intracellular signals, which are either small diffusible second messengers (e.g. calcium, cyclic adenosine monophosphate or the plasma membrane lipid-soluble diacylglycerol), or larger protein complexes that amplify and relay the signal to target control systems. For further reading on growth factors and other signalling molecules, see Epstein (2003).

Some signals are hydrophobic and able to cross the plasma membrane freely. Classic examples are the steroid hormones, thyroid hormones, retinoids and vitamin D. Steroids, for instance, enter cells non-selectively, but elicit a specific response only in those target cells which express specific cytoplasmic or nuclear receptors. Light stimuli also cross the plasma membranes of photoreceptor cells and interact intracellularly, at least in rod cells, with membrane-bound photosensitive receptor proteins. Hydrophobic ligands are transported in the bloodstream or interstitial fluids, generally bound to carrier proteins, and they often have a longer half-life and longer-lasting effects on their targets than do water-soluble ligands.

A separate group of signalling molecules that are able to cross the plasma membrane freely is typified by the gas, nitric oxide. The principal target of short-range nitric oxide signalling is smooth muscle, which relaxes in response. Nitric oxide is released from vascular endothelium as a result of the action of autonomic nerves that supply the vessel wall. It causes local relaxation of smooth muscle and dilation of vessels. In the penis, this mechanism is responsible for penile erection. Nitric oxide is unusual among signalling molecules in having no specific receptor protein; instead, it acts directly on intracellular enzymes of the response pathway.

Receptor proteins

There are some 20 different families of receptor proteins, each with several isoforms responding to different ligands. The great majority of these receptors are transmembrane proteins. Members of each family share structural features that indicate either shared ligand-binding characteristics in the extracellular domain or shared signal transduction properties in the cytoplasmic domain, or both. There is little relationship either between the nature of a ligand and the family of receptor proteins to which it binds and activates, or the signal transduction strategies by which an intracellular response is achieved. The same ligand may activate fundamentally different types of receptor in different cell types.

Cell surface receptor proteins are generally grouped according to their linkage to one of three intracellular systems: ion channel-linked receptors; G-protein-coupled receptors; receptors that link to enzyme systems. Other receptors do not fit neatly into any of these categories. All the known G-protein-coupled receptors belong to a structural group of proteins that pass through the membrane seven times in a series of serpentine loops. These receptors are thus known as seven-pass transmembrane receptors or, because the transmembrane regions are formed from α-helical domains, as seven-helix receptors. The most well-known of this large group of phylogenetically ancient receptors are the odorant-binding proteins of the olfactory system, the light-sensitive receptor protein, rhodopsin, and many of the receptors for clinically useful drugs. A comprehensive list of receptor proteins, their activating ligands and examples of the resultant biological function, is given in Pollard & Earnshaw (2007).

Transport across cell membranes

Lipid bilayers are increasingly impermeable to molecules as they increase in size or hydrophilicity. Transport mechanisms are therefore required to carry essential polar molecules, including ions, nutrients, nucleotides and metabolites of various kinds, across the plasma membrane and into or out of membrane-bound intracellular compartments. Transport is facilitated by a variety of membrane transport proteins, each with specificity for a particular class of molecule, e.g. sugars. Transport proteins fall mainly into two major classes, channel proteins and carrier proteins.

Channel proteins form aqueous pores in the membrane, which open and close under the regulation of intracellular signals, e.g. G-proteins, to allow the flux of solutes (usually inorganic ions) of specific size and charge. Transport through ion channels is always passive and ion flow through an open channel depends only on the ion concentration gradient and its electronic charge, and the potential difference across the membrane. These factors combine to produce an electrochemical gradient, which governs ion flux. Channel proteins are utilized most effectively by the excitable plasma membranes of nerve cells, where the resting membrane potential can change transiently from about −80 mV (negative inside the cell) to +40 mV (positive inside the cell) when stimulated by a neurotransmitter (as a result of the opening and subsequent closure of channels selectively permeable to sodium and potassium).

Carrier proteins bind their specific solutes, such as amino-acids, and transport them across the membrane through a series of conformational changes. This latter process is slower than ion transport through membrane channels. Transport by carrier proteins can occur either passively by simple diffusion, or actively against the electrochemical gradient of the solute. Active transport must therefore be coupled to a source of energy, such as ATP generation, or energy released by the coordinate movement of an ion down its electrochemical gradient. Linked transport can be in the same direction as the solute, in which case the carrier protein is described as a symporter, or in the opposite direction, when the carrier acts as an antiporter.

Exocytosis and endocytosis

Secreted proteins, lipids, mucins, small molecules such as amines and other cellular products destined for export from the cell are transported to the plasma membrane in small vesicles released from the trans face of the Golgi apparatus. This pathway is either constitutive, in which transport and secretion occur more or less continuously, or it is regulated by external signals, as in the control of salivary secretion by autonomic neural stimulation. In regulated secretion, the secretory product is stored temporarily in membrane-bound secretory granules or vesicles. Exocytosis is achieved by fusion of the secretory vesicular membrane with the plasma membrane and release of the vesicle contents into the extracellular domain.

In polarized cells, e.g. most epithelia, exocytosis occurs at the apical plasma membrane and the cells secrete into a duct lumen or onto a free surface such as the lining of the stomach. In hepatocytes, bile is secreted across a very small area of plasma membrane forming the wall of the bile canaliculus. This region is defined as the apical plasma membrane, and is the site of exocrine secretion, whereas secretion of hepatocyte plasma proteins into the bloodstream is targeted to the basolateral surfaces facing the sinusoids. Packaging of different secretory products into appropriate vesicles takes place in the trans-Golgi network. Delivery of secretory vesicles to their correct plasma membrane domains is achieved by sorting sequences in the cytoplasmic tails of vesicular membrane proteins.

There are other mechanisms in which initial delivery of secretory products is less selective, but is followed by selective retention (or degradation) or reprocessing and redistribution by endosomes. Ultimately, secretory vesicles undergo docking, priming (to prepare the vesicle for a regulatory signal, where secretion is regulation-dependent) and fusion with the plasma membrane to release their contents. The process of exocytosis also delivers integral membrane components to the cell surface in the normal turnover and recycling of the plasma membrane. However, excess plasma membrane generated by vesicle fusion during exocytosis is rapidly removed by concurrent endocytosis.

The process of endocytosis involves the internalization of vesicles derived from the plasma membrane. The vesicles may contain: engulfed fluids and solutes from the extracellular interstitial fluid (pinocytosis); larger macromolecules, often bound to surface receptors (receptor-mediated endocytosis); particulate matter, including microorganisms or cellular debris (phagocytosis). Pinocytosis generally involves small fluid-filled vesicles and is a marked property of capillary endothelium, e.g. where vesicles containing nutrients and oxygen dissolved in blood plasma are transported from the vascular lumen to the endothelial basal plasma membrane (see Fig. 6.11). Interstitial fluid containing dissolved carbon dioxide is also taken up by pinocytosis for simultaneous transportation across the endothelial cell wall in the opposite direction, for release into the bloodstream by exocytosis. This shuttling of pinocytotic vesicles is also termed transcytosis. Larger volumes of fluid are engulfed by dendritic cells, e.g. in the process of sampling interstitial fluids by macropinocytosis in immune surveillance for antigens (p. 79). Interstitial fluid is inevitably taken up during receptor-mediated endocytosis when ligands are internalized.

Receptor-mediated endocytosis, also known as clathrin-dependent endocytosis, is initiated at specialized regions of the plasma membrane known as clathrin-coated pits. Clathrin is a protein that cross-links adjacent adaptor protein (adaptin) complexes to form a basket-like structure, bending the membrane inwards into a hemisphere. Much, but not all, fluid-phase pinocytosis also utilizes clathrin-coated pits. Ligands such as the iron-transporting protein, transferrin, and the cholesterol-transporting low-density lipoprotein bind to their receptors, which cluster in clathrin-coated pits through an interaction with adaptins. The pits then invaginate and pinch off from the plasma membrane, internalizing both receptor and ligand. The processing of endocytic vesicles and their contents is described on p. 12. For further details of the molecular mechanisms of endocytosis, see Alberts et al (2002) or Pollard & Earnshaw (2007).

Phagocytosis

Phagocytosis is a property of many cell types, but is most efficient in cells specialized for this activity. The professional phagocytes of the body belong to the monocyte lineage of haemopoietic cells, in particular the tissue macrophages (p. 78). Other effective phagocytes are neutrophil granulocytes and most dendritic cells (p. 79), which are also of haemopoietic origin. Phagocytosis plays an important part in the immune defence system of the body, in which the amoeboid process of ingestion of organisms for nutrition has evolved into a mechanism for the clearance of microorganisms invading the body. Macrophages also ingest particulate material including inorganic matter, such as inhaled dust particles, in addition to debris from dead cells and protein aggregates such as immune complexes in the blood, airways, interstitial spaces and connective tissue matrices.

Phagocytosis is a triggered process, initiated when a phagocytic cell binds to a particle or organism, often through a process of molecular recognition. Typically, a pathogenic microorganism may first be coated by antibodies, which are bound in turn by receptors for the Fc portion of the antibody molecule expressed by macrophages and neutrophils; in this way the microorganism is attached to the cell. This triggers the production of large pseudopodia, which engulf the organism and internalize it, as their pseudopod tips fuse together. The process appears to depend on actin–myosin-based cellular motility and, unlike receptor-mediated endocytosis, it is energy dependent. Phagosomes thus formed are as large as the body they engulf and can be a considerable proportion of the volume of the phagocytic cell. Inside the cell, the phagosome fuses with lysosomes, which degrade its contents.

