The cell (Fig. 3.1) is a highly organized structure and consists of various common organelles held by an adaptive internal scaffolding (the cytoskeleton), which radiates from the nuclear membrane to the cell plasma membrane. The number and finer detail of these common organelles varies according to the specialized function a cell might perform. For example, the mitochondria of muscle cells contain many infolded cristae; they produce large amounts of ATP (from the electron transport chain that sits on the inner membrane), whereas the mitochondria of liver cells are smaller and rounded as they produce low levels of ATP, but many products from the inner matrix feed other metabolic pathways. Each cell is a component unit but can ‘talk’ to an adjacent cell via specific channels and receptors. Within a cell there is a constant flow of traffic between the organelles.

The plasma cell membrane interfaces between the cell’s internal mechanisms and the extracellular environment. It is a bilayer of amphipathic phospholipids that consist of a polar hydrophilic head (e.g. phosphatidyl choline) and an insoluble non-polar lipid hydrophobic tail (commonly two long-chain fatty acids). The phospholipids spontaneously form bilayers (as complete circular structures) that form an effective barrier that is impermeable to most water-soluble molecules. This barrier defines the interior environment of the cell. The exchanges across the plasma membrane are regulated by various proteins that are embedded in the lipid bilayer (Fig. 3.2). The lipids’ hydrophobic structure means that relatively weak bonds hold the plasma membrane together. However, this strongly opposes the transverse movement of hydrophilic molecules but allows considerable freedom for lateral ‘fluid’ movement by molecules such as membrane proteins that are embedded in it. The plasma membrane is thus a very dynamic structure. Cell-to-cell communication is the key to many diseases and chemotherapeutic interventions.
The membrane proteins embedded in the lipid bilayer either traverse the whole membrane or are associated only with the outer or inner leaflet of the bilayer (Fig. 3.2). These proteins are responsible for the cell’s interaction with its external environment.

Cell dynamics

Like all systems, the component proteins – and even organelles – of the cell are continually being formed and degraded. Most of the degradation steps involve ATP-dependent multienzyme complexes. Old cellular proteins are mopped up by a small cofactor molecule called ‘ubiquitin’, which interacts with these worn proteins via their exposed hydrophobic residues. Ubiquitin acts as a signal for destruction or repair, and a complex containing more than five ubiquitin molecules is rapidly degraded by a large proteolytic multienzyme array termed ‘26S proteasome’. The failure to remove worn proteins can result in the development of chronic debilitating disorders. Alzheimer and frontotemporal dementias are associated with the accumulation of ubiquinated proteins (prion-like proteins), which are resistant to ubiquitin-mediated proteolysis. Similar proteolytic-resistant ubiquinated proteins give rise to inclusion bodies found in myositis and myopathies. This resistance can be due to point mutation in the target protein itself (e.g. mutant p53 in cancer; see p. 189) or as a result of an external factor altering the conformation of the normal protein to create a proteolytic-resistant shape, as in the prion protein of variant Creutzfeldt-Jakob disease (vCJD). Nuclear factor kappa B (NFκB) is bound by inhibitory factor IκB. Upon phosphorylation, NFκB, is released and IκB is degraded by the proteasome. Failure to do so results in an accumulation of IκB.

Specialized cells such as macrophages and neutrophils can engulf about 20% of their surface area in the pursuit of large particles such as bacteria. Lysosomes rapidly fuse with phagosomes, giving equally rapid digestion of the contents, and recycling as much of the internalized membrane as possible. Phagocytosis is only triggered when specific cell surface receptors – such as the macrophage Fc receptor – are occupied by their ligand. Pinocytosis is a much smaller-scale model of phagocytosis and is continually occurring in all cells. In contrast to phagocytosis, receptors for smaller molecular complexes, such as low-density lipoprotein (LDL) result in surface clumping and the internal accumulation of a protein called clathrin. Clathrin-coated pits pinch inwards as clathrin-coated vesicles. Clathrin prevents fusion of lysosomes, and thus its removal will result in lysosomal fusion and degradation of the contents. Maintenance of a clathrin coat can result in transcellular transit of the contents and their exocytosis at another side of the plasma membrane, i.e. apical-to-basal surface transcytosis. Similarly, cell organelles bud off vesicles coated in clathrin to prevent lysosomal fusion and degradation. Some of these vesicles rapidly fuse with the plasma membrane and exocytose their contents. Other vesicles do not immediately fuse with the plasma membrane (or indeed any other organelle). The clathrin-coated vesicles have additional lipid bilayer-embedded proteins called v-SNAREs (signal and response elements), which interact with target organelle membrane proteins called t-SNAREs. Vesicle fusion is therefore specific, comprising fusion in the correct place (at a particular organelle or part of the plasma membrane) at the correct time (e.g. the fusion of neuronal transmitter vesicles and release of the transmitter at the synaptic membrane when stimulated).

