This section emphasizes the cell as a dynamic entity. Cells are not simply building blocks that are linked together to create an organism: each cell comprises a dynamic network of interacting macromolecules. Just how dynamic has been brought home by recent advances in cell imaging technologies. A host of multisubunit molecular structures must assemble and disassemble in a highly coordinated, exquisitely regulated, and beautifully choreographed manner in order to ensure the integrity of the cell and to provide its ability to function correctly as a single unit within a large multicellular organism.
The cell is the fundamental unit of all forms of independent life on this planet, from the simplest single-celled prokaryote to the most complex multicellular eukaryote. A limiting membrane, the plasma membrane, encloses the contents of the cell and allows a host of enzymic reactions and intermolecular interactions to occur within a confined, and regulated, environment. This raises the question, ‘What is the limiting membrane composed of and what are the contents of the cell?’ The major component of the limiting membrane is a lipid bilayer and the major components of the lipid bilayer are amphipathic phospholipids. Amphipathic molecules have one part that is water soluble (hydrophilic) and one part that is water insoluble (hydrophobic). A property of amphipathic molecules is that, in an aqueous environment, they spontaneously organize themselves so that the hydrophobic regions face one another (shielding them from the surrounding water) leaving the hydrophilic regions (often referred to as ‘head groups’) exposed. In fact, an appropriate mixture of phospholipids in water will lead to the spontaneous formation of lipid vesicles, with water on the inside and water on the outside (Fig. 4.1.1). This gives us the basic template for the limiting membrane of the cell, but the contents of the cell, even though they are in an aqueous environment, are more than just water! The contents of the cell comprise, in fact, a vast range of biomolecules, from simple building blocks to large macromolecular complexes.
Prokaryotes compared with eukaryotes
Before going further it is probably best to clarify the difference between prokaryotes and eukaryotes. The defining difference between prokaryotes and eukaryotes is that prokaryotes have no nucleus whereas eukaryotes do. Prokaryotes can be divided into two major divisions, or domains, the eubacteria and the archaebacteria, which appear to have diverged from a common ancestor at around about the same time that ancestral eukaryotes evolved as the third domain of life on earth. All three domains use DNA as their hereditary material and information store. In prokaryotes this DNA resides within the cell alongside all the other cellular contents, in eukaryotes it is contained within the nucleus and thereby physically separated from the bulk of the contents of the cell. Just as the cell itself is a membrane-bound structure, so is the nucleus within each eukaryotic cell. In fact the membrane that surrounds the nucleus is a double lipid bilayer and is referred to as the nuclear envelope. The nucleus is just one of several membrane-bound structures within eukaryotic cells. The larger of these structures are referred to as organelles and they serve to compartmentalize the cell, allowing specific processes and reactions to occur within defined and controlled local environments. Smaller membrane-bound compartments are referred to as vesicles and tubules; these are generally involved in transport between organelles or between organelles and the plasma membrane. The contents of a eukaryotic cell are referred to as the cytoplasm; the aqueous part of the cytoplasm outside membrane-bound compartments is referred to as the cytosol and the inside of an organelle, vesicle, or tubule is termed the lumen of that compartment.
Transcription, translation, and macromolecular crowding
In all cells, DNA is transcribed into messenger RNA (mRNA) by RNA polymerase; the mRNA is, in turn, translated into protein by ribosomes. In prokaryotes this all occurs within the same space, since there are essentially no intracellular membrane-bound compartments and transcription and translation can be coupled, i.e. an mRNA can be being translated while still being transcribed. However, things are different in eukaryotes, since (in general) the transcribed mRNA leaves the nucleus (via protein-lined channels, nuclear pores, in the nuclear envelope) before being translated in the cytosol.
It has been estimated that, on average, each mammalian cell contains about 1010 protein molecules of about 10 000 to 20 000 different kinds. On top of this there are multiple copies of a range of other large macromolecules, notably nucleic acids and complex sugars. All of this within a cell that is around 20 µm in diameter (although there is massive variation here). It is, therefore, hardly surprising that there is a very high total concentration of macromolecules within mammalian cells—estimated to be up to 400 g/litre—meaning that anything up to 40% of the cell volume is physically occupied by these molecules. Thus, while the inside of the cell is clearly an aqueous environment, it is a very crowded and highly ordered aqueous environment, and probably has a consistency rather like that of thick soup or porridge.