CYTOPLASM

Compartments and functional organization

The cytoplasm is highly concentrated, with about 200 mg/ml of proteins (about twice the concentration in blood) that must be precisely organized for correct molecular interactions to occur. It normally has extremely low levels of Ca2+, high K+ and low Na+ ions in comparison to extracellular fluid, differences which are important in cell signalling. Cytoplasm is also reductive, a state maintained by a high concentration of thiol-containing glutathione. The cell is able to undertake completely opposite reactions simultaneously (e.g. the synthesis and degradation of proteins; growth at one end of a cell with retraction at another) by partitioning them into different regions of the cytoplasm. The most fundamental divide is the use of oxidative reactions within the reductive cytoplasm, achieved by the compartmentalization of different environments within membranes. For example, the endoplasmic reticulum is comprised of stacks of tubules, whose lumen resembles the extracellular environment in being oxidative and Ca2+-rich, predominantly encrusted on the external (cytoplasmic) face with attached ribosomes (rough endoplasmic reticulum). Ribosomes are macromolecular machines for protein synthesis and those attached to RER are engaged in synthesizing proteins that will undergo post-translational modification to adapt them (in the RER lumen and within the Golgi apparatus and associated vesicles) for exposure to the oxidative extracellular environment. A key development in the evolution of the modern cell was the ability to use oxygen as an energy source. This is possible due to one organelle, the mitochondrion, whose separate genes and dual membranes suggest its origins as a symbiotic bacterium.

The key organizer of the cytoplasm, and thence of the entire cell, is the cytoskeleton (see below). This is composed of three distinct elements. Intermediate filaments are relatively stable cables of approximately 10 nm diameter that provide strength. Actin microfilaments (6–8 nm diameter) form highly branched scaffolds under the cell surface, organizing the shape of the cell surface and its specialized functions, including extracellular interactions such as signalling and adhesion, by binding to the intracellular domains of receptors and adhesive proteins, respectively. The actin scaffold under the cell surface is highly labile, forming, branching and dissolving in response to extracellular signals. Specialized myosin-family motor proteins attach to actin filaments, generating force to move membranes and to relay vesicles between the surface and the tubulin network. The tubulin network, however, is the core organizer of the cell; it is polarized, which allows motor proteins to move directionally along the tubules and convey vesicular traffic around the cell.

Endoplasmic reticulum

Endoplasmic reticulum is a system of interconnecting membrane-lined channels within the cytoplasm (Fig. 1.7). These channels take various forms, including cisternae (flattened sacs), tubules and vesicles. The membranes divide the cytoplasm into two major compartments. The intramembranous compartment includes the space where secretory products are stored or transported to the Golgi complex and cell exterior. The extramembranous cytosol is made up of the colloidal proteins such as enzymes, carbohydrates and small protein molecules, together with ribosomes and ribonucleic acids, and elements of the cytoskeleton.

Structurally, the channel system can be divided into rough or granular endoplasmic reticulum, which has ribosomes attached to its outer cytosolic surface, and smooth or agranular endoplasmic reticulum, which lacks ribosomes. Functionally, the endoplasmic reticulum is compartmentalized into specialized regions with unique functions. For further reading see Levine & Rabouille (2005).

Rough endoplasmic reticulum

The rough endoplasmic reticulum, studded with ribosomes, is a site of protein synthesis (Fig. 1.8). Most proteins pass through its membranes and accumulate within its cisternae, although some integral membrane proteins, e.g. plasma membrane receptors, are inserted into the rough endoplasmic reticulum membrane, where they remain. After passage from the rough endoplasmic reticulum, proteins remain in membrane-bound cytoplasmic organelles such as lysosomes, become incorporated into new plasma membrane, or are secreted by the cell. Some carbohydrates are also synthesized by enzymes within the cavities of the rough endoplasmic reticulum and may be attached to newly formed protein (glycosylation). Vesicles are budded off from the rough endoplasmic reticulum for transport to the Golgi as part of the protein-targeting mechanism of the cell.

Smooth endoplasmic reticulum

The smooth endoplasmic reticulum (Fig. 1.7) is associated with carbohydrate metabolism and many other metabolic processes, including detoxification and synthesis of lipids, cholesterol and other steroids. The membranes of the smooth endoplasmic reticulum serve as surfaces for the attachment of many enzyme systems, e.g. the enzyme cytochrome P450, which is involved in important detoxification mechanisms and is thus accessible to its substrates, which are generally lipophilic. They also cooperate with the rough endoplasmic reticulum and the Golgi apparatus to synthesize new membranes; the protein, carbohydrate and lipid components are added in different structural compartments. Highly specialized types of endoplasmic reticulum are present in some cells. For example, in skeletal muscle cells, the smooth endoplasmic reticulum (sarcoplasmic reticulum) stores calcium ions, which are released into the cytosol to initiate contraction after stimulation initiated by a motor neurone at the neuromuscular junction (p. 62).

Ribosomes

Ribosomes are macromolecular machines that catalyse the synthesis of proteins from amino-acids. They are granules approximately 15 nm in diameter, composed of ribosomal RNA (rRNA) molecules assembled into two unequal subunits. A large number of proteins, mostly small and basic, are applied mainly to the surfaces of the subunit cores of RNA. The subunits can be separated by their sedimentation coefficients (S) in an ultracentrifuge, into larger 60S and smaller 40S components. These are associated with 73 different proteins (40 in the large subunit and 33 in the small), which have structural and enzymatic functions. Three small, highly convoluted rRNA strands (28S, 5.8S and 5S) make up the large subunit, and one strand (18S) is in the small subunit. Their synthesis and assembly into subunits takes place in the nucleolus, and includes association with ribosomal proteins translocated from their site of synthesis in the cytoplasm. The individual subunits are then transported into the cytoplasm, where they remain separate from each other when not actively synthesizing proteins.

A typical cell contains millions of ribosomes. They may be solitary, relatively inactive structures, or may form groups (polyribosomes or polysomes) attached to messenger RNA (mRNA), which they translate during protein synthesis. Polysomes may be attached to the membranes of the rough endoplasmic reticulum (see Fig. 1.8) or may lie free in the cytosol, where they synthesize proteins for use outside the system of membrane compartments, including enzymes of the cytosol and cytoskeletal proteins. Some of the cytosolic products include proteins that can be inserted directly into (or through) membranes of selected organelles, such as mitochondria and peroxisomes.

In a mature polysome, all the attachment sites of the mRNA are occupied as ribosomes move along it, synthesizing protein according to its nucleic acid sequence. Consequently, the number of ribosomes in a polysome indicates the length of the mRNA molecule and hence the size of the protein being made. The two subunits have separate roles in protein synthesis. The 40S subunit is the site of attachment and translation of mRNA. The 60S subunit is responsible for the release of the new protein and, where appropriate, attachment to the endoplasmic reticulum via an intermediate docking protein that directs the newly synthesized protein through the membrane into the cisternal space.

Protein synthesis on ribosomes may be suppressed by a class of RNA molecule known as small interfering RNA (siRNA) or silencing RNA. These molecules are typically 20–25 nucleotides in length and bind (as a complex with proteins) to specific mRNA molecules via their complementary sequence. This triggers the enzymatic destruction of the mRNA or prevents the movement of ribosomes along it. Synthesis of the encoded protein is thus prevented. Their normal function may have anti-viral or other protective effects; there is also potential for developing artificial siRNAs as a therapeutic tool for silencing disease-related genes.

Golgi apparatus (Golgi complex)

The Golgi apparatus is a distinct cytoplasmic region near the nucleus, and is particularly prominent in secretory cells when stained with silver or other metallic salts. The Golgi apparatus forms part of the pathway by which proteins synthesized in the rough endoplasmic reticulum undergo post-translational modification and are targeted to the cell surface for secretion or for storage in membranous vesicles. As with the endoplasmic reticulum, the Golgi apparatus is compartmentalized spatially, in a labile manner, to carry out specific functions.

Ultrastructurally, the Golgi apparatus is a membranous organelle (Fig. 1.8) consisting of a stack of several flattened membranous cisternae, together with clusters of vesicles surrounding its surfaces. Seen in vertical section, it is often cup-shaped. Small transport vesicles from the rough endoplasmic reticulum, generated by a process of budding and pinching off, are received at one face of the Golgi stack, the convex cis-face (entry or forming surface). Here, they deliver their contents to the first cisterna in the series by membrane fusion. From the edges of this cisterna, the protein is transported to the next cisterna by vesicular budding and then fusion, and this process is repeated until the final cisterna at the concave trans face (exit or condensing surface) is reached. Here, larger vesicles are formed for delivery to other parts of the cell.

In addition to these cisternae, there are other membranous structures that form an integral part of the Golgi apparatus, termed the cis-Golgi and trans-Golgi networks. The cis-Golgi network is a region of complex membranous channels interposed between the rough endoplasmic reticulum and the Golgi cis face (Golgi–rough endoplasmic reticulum complex), which receives and transmits vesicles in both directions. Its function is to select appropriate proteins synthesized on the rough endoplasmic reticulum for delivery by vesicles to the Golgi stack, while inappropriate proteins are shuttled back to the rough endoplasmic reticulum.