The plasma membrane is freely permeable to gases such as O2, CO2 and N2, and to small uncharged molecules such as H2O (not H+ and OH-) and urea. Whilst larger hydrophobic lipid-soluble molecules – like steroids – also pass freely through the membrane, large uncharged molecules (glucose, amino acids and nucleotides) and small charged ions (K+, Na+, Ca2+, Cl-, Mg2+ and HCO3-) cannot pass unless via a specific transport protein embedded in the plasma membrane. Two structural types of transport molecules/complexes exist.

- Channel proteins literally open a channel in the lipid membrane to allow a specific solute to pass through.
- Carrier proteins are slower in action, shuttling the solute across and either facilitating diffusion down a gradient across the membrane, or actively pumping solutes against the gradient using ATP as an energy source.

Active carrier pumps and gated ion channels work together in neural transmission. These carrier proteins pump Na+ and K+ across the neuronal cell membrane to create a differential gradient, but ion channels open in response to stimuli to cause a rapid depolarization, allowing the ions to flow back. At synaptic junctions these ion channels open in response to chemical signals such as the release of glutamate, epinephrine (adrenaline) or acetylcholine.

ATP-dependent transport molecules (ATPases) belong to a superfamily called the ‘ABC transporter superfamily’. These include the multidrug-resistance protein (MDR), which pumps out hydrophobic drugs and is overexpressed by tumour cells, and the chloride ion pump coded by the cystic fibrosis gene. All share a common structure of six transmembrane domains interrupted by a cytoplasmic ATPase domain, followed by a further six transmembrane domains and another cytoplasmic ATPase. The cystic fibrosis chloride ion channel is unusual in that it requires the binding and hydrolysis of both ATP and cAMP for activation.

Membrane surface receptors pass their extracellular signal across the plasma membrane to cytoplasmic secondary signalling molecules. Examples of these receptors include the non-lipid-soluble ligands such as growth hormone (GH), insulin, insulin-like growth factor (IGF) and luteinizing hormone (LH). These membrane-bound receptors can be subclassified according to the mechanism by which they activate signalling molecules:

• ion channel linked (see above)
• G-protein linked
• enzyme linked.

Structurally these plasma membrane receptors can be:

• serpentine (seven transmembrane domains, e.g. the LH receptor)
• transmembrane with large extra- and intracellular domains (e.g. the epidermal growth factor (EGF) receptor)
• transmembrane with a large extracellular domain only
• entirely linked onto the outer membrane leaflet by a lipid moiety known as a GPI (glycan phosphatidylinositol) anchor (e.g. T-cell receptor).

The function of these membrane receptors is to initiate a secondary message that ultimately results in activation of a specific enzyme or a DNA-binding protein. This may involve translocation to the nucleus and initiation of transcription of a specific set of genes.

G-protein-linked receptors

The G-protein-linked receptor, once activated by a ligand, binds a trimeric complex (α,β,γ) which is anchored to the inner surface of the plasma membrane. This complex is a GTP-binding protein, or G-protein. The G-protein binds GTP rather than GDP, and then interacts with enzyme complexes anchored into the inner leaflet of the membrane. These complexes in turn activate one or all three of the secondary messengers:

• cyclic AMP (cAMP)
• Ca²⁺ ions
• inositol 1,4,5-trisphosphate/diacylglycerol (IP₃/DAG).