Lipid bilayers and integral membrane proteins
A lipid bilayer, be it the plasma membrane at the cell surface or the defining membrane of an intracellular organelle, is, among other things, a permeability barrier. It is permeable to small lipophilic molecules, partially permeable to water, but impermeable to ions and large molecules. It therefore not only retains the contents of the cell or organelle, but also provides a physical barrier between the contents of the cell or organelle and the exterior environment. A cell, however, clearly has to interact with its exterior environment, whether it be a single-celled prokaryote (and most living organisms are single cells) or a complex multicellular organism such as a human being with an estimated 1013 cells assembled in such a way, and communicating with one another, so that they create a living being that is far more than the sum of the individual parts. This interaction with the exterior environment of the cell is dependent upon proteins that reside in the lipid bilayer. Many of these proteins actually span the lipid bilayer, with part of the protein residing outside the cell, part within the cell and part within the hydrophobic core of the lipid bilayer. In the case of eukaryotic cells, the vast majority of these membrane proteins have been post-translationally modified by the addition of specific sugar residues to create glycoproteins. In many cases the correct sugar modifications are critical for the correct function of the glycoprotein, particularly for those glycoproteins that are involved in cell–cell or cell–substrate interactions (NB: aberrant glycosylation is frequently observed on glycoproteins at the surface of cells present in tumours). Membrane proteins perform a multitude of roles, just about all of which can be considered to be involved, in some way, in communication between the inside and the outside of the cell. They act as transporters of ions, sugars, amino acids, peptides, hormones, and other molecules; they act as receptors for extracellular ligands such as hormones and neurotransmitters, and transmit signals to the inside of the cell; they act to link cells to one another or to the underlying substrate; in short they are the physical link between the inside of the cell and the outside world. It is not surprising, therefore, that it has been estimated that about a third of all the proteins encoded by the human genome are membrane proteins.
What are the main organelles within eukaryotic cells? The major ones are the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus, mitochondria, lysosomes, endosomes, and peroxisomes (and chloroplasts in plants), with a range of specialized organelles occurring in different cells of higher eukaryotes (see Fig. 4.1.2).
The nucleus can be considered to be at the heart of the eukaryotic cell. It harbours the vast majority of DNA within the cell, i.e. the nuclear genome. It is also, among other things, the site of gene transcription, the site of mRNA processing, and the site of ribosome assembly. The control of gene expression, which often occurs at the level of transcriptional regulation, is fundamental to the regulation of cell function and involves a complex interplay between the genomic DNA in the nucleus and a host of cellular proteins. Many of these proteins shuttle, in a controlled manner and in response to specific signals, between the nucleus and the cytosol. They are able to do so because there are gaps, termed nuclear pores, in the membrane that envelopes the nucleus. The nuclear membrane is, in fact not a single lipid bilayer, but a double lipid bilayer. Thus, there is an inner nuclear membrane in contact with the contents of the nucleus and an outer lipid bilayer in contact with the cytosol. The space between the two lipid bilayers is the lumen of the nuclear membrane and is a space that is contiguous with the lumen of the endoplasmic reticulum.
Nuclear pores are complex multiprotein assemblies that allow certain proteins to pass in and out of the nucleus while excluding others. Fully processed mRNA also leaves the nucleus via nuclear pores before being translated in the cytosol by ribosomes. The dynamic traffic within, and in and out of, the nucleus is imperative for cell function.
The mitochondria are also enveloped in two layers of membrane, an inner one and an outer one. They are involved in the oxidation of molecular fuels, including pyruvate derived form sugars and fatty acids, to generate the adenosine triphosphate (ATP) that is needed as an energy source for the reactions of the cell. They are thus fundamental to cell function. Other than chloroplasts, which are also enclosed in a double-layered membrane, mitochondria are the only nonnuclear organelles to contain DNA. It is likely that mitochondria arose by a process related to endosymbiosis (the process whereby certain bacteria enter eukaryotic cells and live within their cytoplasm) in which an aerobic eubacterium was engulfed by a eukaryotic cell—hence the double membrane, one from the engulfing eukaryotic cell and one from the engulfed prokaryote. The biogenesis of chloroplasts also probably occurred via a similar pathway. Mitochondrial disorders are an important cause of neurological diseases and disorders of muscle, including cardiac muscle. Mitochondrial abnormalities may be inherited or acquired. Those specifically affecting the intrinsic genome of this organelle are transmitted in a matrilinear fashion. Descriptions of important mitochondrial disorders are to be found in Chapter 24.24.5.
Peroxisomes are membrane-bound organelles that contain high concentrations of oxidative enzymes such as catalase and are therefore important for a range of oxidative processes necessary for the elimination of multiple substances (e.g. in the breakdown of very long chain fatty acids). Several diseases caused by defects in peroxisomal proteins are described in Chapter 12.9.
Endoplasmic reticulum (ER)
The membranes of the ER are contiguous with those of the nuclear membrane, thus the lumen of the ER is contiguous with the space between the two membranes of the nuclear envelope. The lumen of the ER serves as a calcium store for the cell, with concentrations of calcium in the ER lumen being around 10–3 M, compared with 10–8 M to 10–6 M in the cytosol. Regulated release of calcium from the ER, in response to extracellular signals detected by membrane proteins at the cell surface and transmitted via specific intracellular second-messenger molecules, leads to changes in the activity of a host of cellular processes. This is because many intracellular proteins bind calcium and their activities and/or interactions with other proteins are dependent upon whether or not they are calcium bound. In fact, the calcium concentration in the cytosol has to be very carefully controlled because it is an important regulator of many intracellular processes including muscle contraction and secretion. Excess calcium within the cytosol can rapidly lead to cell death. There is, therefore a complex array of transporters operating to ensure that calcium levels remain high in the lumen of the ER and are only transiently raised in the cytosol in response to specific stimuli. Some of these transporters are in the ER membrane, others in the plasma membrane, others in the mitochondria.