The trans-Golgi network, at the other side of the Golgi stack, is also a region of interconnected membrane channels engaged in protein sorting. Here, modified proteins processed in the Golgi cisternae are packaged selectively into vesicles and dispatched to different parts of the cell. The packaging depends on the detection, by the trans-Golgi network, of particular amino-acid signal sequences, leading to their enclosure in membranes of appropriate composition that will further modify their contents, e.g. by extracting water to concentrate them or by pumping in protons to acidify their contents. The membranes contain specific signal proteins, which may allocate them to microtubule-based transport pathways and allow them to dock with appropriate targets elsewhere in the cell, e.g. the plasma membrane in the case of secretory vesicles. Vesicle formation and budding at the trans-Golgi network involves the addition of clathrin on their external surface, to form coated pits.

Within the Golgi stack proper, proteins undergo a series of sequential chemical modifications that started in the rough endoplasmic reticulum. These include: changes in glycosyl groups, e.g. removal of mannose, addition of N-acetyl glucosamine and sialic acid; sulphation of attached glycosaminoglycans; protein phosphorylation. Lipids formed in the endoplasmic reticulum are also routed for incorporation into vesicles.

The role of the Golgi apparatus in the synthesis of primary lysosomes is a major activity in cells with abundant lysosomes, such as those with phagocytic roles. In glandular cells with an apical secretory zone, the Golgi apparatus lies between the secretory surface and the nucleus. In fibroblasts, there are two or more groups of Golgi stacks; up to 50 groups are found in liver cells. The Golgi apparatus is often closely associated with the centrosome (a region of the cell containing a centriole pair and related microtubules), reflecting a link with the microtubule-mediated vesicle transport system.

Endosomes, lysosomes, proteasomes and peroxisomes

The endosome system of vesicles originates in small endocytic vesicles (clathrin-coated vesicles and caveolae) or larger phagosomes and macropinocytic vesicles taken up by the cell from the exterior. Clathrin-dependent endocytosis occurs at specialized patches of plasma membrane called coated pits; this mechanism is also used to internalize ligands bound to surface receptor molecules and is also termed receptor-mediated endocytosis. Caveolae (little caves) are structurally distinct vesicles most widely used by endothelial and smooth muscle cells, where they are involved in transcytosis, signal transduction and possibly other functions. For further reading, see Pollard & Earnshaw (2007).

The endocytic system is linked functionally to a second series of membranous structures, the lysosomes. Lysosomes contain acid hydrolases, which process or degrade exogenous materials (heterophagy), and intracellular organelles that are exhausted, damaged or no longer required (autophagy). There is a continual exchange of vesicles between this system and the Golgi–rough endoplasmic reticulum complex, so that the endosomal/lysosomal system is provided with hydrolytic enzymes and the Golgi receives depleted vesicles for recharging. Once internalized, endocytic vesicles shed their coat of adaptin and clathrin, and fuse with a tubular cisterna termed an early endosome, where the receptor molecules release their bound ligands. Membrane and receptors from the early endosomes can be recycled to the cell surface as exocytic vesicles.

Lysosomes

Lysosomes are dense, spheroidal, membrane-bound bodies 80–800 nm in diameter (Fig. 1.8, Fig. 1.9), often with complex inclusions of material undergoing hydrolysis (secondary lysosomes). They contain acid hydrolases able to degrade a wide variety of substances. To date, more than 40 lysosomal enzymes have been described, including proteases, lipases, carbohydrases, esterases and nucleases. The enzymes are heavily glycosylated, and are maintained at a low pH by proton pumps in the lysosomal membranes.

Lysosomes are numerous in actively phagocytic cells, e.g. macrophages and neutrophil granulocytes, in which lysosomes are responsible for destroying phagocytosed bacteria. In these cells, the phagosome containing the bacterium may fuse with several lysosomes. Lysosomes are also frequent in cells with a high turnover of organelles, e.g. exocrine gland cells and neurones. Effete organelles are targeted for demolition by a process that is not fully understood, but which results in engulfment of areas of cytoplasm, including entire organelles, in a membranous cisterna. The structure then fuses with lysosomes and the contents are rapidly degraded.

Material that has been hydrolysed within late endosomes and lysosomes may be completely degraded to soluble products, e.g. amino-acids, which are recycled through metabolic pathways. However degradation is usually incomplete, and some debris remains. A debris-laden vesicle is called a residual body or tertiary lysosome (Fig. 1.8B), and may be passed to the cell surface, where it is ejected by exocytosis; alternatively, it may persist inside the cell as an inert residual body. Considerable numbers of residual bodies can accumulate in long-lived cells, often fusing to form larger dense vacuoles with complex lamellar inclusions. As their contents are often darkly pigmented, this may change the colour of the tissue, e.g. in neurones the end-product of lysosomal digestion, lipofuscin (neuromelanin or senility pigment), gives ageing brains a brownish-yellow colouration.

Lysosomal enzymes may also be secreted – often as part of a process to alter the extracellular matrix, as in osteoclast erosion of bone (p. 88). Abnormal release of enzymes can cause tissue damage, as in certain types of arthritis. Some drugs, e.g. cortisone, can stabilize lysosomal membranes and may therefore inhibit many lysosomal activities, including the secretion of enzymes, and their fusion with phagocytic vesicles.

Mitochondria

Mitochondria are membrane-bound organelles (Fig. 1.9). They are the principal source of chemical energy in most cells. Mitochondria are the site of the citric acid (Krebs’, tricarboxylic acid) cycle and the electron transport (cytochrome) pathway by which complex organic molecules are finally oxidized to carbon dioxide and water. This process provides the energy to drive the production of ATP from ADP and inorganic phosphate (oxidative phosphorylation). The various enzymes of the citric acid cycle are located in the mitochondrial matrix, whereas those of the cytochrome system and oxidative phosphorylation are localized chiefly in the inner mitochondrial membrane. It is now known that in many tissues, especially smooth muscle, mitochondria also play an important role in cell signalling, especially intracellular calcium homeostasis. They are also major producers of reactive oxygen species and oxidant stress, and are involved in activation of apoptosis.

The numbers of mitochondria in a particular cell reflect its general energy requirements; e.g. in hepatocytes there may be as many as 2000, whereas in resting lymphocytes there are usually very few. Mature erythrocytes lack mitochondria altogether. Cells with few mitochondria generally rely largely on glycolysis for their energy supplies. These include some very active cells, e.g. fast twitch skeletal muscle fibres, which are able to work rapidly, but for only a limited duration. Mitochondria appear in the light microscope as long thin threads in the cytoplasm of most cells, particularly those with a high metabolic rate, e.g. secretory cells in exocrine glands. In living cells, mitochondria constantly change shape and intracellular position; they multiply by growth and fission and may undergo fusion.

In the electron microscope, mitochondria usually appear as round or elliptical bodies 0.5–2.0 μm long. Each mitochondrion is lined by an outer and an inner unit membrane, separated by a variable gap termed the intermembrane space. The lumen is surrounded by the inner membrane and contains the mitochondrial matrix. The outer membrane is smooth and sometimes attached to other organelles, particularly microtubules. The inner membrane is deeply folded to form incomplete transverse or longitudinal tubular invaginations, cristae, which create a relatively large surface area of membrane. Mitochondrial shape, and the shape and organization of the cristae, vary with the cell type. Cristae are most numerous and complex in cells with a high metabolic rate, e.g. cardiac muscle cells. The permeabilities of the two mitochondrial membranes differ considerably: the outer membrane is freely permeable to many substances because of the presence of large non-specific channels formed by proteins (porins), whereas the inner membrane is permeable to only a narrow range of molecules. The presence of cardiolipin, a phospholipid, in the inner membrane may contribute to this relative impermeability.

The mitochondrial matrix is an aqueous environment. It contains a variety of enzymes, and strands of mitochondrial DNA with the capacity for transcription and translation of a unique set of mitochondrial genes (mitochondrial mRNAs and transfer RNAs, mitochondrial ribosomes with rRNAs). The DNA forms a closed loop, about 5 μm across; several identical copies are present in each mitochondrion. The ratio between its bases differs from that of nuclear DNA, and the RNA sequences also differ in the precise genetic code used in protein synthesis. At least 13 respiratory chain enzymes of the matrix and inner membrane are encoded by the small number of genes along the mitochondrial DNA. The great majority of mitochondrial proteins are encoded by nuclear genes and made in the cytosol, then inserted through special channels in the mitochondrial membranes to reach their destinations. Their membrane lipids are synthesized in the endoplasmic reticulum.

Mitochondrial ribosomes are smaller and quite distinct from those of the rest of the cell; they (and mitochondrial nucleic acids) resemble those of bacteria. This similarity underpins the theory that mitochondrial ancestors were oxygen-utilizing bacteria that existed in a symbiotic relationship with eukaryotic cells unable to metabolize the oxygen produced by early plants. As mitochondria are formed only from previously existing ones, it follows that all mitochondria in the body are descended from those in the cytoplasm of the fertilized ovum. It has also been shown that mitochondria are of maternal origin because the mitochondria of the sperm are not generally incorporated into the ovum at fertilization. Thus mitochondria (and mitochondrial genetic variations and mutations) are passed only through the female line.

Mitochondria are distributed within a cell according to regional energy requirements, e.g. near the bases of cilia in ciliated epithelia, in the basal domain of the cells of proximal convoluted tubules in the renal cortex (where considerable active transport occurs) and around the proximal end of the flagellum in spermatozoa. They may be involved with tissue-specific metabolic reactions, e.g. various urea-forming enzymes in liver cell mitochondria. Moreover, a number of genetic diseases of mitochondria affect particular tissues exclusively, e.g. mitochondrial myopathies (skeletal muscle) and mitochondrial neuropathies (nervous tissue). For further information see Graff et al (2002).