Enzyme-linked surface receptors

These receptors usually have a single transmembrane-spanning region, and a cytoplasmic domain that has intrinsic enzyme activity or will bind and activate other membrane-bound or cytoplasmic enzyme complexes. Four classes of enzymes have been designated:

• Guanylyl cyclase-linked receptors (e.g. the atrial natriuretic peptide receptor), which produce cyclic GMP. This in turn activates a cGMP-dependent kinase (G-kinase), which binds to and phosphorylates serine and threonine residues of specific secondary messengers.
• Tyrosine kinase receptors (e.g. the platelet-derived growth factor (PDGF) receptor), which either specifically phosphorylate kinases on a small set of intracellular signalling proteins, or associate with proteins that have tyrosine kinase activity.
• Tyrosine phosphatase receptors (e.g. CD45), which remove phosphates from tyrosine residues of specific intracellular signalling proteins.
• Serine/threonine kinase receptors (e.g. the transforming growth factor-beta (TGF-β) receptor), which phosphorylate specific serine and threonine residues of intracellular signalling proteins.

There are many intracellular receptors that bind lipid-soluble ligands such as steroid hormones (e.g. progesterone, cortisol, T3 and T4). These cytoplasmic receptors often change shape in response to binding their ligands, form dimers, enter the nucleus and interact directly with specific DNA sequences.

This is the fluid component inside the cell membrane and contains many specialized organelles. It contains a scaffolding or cytoskeleton that regulates the passage and direction in which the interior solutes and storage granules flow. The cytoplasm contains:

• Endoplasmic reticulum (ER). This consists of interconnecting tubules or flattened sacs (cisternae) of lipid bilayer membrane. It may contain ribosomes on the surface (termed rough endoplasmic reticulum (RER) when present, or smooth endoplasmic reticulum (SER) when absent). The ER is involved in the processing of proteins: the ribosomes translate mRNA into a primary sequence of amino acids of a protein peptide chain. This chain is synthesized into the ER where it is first folded and modified into mature peptides. ER is the major site of drug metabolism.
• Golgi apparatus. This consists of flattened cisternae similar to the ER. It is characterized as a stack of cisternae from which vesicles bud off from the thickened ends. The primary processed peptides of the ER are exported to the Golgi apparatus for maturation into functional proteins (e.g. glycosylation of proteins which are to be excreted occurs here) before packaging into secretory granules and cellular vesicles that bud off the end.
• Lysosomes. These are dense cellular vesicles containing acidic digestive enzymes. They fuse with phagocytotic vesicles from the outer cell membrane, digesting the contents into small biomolecules that can cross the lysosomal lipid bilayer into the cell cytoplasm. Lysosomal enzymes can also be released outside the cell by fusion of the lysosome with the plasma membrane. Lysosomal action is crucial to the function of macrophages and polymorphs in killing and digesting infective agents, tissue remodelling during development and osteoclast remodelling of bone. Not surprisingly, many metabolic disorders result from impaired lysosomal function.
• Peroxisomes. These are dense cellular vesicles so named because they contain enzymes that catalyse the breakdown of hydrogen peroxide. They are involved in the metabolism of bile and fatty acids, and are primarily concerned with detoxification, e.g. d-amino acid oxidase and H₂O₂ catalase. The inability of the peroxisomes to function correctly can lead to rare metabolic disorders such as Zellweger’s syndrome and rhizomelic dwarfism.
• Mitochondria. These organelles are the powerhouse of the cell. Each mitochondrion comprises two lipid bilayer membranes and a central matrix. It also possesses several copies of its own DNA in a circular genome. The outer membrane contains many gated receptors responsible for the import of raw materials like pyruvate and ADP, and the export of products such as oxaloacetate (precursor of amino acids and sugars) and ATP. An interesting caveat to our symbiotic relationship is that proteins of the Bcl-2/Bax family are incorporated in this outer membrane and can release mitochondrial enzymes that trigger apoptosis. The inner membrane is often highly infolded to form cristae to increase its effective surface area. It contains transmembrane enzyme complexes of the electron transport chain, which generate an H⁺ ion gradient. This gradient then drives the adjacent transmembrane ATPase complex to form ATP from ADP and P_i. The inner matrix contains the enzymes of the Krebs cycle that generate the substrates of both the electron transport chain (FADH₂ and NADH) and central metabolism.