Cytosolic organelles

The aqueous cytosol surrounds the membranous organelles described above. It also contains various non-membranous organelles, including free ribosomes, a system of filamentous proteins known as the cytoskeleton, and other inclusions, such as storage granules (e.g. glycogen) and lipid vacuoles.

Cytoskeleton

The cytoskeleton is a system of filamentous intracellular proteins of different shapes and sizes that form a complex, often interconnected, network throughout the cytoplasm. It provides mechanical support, maintains cell shape and rigidity, and enables cells to adopt highly asymmetric or irregular profiles, e.g. in neurones. The cytoskeleton plays an important part in establishing structural polarity and different functional domains within a cell. It also provides mechanical support for projections from the cell surface such as microvilli and cilia, and anchors them into the cytoplasm.

The cytoskeleton restricts specific organelles to particular cellular locations, e.g. the Golgi apparatus is near the nucleus and endoplasmic reticulum, and mitochondria are near sites of energy requirement. Most specifically, the cytoskeleton is concerned with motility, either within the cell (e.g. shuttling vesicles and macromolecules between cytoplasmic sites, or the movement of chromosomes during mitosis), or of the entire cell (e.g. in embryonic morphogenesis or the chemotactic migration of leukocytes). One of the most highly developed and specialized functions of the cytoskeleton is seen in the contractility of muscle cells.

The catalogue of cytoskeletal structural proteins is extensive and still increasing. The major filamentous structures found in non-muscle cells are microfilaments (actin), microtubules (tubulin), and intermediate filaments (assemblies of cell type-specific intermediate filament proteins). Other important components are proteins that bind to the principal filamentous types to link them together or to generate movement. These include actin-binding proteins such as myosin, which in some cells can assemble into thick filaments, and microtubule-associated proteins. Pathologies involving cytoskeletal abnormalities are reviewed in Ramaekers & Bosman (2004).

Actin filaments (microfilaments)

Actin filaments are flexible filaments with a width of 6–8 nm (Fig. 1.10), and a solid cross-section. Within most cell types, actin constitutes the most abundant protein and in some motile cells its concentration may exceed 200 μM (10 mg protein per ml cytoplasm). The filaments are formed by the ATP-dependent polymerization of actin monomer (with a molecular mass of 43 kDa) into a characteristic linear form in which the subunits are arranged in a single tight helix with a distance of 13 subunits between turns. The polymerized form is termed F-actin (fibrillar actin) and the unpolymerized form is G-actin (globular actin). Each monomer has an asymmetric structure. When monomers polymerize, they confer a defined polarity on the filament: the plus end favours monomer addition, and the minus end favours monomer dissociation. Myosins bind to filamentous actin at an angle to give the appearance of a series of arrowheads pointing towards the minus end of the filament, and the barbs point towards the plus end. There is a dynamic equilibrium between G-actin and F-actin: in most cells about 50% of the actin is estimated to be in the polymerized state.

Actin-binding proteins

A wide variety of actin-binding proteins are capable of modulating the form of actin within the cell. These interactions are fundamental to the organization of cytoplasm and to cell shape. Actin-binding proteins can be grouped into bundling proteins, gel-forming proteins and filament severing proteins.

Bundling proteins tie actin filaments together in longitudinal arrays to form cables or core structures. The bundles may be closely spaced, e.g. in microvilli, microspikes and filopodia, where parallel filaments are tied tightly together to form stiff bundles orientated in the same direction. Proteins with this function include fimbrin and villin (also classified as a severing protein). Other actin-bundling proteins form rather looser bundles of filaments that run anti-parallel to each other with respect to their plus and minus ends. They include myosin II, which can form cross-links with ATP-dependent motor activity, and cause adjacent actin filaments to slide on each other, and either change the shape of cells or (if the actin bundles are anchored into the cell membrane at both ends), maintain a degree of active rigidity.

Gel-forming proteins, such as filamin, interconnect adjacent actin filaments to produce loose filamentous meshworks (gels) composed of randomly orientated F-actin. These networks are frequently found in the outer cortical regions of cells, e.g. fibroblasts. They form a semi-rigid zone from which most other organelles are excluded. Severing proteins, such as gelsolin and severin, bind to F-actin filaments and sever them, which produces profound changes within the actin cytoskeleton and in its coupling to the cell surface.

Other classes of actin-binding protein link the actin cytoskeleton to the plasma membrane either directly or indirectly through a variety of membrane-associated proteins. The latter may also create links via transmembrane proteins to the extracellular matrix. Best known of these is the family of spectrin-like molecules, which can bind to actin and also to each other and various membrane-associated proteins to create supportive networks beneath the plasma membrane. Defects in such molecules are linked to a number of inherited diseases (reviewed in Bennett & Healy 2008). Spectrin is found in erythrocytes, and closely related molecules are present in many other cells; for instance, fodrin is found in nerve cells, and dystrophin occurs in muscle cells, linking the contractile apparatus with the extracellular matrix via integral membrane proteins. Proteins such as ankyrin (which also binds actin directly), vinculin, talin, zyxin and paxillin connect actin-binding proteins to integral plasma membrane proteins such as integrins (directly or indirectly), and thence to focal adhesions. Myosin I and other unconventional myosins connect actin filaments to membranous structures, including the plasma membrane and transport vesicle membranes. Tropomyosin, an important regulatory protein of muscle fibres, is also present in non-muscle cells, where its function may be primarily to stabilize actin filaments against depolymerization. For further reading see Pollard & Earnshaw (2007).

Myosins – the motor proteins

The myosin family of microfilaments is often classified within a distinct category of motor proteins. Myosin proteins have a globular head region consisting of a heavy and a light chain. The heavy chain bears an α-helical tail of varying length. The head has an ATPase activity and can bind to and move along actin filaments – the basis for myosin function as a motor protein. The best-known class is myosin II, which occurs in muscle and in many non-muscle cells. Its molecules have two heads and two tails, intertwined to form a long rod. The rods can bind to each other to form long, thick filaments, as seen in striated and smooth muscle fibres, myoepithelial cells and myofibroblasts. Myosin II molecules can also assemble into smaller groups, especially dimers, which can cross-link individual actin microfilaments in stress fibres and other F-actin arrays. The ATP-dependent sliding of myosin on actin forms the basis for muscle contraction and the extension of microfilament bundles, as seen in cellular motility or in the contraction of the ring of actin and myosin around the cleavage furrow of dividing cells. There are a number of known subtypes of myosin II: they assemble in different ways and have different dynamic properties. In skeletal muscle the myosin molecules form bipolar filaments 15 nm thick. Because these filaments have a symmetric anti-parallel arrangement of subunits, the midpoint is bare of head regions. In smooth muscle the molecules form thicker, flattened ribbons and are orientated in different directions on either face of the ribbon. These arrangements have important consequences for the contractile force characteristics of the different types of muscle cell.

Related molecules include the myosin I subfamily of single-headed molecules with tails of varying length. Functions of myosin I include the movements of membranes in endocytosis, microspike formation in neuronal growth cones, actin–actin sliding and attachment of actin to membranes, e.g. of microvilli. Myosin V is implicated in the movements of membranous organelles on actin filaments. So, for example, vesicles track along F-actin in a similar manner to kinesin and dynein-related movements along microtubules. Other myosins have been isolated; the significance of their diversity is not fully understood.

Intermediate filaments

Intermediate filaments are about 10 nm thick and formed by a heterogeneous group of filamentous proteins. They are found in different cell types and are often present in large numbers, either where structural strength is needed (Fig. 1.10B,C), or to provide scaffolding for the attachment of other structures. It is likely that more complex, non-mechanical functions of intermediate filaments, with implications for human disease, remain to be discovered (see Toivola et al 2005). Intermediate filaments of different molecular classes are characteristic of particular tissues or states of maturity. They are therefore important indicators of the origins of cells or levels of differentiation, and are of considerable value in histopathology.

Of the different classes of intermediate filaments, keratin (cytokeratin) proteins are found in epithelia, where keratin filaments are always composed of equal ratios of types I (acidic) and II (basic to neutral) keratins. About 20 types of each of the acidic and basic/neutral keratin proteins are known. Within the epidermis, expression of keratin heterodimer combinations changes as keratinocytes mature during their transition from basal to superficial layers. Genetic abnormalities of keratins are known to affect the mechanical stability of epithelia. For example, the disease epidermolysis bullosa simplex causes lysis of epidermal basal cells and blistering of the skin after mechanical trauma. It is caused by defects in genes encoding keratins 5 and 14, which produce cytoskeletal instability and thus cellular fragility in the basal cells. When keratins 1 and 10 are affected, cells in the prickle cell layer of the epidermis lyse, and this produces the intraepidermal blistering of epidermolytic hyperkeratosis. For a review, see Porter & Lane (2003).

Vimentins occur in mesenchyme-derived cells of connective tissue, desmins in muscle cells, glial fibrillary acidic protein in glial cells, and peripherin in peripheral axons. Neurofilaments are a major cytoskeletal element in neurones, particularly in axons (Fig. 1.10C), where they are the dominant protein. They are heteropolymers of low, medium and high molecular weight neurofilament proteins; the low molecular weight form is always present in combination with either the medium- or the high-molecular weight neurofilament. Abnormal accumulations of neurofilaments (neurofibrillary tangles) are characteristic features of a number of neuropathological conditions.

Other intermediate filament proteins include nestin, a molecule resembling neurofilament protein which forms intermediate filaments in neurectodermal stem cells in particular. Nuclear lamins are intermediate filaments that line the inner surface of the nuclear envelope of all nucleated cells. They provide a mechanical framework for the nucleus and act as attachment sites for a number of proteins that organize the chromatin at the periphery of the nucleus. They are unusual in that they form an irregular anastomosing network of filaments rather than linear bundles.

The exact manner in which intermediate filament proteins polymerize to form linear filaments is much more complex than that of tubulin or actin, and has not been fully determined. The individual intermediate filament proteins are chains with a middle α-helical region flanked on either side by non-helical domains. The proteins associate as coiled coil dimers that form short rods about 48 nm long. These assemble in pairs in a staggered antiparallel formation to form soluble tetramers, eight of which pack together laterally and twist into the rope-like 10 nm intermediate filament. The 32 α-helices in parallel give the filaments their tensile strength. However, unlike actin and myosin, the antiparallel arrangement of the dimers produces a filamentous protein with no intrinsic polarity. The non-coiled regions of the subunits project outwards as side arms that can link intermediate filaments into bundles or attach them to other structures. The existence of different combinations of subunit proteins within one filament is the basis of their functional diversity. In the living cell they have been shown to be quite dynamic structures, possibly as a result of reversible phosphorylation.

Microtubules

Microtubules are polymers of tubulin with the form of hollow, relatively rigid cylinders, approximately 25 nm in diameter and of varying length (up to 70 μm in spermatozoan flagella). They are present in most cell types, and are particularly abundant in neurones (Fig. 1.10C), leukocytes and blood platelets. They are the predominant constituent of the mitotic spindles of dividing cells. They also form part of the structure of cilia, flagella and centrioles.

There are two major classes of tubulin: α- and β-tubulins. Before microtubule assembly, tubulins are associated as dimers with a combined molecular mass of 100 kDa (50 kDa each). Each protein subunit is approximately 5 nm across and arranged along the long axis in straight rows of alternating α- and β-tubulins, forming protofilaments. Typically, 13 protofilaments (the number can vary between 11 and 16), associate in a ring to form the wall of a hollow cylindrical microtubule. Each longitudinal row is slightly out of alignment with its neighbour, so that a spiral pattern of alternating α and β subunits appears when the microtubule is viewed from the side. There is a dynamic equilibrium between the dimers and assembled microtubules: dimeric asymmetry creates polarity (α-tubulins are all orientated towards the minus end, β-tubulins towards the plus end). Tubulin is added preferentially to the plus end; the minus end is relatively slow growing. Microtubules exhibit a dramatic behaviour, known as dynamic instability, in which growing tubules can undergo a ‘catastrophe’, abruptly shifting from net growth to rapid shrinkage. This can result in disappearance of the microtubule, or the catastrophe can be rescued and growth resumed. Tubulins are guanosine triphosphate (GTP)-binding proteins, and growth is accompanied by hydrolysis of GTP. This may regulate the dynamic behaviour of the tubules. Microtubule growth is initiated at specific sites known as microtubule-organizing centres, the best known of which are centrosomes, from which most cellular microtubules polymerize and basal bodies, from which cilia grow.

Various drugs (e.g. colcemid, vinblastine, griseofulvin, nocodazole) cause microtubule depolymerization by binding the soluble tubulin dimers and so shifting the equilibrium towards the unpolymerized state. Microtubule disassembly causes a wide variety of effects, including the inhibition of cell division by disruption of the mitotic spindle. Conversely, the drug taxol stabilizes microtubules and promotes abnormal microtubule assembly. This can cause a peripheral neuropathy, but taxol is widely used as an effective chemotherapeutic agent in the treatment of cancer.

Different microtubules possess varying degrees of stability, e.g. microtubules in cilia are generally unaffected by many drugs that cause microtubular demolition. There are also differences between tissues, e.g. neurones have a special tubulin subclass. Microtubule organizing centres include a specialized tubulin isoform known as γ-tubulin, that is essential for the nucleation of microtubule growth.

Microtubule-associated proteins

Various proteins that can bind to assembled tubulins may be concerned with structural properties or associated with motility. One important class of microtubule-associated proteins (MAPs) consists of proteins that associate with the plus ends of microtubules. They regulate the dynamic instability of microtubules as well as interactions with other cellular substructures. Structural MAPs form cross-bridges between adjacent microtubules or between microtubules and other structures such as intermediate filaments, mitochondria and the plasma membrane. Microtubule-associated proteins found in neurones include: MAPs 1A and 1B, which are present in neuronal dendrites and axons; MAPs 2A and 2B, found chiefly in dendrites; and tau, found only in axons. MAP 4 is the major microtubule-associated protein in many other cell types. Structural microtubule-associated proteins are implicated in microtubule formation, maintenance and demolition, and are therefore of considerable significance in cell morphogenesis, mitotic division, and the maintenance and modulation of cell shape. Motility-associated microtubule-associated proteins are found in situations in which movement occurs over the surfaces of microtubules, e.g. the transport of cytoplasmic vesicles, bending of cilia and flagella, and some movements of mitotic spindles. They include a large family of motor proteins, the best known of which are the dyneins and kinesins. Another protein, dynamin, is involved in endocytosis. The kinetochore proteins assemble at the chromosomal centromere during mitosis and meiosis. They attach (and thus attach chromosomes) to spindle microtubules; some of the kinetochore proteins are responsible for chromosomal movements in mitotic and meiotic anaphase.

All of these microtubule-associated proteins bind to microtubules and either actively slide along their surfaces or promote microtubule assembly or disassembly. Kinesins and dyneins can simultaneously attach to membranes such as transport vesicles and convey them along microtubules for considerable distances, thus enabling selective targeting of materials within the cell. Such movements occur in both directions along microtubules. Kinesin-dependent motion is usually towards the plus ends of microtubules, e.g. from the cell body towards the axon terminals in neurones, and away from the centrosome in other cells. Conversely, dynein-related movements are in the opposite direction, i.e. to the minus ends of microtubules. Dyneins also form the arms of peripheral microtubules in cilia and flagella, where they make dynamic cross-bridges to adjacent microtubule pairs. When these tethered dyneins try to move, the resulting shearing forces cause the axonemal array of microtubules to bend, generating ciliary and flagellar beating movements. Kinesins form a large and diverse family of related microtubule-stimulated ATPases. Some kinesins are motors that move cargo, others cause microtubule disassembly, whilst still others cross-link mitotic spindle microtubules to push the two centriolar poles apart during mitotic prophase.

Centrioles, centrosomes and basal bodies

Centrioles are microtubular cylinders 0.2 μm in diameter and 0.4 μm long (Fig. 1.11). They are formed by a ring of nine microtubule triplets linked by a number of other proteins. At least two centrioles occur in all animal cells that are capable of mitotic division (eggs, which undergo meiosis instead of mitosis, lack centrioles). They usually lie close together, at right angles or, most usually, at an oblique angle to each other (an arrangement often termed a diplosome), within the centrosome, a densely filamentous region of cytoplasm at the centre of the cell. The centrosome is the major microtubule-organizing centre of most cells; it is the site at which new microtubules are formed and the mitotic spindle is generated during cell division. Centriole biogenesis is a complex process that takes more than a single cell cycle to complete. At the beginning of the S phase (DNA replication phase) of the cell cycle, a new daughter centriole forms at right angles to each separated maternal centriole. Each mother–daughter pair forms one pole of the next mitotic spindle, and the daughter centriole becomes fully mature only as the progeny cells are about to enter the next mitosis. Because centrosomes are microtubule organizing centres, they lie at the centre of a network of microtubules all of which have their minus ends proximal to the centrosome. The association of membrane vesicles with dynein motors means that certain membranes (including the Golgi apparatus) concentrate near the centrosome. This is convenient, as the microtubules provide a means of targeting Golgi vesicular products to different parts of the cell.

The microtubule-organizing centre contains complexes of γ-tubulin that nucleate microtubule polymerization at the minus ends of microtubules. Basal bodies are microtubule-organizing centres that are closely related to centrioles, and are believed to be derived from them. They are located at the bases of cilia and flagella, which they anchor to the cell surface. The outer microtubule doublets of cilia and flagella originate from two of the microtubules in each triplet of the basal body.

Cell surface projections

The surfaces of many different types of cell are specialized to form structures that project from the surface. These projections may permit movement of the cell itself (flagella), or of fluids across the apical cell surface (cilia), or increase the surface area available for absorption (microvilli). Infoldings of the basolateral plasma membrane also increase the area for transport across this surface of the cell. In most non-dividing epithelial cells, the centriole gives rise to a non-motile primary cilium, which has an important sensory role.

Cilia and flagella

Cilia and flagella are motile, hair-like projections of the cell surface which create currents in the surrounding fluid, movements of the cell to which they are attached, or both. Cilia occur on many internal surfaces of the body, in particular: the epithelia of most of the respiratory tract; parts of the male and female reproductive tracts; the ependyma that line the central canal of the spinal cord and ventricles of the brain. They also occur at the endings of olfactory receptors and vestibular hair cells, and, in modified form, as portions of the rods and cones of the retina. A single cell may bear many cilia, e.g. in bronchial epithelium, or only one or two. Each male gamete possesses a single flagellum 70 μm long.