Secondary messengers are molecules that transduce a signal from a bound receptor to its site of action (e.g. the nucleus). There are essentially four mechanisms by which secondary messengers act but they cross talk and are rarely activated independently of each other. These mechanisms are cyclic AMP, IP3/DAG, Ca2+ ions and protein phosphorylation.

Cyclic AMP, IP3/DAG and Ca2+ ions

The generation of cAMP by G-protein-linked receptors results in an increase in cellular cAMP, which binds and activates specific cAMP-binding proteins. These dimerize and enter the cell nucleus to interact with set DNA sequences (the cAMP response elements). In addition, cofactors in the cAMP-binding proteins are co-activated and interact with the phosphorylation pathway.

Other G-protein complexes activate inner membrane-bound phospholipase complexes. These in turn cleave membrane phospholipid-polyphosphoinositide (PIP2) into two components. The first is the water-soluble molecule inositol trisphosphate, IP3. This floats off into the cytoplasm and interacts with gated ion channels in the endoplasmic reticulum (or sarcoplasmic reticulum in muscle cells), causing a rapid release of Ca2+. The lipid-soluble component diacylglycerol (DAG) remains at the membrane, but activates a serine/threonine kinase, protein kinase C (see phosphorylation section further).

Although the cellular calcium-binding proteins and ion pumps rapidly remove Ca2+ from the cytoplasm back into a storage compartment (such as the endoplasmic reticulum), free Ca2+ interacts with target proteins in the cytoplasm, inducing a phosphorylation/dephosphorylation cascade, resulting in activated DNA-binding proteins entering the nucleus.

Protein phosphorylation

Although phosphorylation of the cytoplasmic secondary messengers is often a consequence of secondary activation of cAMP, Ca2+ and DAG, the principal route for the protein phosphorylation cascades is from the dimerization of surface protein kinase receptors, which have bound their ligands. The tyrosine kinase receptors phosphorylate each other when ligand binding brings the intracellular receptor components into close proximity. The inner membrane and cytoplasmic targets of these activated receptor complexes are ras, protein kinase C and ultimately the MAP (mitogen activated protein) kinase, Janus-Stat pathways or phosphorylation of IκB causing it to release its DNA-binding protein, nuclear factor kappa B (NFκB). These intracellular signalling proteins usually contain conserved non-catalytic regions called SH2 and SH3 (serc homology regions 2 and 3). The SH2 region binds to phosphorylated tyrosine. The SH3 domain has been implicated in the recruitment of intermediates that activate ras proteins. Like G-proteins, ras (and its homologous family members rho and rac) switches between an inactive GDP-binding state and an active GTP-binding state. This starts a phosphorylation cascade of the MAP kinase, Janus-Stat protein pathways, which ultimately activate a DNA-binding protein (NFκB). NFκB undergoes a conformational change, enters the nucleus and initiates transcription of specific genes.

Lipid-soluble ligands (e.g. steroids) do not need secondary messengers; their cytoplasmic receptors, once activated, enter the nucleus as DNA-binding proteins and alter gene expression directly.

This is a complex network of structural proteins which regulates not only the shape of the cell, but also its ability to traffic internal cell organelles and even move in response to external stimuli. The major components are microtubules, intermediate filaments and microfilaments.

• Microtubules. These are made up of two protein subunits, α and β tubulin (50 kDa), and are continuously changing length. They form a ‘highway’, transporting organelles through the cytoplasm. There are two motor microtubule-associated proteins – dynein and kinesin – allowing antegrade and retrograde movement. Dynein is also responsible for the beating of cilia. During interphase the microtubules are rearranged by the microtubule-organizing centre (MTOC), which consists of centrosomes containing tubulin and provides a structure on which the daughter chromosomes can separate. Another protein involved in the binding of organelles to microtubules is the cytoplasmic linker protein (CLIP). Drugs that disrupt the microtubule assembly (e.g. colchicine and vinblastine) affect the positioning and morphology of the organelles. The anticancer drug paclitaxel causes cell death by binding to microtubules and stabilizing them so much that organelles cannot move, and thus mitotic spindles cannot form.
• Intermediate filaments. These form a network around the nucleus and extend to the periphery of the cell. They make cell-to-cell contacts with the adjacent cells via desmosomes, and with basement matrix via hemidesmosomes . Their function appears to be in structural integrity; they are prominent in cellular tissues under stress. The intermediate filament fibre proteins are specific to the embryonic lineage of the cell concerned, for example keratin intermediate fibres are only found in epithelial cells whilst vimentin is only found in mesothelial (fibroblastic) cells.
• Microfilaments. Muscle cells contain a highly ordered structure of actin (a globular protein, 42-44 kDa) and myosin filaments, which form the contractile system. These filaments are also present throughout the non-muscle cells as truncated myosins (e.g. myosin 1), in the cytosol (forming a contractile actomyosin gel), and beneath the plasma membrane. Cell movement is mediated by the anchorage of actin filaments to the plasma membrane at adherent junctions between cells. This allows a non-stressed coordination of contraction between adjacent cells of a tissue. Similarly, vertical contraction of tissues is anchored across the cell membrane to the basement matrix at focal adhesion
  junctions where actin fibres converge . Actin-binding proteins (e.g. fimbria) modulate the behaviour of microfilaments, and their effects are often calcium-dependent. The actin-associated proteins can be tissue type specific, for example actin-binding troponin is a complex of three subunits and two of these have isomers which are only found in cardiac muscle. Cardiac troponin I and T are released into the blood circulation after the onset of a myocardial infarction.

Alterations in the cell’s actin architecture are also controlled by the activation of small ras-like GTP-binding proteins rho and rac. These are involved in rearrangement of the cell during division, and thus dysfunctions of these proteins are associated with malignancy.

The cytoskeleton and plasma membrane interconnect, and extracellular domains form junctions between cells to form tissues. There are three types of junction between cells: tight junctions, adherent junctions and gap junctions.

Tight junctions

Tight junctions (zonula occludens) hold cells together. They are situated at the ends of margins adjacent to epithelial cells (e.g. intestinal and renal cells) and form a barrier to the movement of ions and solutes across the epithelium, although they can be variably ‘leaky’ to certain solutes. The proteins responsible for the intercellular tight junction closure are called claudins. They show selective expression within tissue and regulate what small ions may pass through the gaps between cells. For example, the kidney displays a differential expression of these claudin proteins. Mutations of claudin-16 (which is expressed in the distal convoluted tubule in the kidney, where magnesium is reabsorbed) are responsible for some forms of Gitelman’s syndrome. Since magnesium reabsorption is paracellular, tight junctions (which contain claudin-16) prevent these divalent ions rapidly diffusing back between the cells into renal tubules.

Adherent junctions

Adherent junctions (zonula adherens) are continuous on the basal side of cells. They contain cadherins and are the major site of attachment of intracellular microfilaments. Intermediate filaments attach to desmosomes, which are apposed areas of thickened membranes of two adjacent cells. Hemidesmosomes attach cells to the basal lamina and are also connected to intermediate filaments. Transmembrane integrins link the extracellular matrix to microfilaments at focal areas where cells also attach to their basal laminae. In blistering dermatological disorders autoantibodies cause damage by attacking tight junction desmosomal proteins such as desmoglein-3 in pemphigus vulgaris and desmoglein-1 in pemphigus foliaceus.

Gap junctions

Gap junctions allow substances to pass directly between cells without entering the extracellular fluids. Protein channels (connexons) are lined up between two adjacent cells and allow the passage of solutes up to molecular weight 1000 kDa (e.g. amino acids and sugars), as well as ions, chemical messengers and other factors. The diameter of these channels is regulated by intracellular Ca2+, pH and voltage. Connexons are made up of six subunits surrounding a channel and their isoforms in tissues are encoded by different genes. Mutant connexons can cause disorders, such as the X-linked form of Charcot-Marie-Tooth disease.

Newer Posts »