A cilium or flagellum consists of a shaft (0.25 μm diameter) constituting most of its length, a tapering tip and a basal body at its base, which lies within the surface cytoplasm of the cell (Fig. 1.12). Other than at its base, the entire structure is covered by plasma membrane. The core of the cilium is the axoneme, a cylinder of nine microtubule doublets that surrounds a central pair of single microtubules (Fig. 1.12). However, there is a class of cilia (primary cilia and nodal cilia) that are composed of nine microtubule doublets and no central microtubules.

Several filamentous structures are associated with the microtubules in the shaft, e.g. radial spokes extend inwards from the outer microtubules towards the central pair. The outer doublet microtubules bear two rows of tangential dynein arms attached to the A subfibre of the doublet, which point towards the B subfibre of the adjacent doublet. Adjacent doublets are also linked by thin filaments. Other filaments partially encircle the central pair of microtubules, which are also united by ladder-like spokes.

Movements of cilia and flagella are broadly similar. Flagella move by rapid undulation, which passes from the attached to the free end. In human spermatozoa there is an additional helical component to this motion. In cilia, the beating is planar, but asymmetric. In the effective stroke, the cilium remains stiff except at the base, where it bends to produce an oar-like stroke. The recovery stroke follows, during which the bend passes from base to tip, returning the cilium to its initial position for the next cycle. The activity of groups of cilia is usually coordinated so that the bending of one is rapidly followed by the bending of the next and so on, resulting in long travelling waves of metachronal synchrony. These pass over the tissue surface in the same direction as the effective stroke.

When a cilium bends, the microtubules do not change in length, but slide on one another. The dynein arms of peripheral doublets slant towards the base of the cilium from their attached ends. Dynein has an ATPase activity, which causes mutual sliding of adjacent doublets by initially attaching sideways to the next pair, then swinging upwards towards the tip of the cilium. There is a group of genetic diseases (reviewed in Afzelius 2004) in which cilia beat either ineffectively or not at all, e.g. Kartagener’s immotile cilia syndrome. Affected cilia exhibit various ultrastructural defects, such as a lack of dynein arms or missing spokes. Patients with this syndrome suffer various respiratory problems caused by the accumulation of particles in the lungs; males are typically sterile because of the loss of sperm motility, and 50% have an alimentary tract that is a mirror image of the usual pattern (situs inversus) – i.e. it rotates in the opposite direction during early development.

Microvilli

Microvilli are finger-like cell surface extensions usually 0.1 μm in diameter and up to 2 μm long (Fig. 1.13). When arranged in a regular parallel series, they constitute a striated border, as typified by the absorptive surfaces of the epithelial enterocytes of the small intestine. When they are less regular, as in the gallbladder epithelium and proximal kidney tubules, the term brush border is used.

Microvilli are covered by plasma membrane and supported internally by closely packed bundles of actin filaments linked by cross-bridges of the actin-bundling proteins, fascin and fimbrin. Other bridges composed of myosin I and calmodulin connect the filament bundles to the plasma membrane. At the tip of each microvillus, the free ends of microfilaments are inserted into a dense mass that includes the protein, villin. The actin filament bundles of microvilli are embedded in the apical cytoplasm amongst a meshwork of transversely running actin filaments stabilized by spectrin to form the terminal web, which is underlain by keratin intermediate filaments. The web is anchored laterally to the zonula adherens of the apical epithelial junctional complex. Myosin II and tropomyosin are also found in the terminal web, which may explain its contractile activity.

Microvilli greatly increase the area of cell surface (up to 40 times), particularly at sites of active absorption. In the small intestine, they have a very thick cell coat or glycocalyx, which reflects the presence of integral membrane glycoproteins, including enzymes concerned with digestion and absorption. Irregular microvilli, filopodia, are also found on the surfaces of many types of cell, particularly free macrophages and fibroblasts, where they may be associated with phagocytosis and cell motility.

Long, regular microvilli are called stereocilia, an early misnomer, as they are not motile and lack microtubules. They are found on cochlear and vestibular receptor cells, where they act as sensory transducers, and also in the absorptive epithelium of the epididymis.

NUCLEUS

The nucleus (Figs 1.1, 1.2) is generally the largest intracellular structure and is usually spherical or ellipsoid in shape, with a diameter of 3–10 μm. Histological stains used to identify nuclei in tissue sections mainly detect the acidic molecules of deoxyribonucleic acid (DNA), which are largely confined to the nucleus.

Nuclear membrane

The nucleus is surrounded by two concentric lipid bilayers which together form the nuclear membrane or envelope. The outer membrane bilayer and the lumen between the two bilayers are continuous with the rough endoplasmic reticulum. Like the rough endoplasmic reticulum, the outer membrane of the nuclear envelope is studded with ribosomes that are active in protein synthesis; the newly synthesized proteins pass into the perinuclear space between the two membrane layers.

A special class of intermediate filaments known as lamins is associated with the inner surface of the nuclear membrane. The lamins form a dense meshwork beneath the membrane, the nuclear lamina. The lamin filaments cross each other at right angles to create an irregular anastomosing network that covers the interior surface of the nuclear membrane. In so doing, they reinforce the nuclear membrane mechanically, determine the shape of the nucleus and provide a binding site for a range of proteins that anchor chromatin. Nuclear lamin A, with over 350 mutations, is the most mutated protein linked to human disease. Lamin A mutations cause a surprisingly wide range of diseases, from progeria to various dystrophies (reviewed in Mattout et al 2006 and Pollard and Earnshaw 2007).

Condensed chromatin (heterochromatin) also tends to aggregate near the nuclear membrane during interphase. At the end of mitotic and meiotic prophase (see below), the lamin filaments disassemble, causing the nuclear membranes to vesiculate and disperse into the endoplasmic reticulum. During the final stages of mitosis (telophase), proteins of the nuclear periphery, including lamins, associate with the surface of the chromosomes, providing docking sites for membrane vesicles. Fusion of these vesicles reconstitutes the nuclear compartment.

The transport of molecules between the nucleus and the cytoplasm occurs via specialized nuclear pore structures that perforate the nuclear membrane (Fig. 1.14A). They act as highly selective directional molecular filters, permitting proteins such as histones and gene regulatory proteins (which are synthesized in the cytoplasm but function in the nucleus) to enter the nucleus, and molecules that are synthesized in the nucleus but destined for the cytoplasm (e.g. ribosomal subunits, transfer RNAs and messenger RNAs), to leave the nucleus.

Ultrastructurally, nuclear pores appear as disc-like structures with an outer diameter of 130 nm and an inner pore with an effective diameter for free diffusion of 9 nm (Fig. 1.14B). The nuclear membrane of an active cell is bridged by up to 4000 such pores. The nuclear pore complex has an octagonal symmetry and is formed by an assembly of more than 50 proteins, the nucleoporins. The inner and outer nuclear membranes fuse around the pore complex (Fig. 1.14A). Nuclear pores are freely permeable to small molecules, ions and proteins up to about 17 kDa. Most proteins that enter the nucleus do so as complexes with specific transport receptor proteins known as importins. Importins shuttle back and forth between the nucleus and cytoplasm. Binding of the cargo to the importin requires a short sequence of amino acids known as a nuclear localization sequence (NLS), and can be either direct or via an adapter protein. Interactions of the importin with components of the nuclear pore move it together with its cargo through the pore by an energy-independent process still not understood. A complementary cycle functions in export of proteins and RNA molecules from the nucleus to the cytoplasm using transport receptors known as exportins. For further explanation, see Pollard & Earnshaw (2007).

Chromatin

DNA is organized within the nucleus in a DNA–protein complex known as chromatin. The protein constituents of chromatin are the histones and the non-histone proteins. Non-histone proteins are an extremely heterogeneous group that includes structural proteins, DNA and RNA polymerases and gene regulatory proteins. Histones are the most abundant group of proteins in chromatin, primarily responsible for the packaging of chromosomal DNA into its primary level of organization, the nucleosome. There are four core histone proteins: H2A, H2B, H3 and H4 that combine in equal ratios to form a compact octameric nucleosome core. A fifth histone, H1, is involved in further compaction of the chromatin. The DNA molecule (one per chromosome) winds twice around each nucleosome core, taking up 165 nucleotide pairs. This packaging organizes the DNA into a chromatin fibre 11 nm in diameter, and imparts to this form of chromatin the electron microscopic appearance of beads on a string, in which each bead is separated by a variable length of DNA, typically about 35 nucleotide pairs long. The nucleosome core region and one of the linker regions constitute the nucleosome proper, which is typically about 200 nucleotide pairs in length. However, chromatin rarely exists in this simple form and is usually packaged further into a 30 nm thick fibre, involving a single H1 histone per nucleosome, which interacts with both DNA and protein to impose a higher order of nucleosome packing. Usually, 30 nm fibres are further coiled or folded into larger domains. Individual domains are believed to decondense and extend during active transcription. In a typical interphase nucleus, euchromatin (nuclear regions that appear pale in appropriately stained tissue sections, or relatively electron-lucent in electron micrographs; Fig. 1.2) is likely to consist mainly of 30 nm fibres and loops, and contains the transcriptionally active genes. Transcriptionally active cells, such as most neurones, have nuclei that are predominantly euchromatic and often described as ‘open face’ nuclei.

Heterochromatin (nuclear regions that appear dark in appropriately stained tissue sections or electron-dense in electron micrographs) is characteristically located mainly around the periphery of the nucleus, except over the nuclear pores (Fig. 1.14A), and around the nucleolus (Fig. 1.2). It is a relatively compacted form of chromatin in which the histone proteins carry a specific set of post-translational modifications, including methylation at characteristic residues. This facilitates the binding of specific heterochromatin-associated proteins. Heterochromatin includes non-coding regions of DNA, such as centromeric regions, which are known as constitutive heterochromatin. DNA that is inactivated (becoming resistant to transcription) in some cells as they differentiate during development or cell maturation contributes to heterochromatin, and is known as facultative heterochromatin. The inactive X chromosome in females is an example of facultative heterochromatin and can be identified in the light microscope as the deeply staining Barr body (drumstick chromosome), often located near the nuclear periphery.

In transcriptionally inactive cells, chromatin is predominantly in the condensed, heterochromatic state, and may comprise as much as 90% of the total. Examples of such cells are mature neutrophil leukocytes (in which the condensed chromatin is present in a multilobed, densely staining nucleus), and the highly condensed nuclei of orthochromatic erythroblasts (late-stage erythrocyte precursors). In most mature cells, a mixture of the two occurs, indicating that only a proportion of the DNA is being transcribed. A particular instance of this is seen in the mature B lymphocyte (plasma cell), in which much of the chromatin is in the condensed condition and is arranged in regular masses around the perimeter of the nucleus, producing the so-called ‘clock-face’ nucleus (Figs 4.6, 4.12). Although this cell is actively transcribing, much of its protein synthesis is of a single immunoglobulin type, and consequently much of its genome is in an inactive state.

During mitosis, the chromatin is further reorganized and condensed to form the much shortened chromosomes characteristic of metaphase. This shortening is achieved through further levels of close packing of the chromatin. The mechanism of condensation is unknown, but the condensed chromosomes are stabilized by protein complexes known as condensins. Progressive folding of the chromosomal DNA by interactions with specific proteins can reduce 5 cm of chromosomal DNA by 10,000 fold, to a length of 5 μm in the mitotic chromosome.

Chromosomes and karyotypes

The nuclear DNA of eukaryotic cells is organized into linear units called chromosomes. The DNA in a normal human diploid cell contains 6 × 109 nucleotide pairs organized in the form of 46 chromosomes (44 autosomes and two sex chromosomes). The largest human chromosome (number 1) contains 2.5 × 108 nucleotide pairs, and the smallest (the Y chromosome) 5 × 107 nucleotide pairs.

Each chromosomal DNA molecule contains a number of specialized nucleotide sequences that are associated with its maintenance. One is the centromere. During mitosis, a disc-shaped structure composed of a complex array of proteins, the kinetochore, forms as a substructure of the centromeric region of DNA in order to attach it to the microtubular spindle. Another sequence, the telomere, defines the end of each chromosomal DNA molecule. Telomeres consist of hundreds of repeats of the nucleotide sequence (TTAGGG)n. The very ends of the chromosomes cannot be replicated by the same DNA polymerase as the rest of the chromosome, and are maintained by a specific enzyme called telomerase which contains an RNA subunit which acts as the template for lengthening the TTAGGG repeats. Thus telomerase is a specialized type of polymerase known as a reverse transcriptase that turns sequences in RNA back into DNA. The number of tandem repeats of the telomeric DNA sequence varies. It appears to shorten with successive cell divisions, because telomerase activity reduces or is absent in differentiated cells with a finite lifespan. It is believed that this mechanism contributes to regulation of cell senescence and may protect against proliferative disorders, including cancer (reviewed in Flores et al 2006).

Classification of human chromosomes

A number of genetic abnormalities can be directly related to the chromosomal pattern. The characterization or karyotyping of chromosome number and structure is therefore of considerable diagnostic importance. The identifying features of individual chromosomes are most easily seen during metaphase, although prophase chromosomes can be used for more detailed analyses.

Lymphocytes separated from blood samples, or cells taken from other tissues, are used as a source of chromosomes. Diagnosis of fetal chromosome patterns is generally carried out on samples of amniotic fluid containing fetal cells aspirated from the uterus by amniocentesis, or on a small piece of chorionic villus tissue removed from the placenta. Whatever their origin, the cells are cultured in vitro and stimulated to divide by treatment with agents that stimulate cell division. Mitosis is interrupted at metaphase with spindle inhibitors. The chromosomes are dispersed by first causing the cells to swell in a hypotonic solution, then the cells are gently fixed and mechanically ruptured on a slide to spread the chromosomes. They are subsequently stained in various ways to allow the identification of individual chromosomes by size, shape and distribution of stain (Fig. 1.15). General techniques show the obvious landmarks, e.g. lengths of arms and positions of constrictions. Banding techniques demonstrate differential staining patterns, characteristic for each chromosome type. Fluorescence staining with quinacrine mustard and related compounds produces Q bands, and Giemsa staining (after treatment that partially denatures the chromatin) gives G bands (Fig. 1.15A). Other less widely used methods include: reverse-Giemsa staining, in which the light and dark areas are reversed (R bands); the staining of constitutive heterochromatin with silver salts (C-banding); T-banding to stain the ends (telomeres) of chromosomes. Collectively, these methods permit the classification of chromosomes into numbered autosomal pairs in order of decreasing size, from 1 to 22, plus the sex chromosomes.

A summary of the major classes of chromosomes is given below:

Group Features
1–3 (A) Large metacentric chromosomes
4–5 (B) Large submetacentric chromosomes
6–12 + X (C) Metacentrics of medium size
13–15 (D) Medium-sized acrocentrics with satellites
16–18 (E) Shorter metacentrics (16) or submetacentrics (17,18)
19–20 (F) Shortest metacentrics
21–22 + Y (G) Short acrocentrics; 21, 22 with satellites, Y without

Methodological advances in banding techniques improved the recognition of abnormal chromosome patterns. The use of in situ hybridization with fluorescent DNA probes specific for each chromosome (Fig. 1.15B) permits the identification of even very small abnormalities.

Nucleolus

Nucleoli are a prominent feature of an interphase nucleus (Fig. 1.2). They are the site of most of the synthesis of rRNA and assembly of ribosome subunits. Ultrastructurally, the nucleolus appears as a pale fibrillar region (non-transcribed DNA), containing dense fibrillar cores (sites of rRNA gene transcription) and granular regions (sites of ribosome subunit assembly) within a diffuse nucleolar matrix. Five pairs of chromosomes carry rRNA genes organized in clusters of tandemly repeated units on each chromosome. Each rRNA unit is transcribed individually and encodes a large precursor RNA that is processed to yield the 28S, 18S and 5.8S rRNA molecules. This processing takes place in the nucleolus, as does the processing of a number of other stable RNAs, including the RNA component of the signal recognition particle (SRP), which is essential for protein secretion. During mitosis the nucleolus breaks down. It reforms after nuclear envelope reformation in telophase, in a process associated with the onset of transcription in nucleolar organizing centres on each chromosome. The 28S, 18S and 5.8S rRNA molecules are assembled into their ribosomal subunits in the granular region of the nucleolus together with the 5S rRNA, which is not synthesized in the nucleolus. The newly formed ribosomal subunits are then translocated to the cytoplasm through the nuclear pores.

CELL DIVISION AND THE CELL CYCLE

During prenatal development, most cells undergo repeated division as the body grows in size and complexity. As cells mature, they differentiate structurally and functionally. Some cells, such as neurones, lose the ability to divide. Others may persist throughout the lifetime of the individual as replication-competent stem cells, e.g. cells in the haemopoietic tissue of bone marrow. Many stem cells divide infrequently, but give rise to daughter cells that undergo repeated cycles of mitotic division as transit (or transient) amplifying cells. Their divisions may occur in rapid succession, as in cell lineages with a short lifespan and similarly fast turnover and replacement time. Transit amplifying cells are all destined to differentiate and ultimately to die and be replaced, unlike the population of parental stem cells, which self-renews.

Patterns and rates of cell division within tissues vary considerably. In many epithelia, such as the crypts between intestinal villi, the replacement of damaged or effete cells by division of stem cells can be rapid. Rates of cell division may also vary according to demand, as occurs in the healing of wounded skin, in which cell proliferation increases to a peak and then returns to the normal replacement level (see Ch. 7). The rate of cell division is tightly coupled to the demand for growth and replacement. Where this coupling is faulty, tissues either fail to grow or replace their cells, or they can overgrow, producing neoplasms.

The cell cycle is the period of time between the birth of a cell and its own division to produce two daughter cells. It generally lasts a minimum of 12 hours, but in most adult tissues can be considerably longer, and is divided into four distinct phases, which are known as G1, S, G2 and M. The combination of G1, S and G2 phases is known as interphase. M is the mitotic phase. G1 is the period when cells respond to growth factors directing the cell to initiate another cycle; once made, this decision is irreversible. It is also the phase in which most of the molecular machinery required to complete another cell cycle is generated. Cells that retain the capacity for proliferation, but which are no longer dividing, have entered a phase called G0 and are described as quiescent even though they may be quite active physiologically. Growth factors can stimulate quiescent cells to leave G0 and re-enter the cell cycle, whereas the proteins encoded by certain tumour suppressor genes (e.g. the gene mutated in retinoblastoma, Rb) block the cycle in G1. DNA synthesis (replication of the genome) occurs during S phase, at the end of which the DNA content of the cell has doubled. During G2, the cell prepares for division; this period ends with the onset of chromosome condensation and breakdown of the nuclear membrane. The times taken for S, G2 and M are similar for most cell types, and occupy 6–8, 2–4 and 1–2 hours respectively. In contrast, the duration of G1 shows considerable variation, sometimes ranging from less than 2 hours in rapidly dividing cells, to more than 100 hours within the same tissue.

Cell cycle progression is driven in part by changes in the activity of cyclin-dependent protein kinases, CDKs (protein kinases which are activated by binding of a cyclin subunit). Each cell cycle stage is characterized by the activity of one or more CDK-cyclin pairs. Transitions between cell cycle stages are triggered by highly specific proteolysis of the cyclins and other key components. The targets for proteolysis are marked for destruction by E3 ubiquitin ligases, which decorate them with polymers of the small protein ubiquitin, a sign for recognition by the proteasome. To give one example, the transition from G2 to mitosis is driven by activation of CDK1 by its partners the A- and B-type cyclins: the characteristic changes in cellular structure that occur as cells enter mitosis are largely driven by phosphorylation of proteins by active CDK1-cyclin A and CDK1-cyclin B (further details are beyond the scope of this book). Cells exit from mitosis when the anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase, marks the cyclins for destruction. The APC/C also triggers for destruction another protein whose function is to protect the cohesion between sister chromatids. An analogous process of protein phosphorylation coupled with targeted destruction drives the transition between G1 and S phase as cells commit to another cycle of proliferation.

There are important checkpoints in the cell cycle at which progress will be arrested if, for instance, DNA replication or mitotic spindle assembly and chromosome attachment are incomplete. Negative regulation systems also operate to delay cell cycle progression when DNA has been damaged by radiation or chemical mutagens. Cells with checkpoint defects, such as loss of the protein p53, which is a major negative control element in the division cycle of all cells, are commonly associated with the development of malignancy. Cells lacking one of the critical checkpoint functions are able to progress through the cycle carrying defects, thus increasing the probability that further abnormalities will accumulate in their progeny. The p53 gene is an example of a tumour suppressor gene. For further reading, see Blow & Tanaka (2005); Pollard & Earnshaw (2007).

MITOSIS AND MEIOSIS

Mitosis is the process that results in the distribution of identical copies of the parent cell genome to the two daughter somatic cells. In meiosis, the divisions immediately before the final production of gametes halve the number of chromosomes to the haploid number, so that at fertilization the diploid number is restored. Moreover, meiosis includes a phase in which exchange of genetic material occurs between homologous chromosomes. This allows a reassortment of genes to take place, which means that the daughter cells differ from the parental cell in both their precise genetic sequence and their haploid state. Mitosis and meiosis are alike in many respects, and differ principally in chromosomal behaviour during the early stages of cell division. In meiosis, two divisions occur in succession, without an intervening S phase. Meiosis I is distinct from mitosis, whereas meiosis II is more like mitosis.

Mitosis

New DNA is synthesized during the S phase of the cell cycle interphase. This means that the amount of DNA in diploid cells has doubled to the tetraploid value by the onset of mitosis, although the chromosome number is still diploid. During mitosis, this amount is halved between the two daughter cells, so that DNA quantity and chromosome number are diploid in both cells. The cellular changes that achieve this distribution are conventionally divided into four phases called prophase, metaphase, anaphase and telophase (Fig. 1.16, Fig. 1.17).

Telophase

During telophase the nuclear membranes reform, beginning with the association of membranous vesicles with the surface of the chromosomes. Later, after the vesicles have fused and the nuclear envelope is complete, the chromosomes decondense and the nucleoli reform. At the same time, cytoplasmic division, which usually begins in early anaphase, continues until the new cells separate, each with its derived nucleus. The spindle remnant now disintegrates. While the cleavage furrow is active, a peripheral band or belt of actin and myosin appears in the constricting zone: contraction of this band is responsible for furrow formation.

Failure of disjunction of chromatids, so that sister chromatids pass to the same pole, may sometimes occur. Of the two new cells, one will have more, and the other fewer, chromosomes than the diploid number. Exposure to ionizing radiation promotes non-disjunction and may, by chromosomal damage, inhibit mitosis altogether. A typical symptom of radiation exposure is the failure of rapidly dividing epithelia to replace lost cells, with consequent ulceration of the skin and mucous membranes. Mitosis can also be disrupted by chemical agents, particularly colchicine, taxol and their derivatives. These compounds either disassemble spindle microtubules or interfere with their dynamics. As a result, mitosis is arrested in metaphase. Taxol and its derivatives are widely used in the treatment of breast cancer. Colchicine is widely used for the treatment of gout, but the mechanism is not known and may have nothing to do with mitotic regulation.

Meiosis

There are two cell divisions during meiosis (Fig. 1.18). Details of this process differ at a cellular level for male and female lineages.

Meiosis I

Prophase I

Meiotic prophase I is a long and complex phase that differs considerably from mitotic prophase and is customarily divided into five substages, called leptotene, zygotene, pachytene, diplotene and diakinesis (see Pollard & Earnshaw 2007).

CELL DIFFERENTIATION

As the embryo develops, its cells pass through a series of changes in gene expression, reflected in alterations of cell structure and behaviour. They begin to diversify, separating first into two main tissue arrangements, epithelium and embryonic mesenchyme, then into more restricted subtypes of tissue, until finally they mature into cells of their particular adult lineage. In this process, and in the maturation of functioning cells of the different lineages from their stem cells, there is a sequential pattern of gene expression that changes and limits the cell to a particular specialized range of activities. Such changes involve alterations in cell structure and biochemical characteristics, particularly in the types of proteins that are synthesized. At the genetic level, differentiation is based on a change in the pattern of repression and activation of the DNA sequences encoding proteins specific to that stage of development.

A cell may be committed to a particular differentiated fate without manifesting its commitment until later. Once switched in this way, cells are not usually able to revert to an earlier stage of commitment to a differentiation pathway, so that an irreversible repression of some gene sequences must have occurred. Differentiation signals include interactions between cells that are mediated by diffusible signalling molecules elaborated by one cell and detected by another, and by contactmediated signalling (such as Delta–Notch signalling). The latter is particularly important in establishing boundaries between different cell populations in development.

Differentiation may also depend in some instances on a temporal sequence, but probably not the number of previous cell divisions. In mature tissues in which cell turnover occurs, similar mechanisms appear to ensure the final differentiation to a functional end cell. This may be linked to the presence of a physiological stimulus, e.g. B lymphocytes respond to exposure to an antigen by differentiating into plasma cells that secrete a neutralizing antibody. In other cases, particularly where a cell is part of a highly organized tissue system, more subtle mechanisms exist to ensure a balance between cell proliferation, differentiation and programmed cell death (apoptosis). This balance is disturbed when tissue is damaged and different cell types respond differently to repair the damage. Liver hepatocytes are able to revert to a functionally less well-differentiated phenotype and re-enter the cell cycle, in order to restore cell numbers and tissue mass. Other cell types (such as skeletal muscle fibres) are unable to do so and depend on the proliferation of precursor cells (stem cells) for repair. In many tissues such as skin, where normal cell turnover is continuous, wound repair includes up-regulation of proliferation in the stem cell and transit amplifying cell compartments.

There are few instances of the transdifferentiation of one differentiated cell type into another (metaplasia, see Ch. 2), but there is evidence that stem cells in the developing embryo and in certain mature tissues (e.g. bone marrow) may have the potential to differentiate into more diverse phenotypes than was once believed. This plasticity depends on environmental cues and offers the prospect of engineering tissues for clinical therapy. For further reading, see Alberts et al (2002).

APOPTOSIS

Cells die as a result of either tissue injury (necrosis) or the internal activation of a ‘suicide’ programme (apoptosis) in response to extrinsic or intrinsic cues. Apoptosis (programmed cell death, regulated cell suicide) is a central mechanism controlling multicellular development. During morphogenesis, apoptosis mediates activities such as the separation of the developing digits, and has an important role in regulating the number of neurones in the nervous system (the majority of neurones die during development). Apoptosis also ensures that inappropriate or inefficient cells of the acquired immune system are eliminated.

The morphological changes exhibited by necrotic cells are very different from those seen in apoptotic cells (Fig. 1.19). Necrotic cells swell and subsequently rupture; the resulting debris may induce an inflammatory response (see Ch. 4, Ch. 7). Apoptotic cells shrink, their nuclei and chromosomes fragment, forming apoptotic bodies, and their plasma membranes undergo conformational changes that act as a signal to local phagocytes. The dead cells are removed rapidly, and as their intracellular contents are not released into the extracellular environment, inflammatory reactions are avoided; the apoptotic fragments also stimulate macrophages to release anti-inflammatory cytokines.

Apoptosis and cell proliferation are intimately coupled: several cell cycle regulators can influence both cell division and apoptosis. The signals that trigger apoptosis include withdrawal of survival factors or exposure to inappropriate proliferative stimuli. The current model of the intracellular pathway(s) that leads to apoptosis implicates permeabilization of the mitochondrial membrane, the release of cytochrome c (from the space between the inner and outer mitochondrial membranes) into the cytosol, and subsequent activation of a family of cysteine proteases known as caspases. Caspases are the intracellular mediators of apoptosis: when activated, they initiate a cascade of degradative processes targeting proteins throughout the cell. Caspase cleavage inactivates many systems that normally promote damage repair and support cell viability in general. They also activate a number of proteins which promote the death and disassembly of the cell.

Subversion of the apoptotic response is a key characteristic of many cancer cells. Thus the tumour suppressor gene p53 (which functions in cell-cycle control, regulation of apoptosis and the maintenance of genetic stability), is mutated in about 50% of all human cancers. For further details, see Pollard & Earnshaw (2007).

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