The ER is, however, not simply a calcium store. It is also a major site of lipid biosynthesis within the cell. Furthermore, it is also the site of synthesis for proteins that are destined to be secreted from the cell or to be membrane proteins. Such proteins are synthesized by ribosomes that become attached to the cytosolic face of the ER membrane soon after they have started to translate an mRNA encoding a secretory or integral membrane protein. Before folding up as a secretory protein in the lumen of the ER, or passing laterally from the translocation channel into the ER membrane if it is destined to be an integral membrane protein, the nascent protein is translocated through a protein-lined channel in the ER membrane. This channel opens only when a ribosome synthesizing a secretory or integral membrane protein is bound to its cytosolic face. The ER is thus the start of the secretory pathway in eukaryotic cells. The fact that the translocation channel opens only when a ribosome is bound to it preserves the integrity of the ER membrane as a permeability barrier and ensures no leakage of ions, such as calcium, from the ER lumen. Some of the most abundant proteins in eukaryotic cells reside in the lumen of the ER; these are proteins that are involved in assisting the correct folding of newly synthesized proteins in the secretory pathway and include proteins such as the enzyme protein disulphide isomerase (which ensures that the correct disulphide bonds are formed in proteins), calnexin, and calreticulin. The latter two are also calcium-binding proteins. Thus, although the majority of proteins that enter the secretory pathway at the ER are destined to be secreted or to become integral membrane proteins in the plasma membrane, certain proteins (both soluble proteins within the lumen of organelles along the secretory pathway and integral membrane proteins within the membranes of organelles along the secretory pathway) are primarily localized to specific compartments along the secretory pathway.
It has become increasingly clear over recent years that a combination of retention and retrieval signals within proteins serve to ensure these localizations, with retention signals serving to hold proteins in place and retrieval signals operating to bring proteins back to their steady-state localization from a point further along the secretory pathway.
Most diagrams of eukaryotic cells in textbooks, including Fig. 4.1.2 here, show the ER as a membranous organelle linked to the nucleus; this is correct, but it fails to illustrate the extent of the ER since, in most cells, it pervades much of the extranuclear space of the cell and is a highly dynamic organelle. More than half of the total membrane area of a mammalian cell can be ER.
The step beyond the ER in the secretory pathway is the Golgi apparatus. The Golgi apparatus has been likened to a small stack of pitta bread, with each pitta corresponding to a cisterna (segment) of the Golgi. Vesicles, containing soluble and integral membrane protein cargo, bud off from the ER and are delivered to the cis face of the Golgi apparatus where they fuse with the Golgi membrane and deliver their contents. The recruitment of specific cargo into the vesicles, the budding of the vesicles and their fusion with the Golgi are all steps that involve discrete, and transient, assemblies of proteins. The different cisternae of the Golgi apparatus—known as cis, medial, and trans, although there may well be many more than three in certain cell types—represent the next steps along the secretory pathway. It now appears that passage through this part of the pathway can be quite complex, with both forward (anterograde) and backward (retrograde) vesicular traffic occurring. The anterograde traffic moves cargo towards the trans side of the Golgi apparatus and the retrograde traffic retrieves material that is required earlier in the secretory pathway, i.e. in the medial or cis cisternae of the Golgi apparatus or in the ER. Vesicular traffic within the Golgi apparatus also seems to be complemented by a process that has been termed cisternal maturation. This describes the maturation of a cis cisterna into a medial cisterna by the vesicular retrieval of material that should not be present in a medial cisterna. The retrieved vesicles fuse with newly arrived vesicles from the ER to form a new cis cisterna; meanwhile the medial cisterna matures into a trans cisterna via the same process. As all of this is happening, the proteins that are passing along the secretory pathway are being sequentially post-translationally modified, primarily by the addition of a series of sugar residues to generate glycoproteins, by specific enzymes with discrete steady-state localizations maintained by retention and retrieval signals, within specific cisternae of the Golgi apparatus.
Beyond the trans cisterna of the Golgi apparatus lies the trans Golgi network (TGN) from which a range of vesicles and tubules bud to deliver their cargo to its destination. This may be the cell surface, for proteins that are to be secreted or to become integral membrane proteins in the plasma membrane, but may also be an intracellular organelle. Thus, for example, lysosomal enzymes have to be delivered to the lysosome and this is done via the secretory pathway.
Lysosomes can be considered as the recycling centres of the cell. Macromolecules are delivered to lysosomes to be broken down into their constituent building blocks (e.g. proteins to amino acids, polysaccharides to monosaccharides) by a host of acid hydrolases (proteases, glycosidases, nucleases, lipases, etc.). The building blocks are then exported from the lysosome and used by the cell to make new macromolecules. It is clearly important that the hydrolysis of macromolecules is strictly compartmentalized, otherwise the cell would destroy itself. In fact the cell not only compartmentalizes lysosomal enzymes within the lysosome, but also ensures that these enzymes only become fully active once they have been delivered into the lysosome. The lysosomal enzymes are acid hydrolases, that is to say that they function at low pH. The pH of the lumen of the lysosome is about 4.5. This is in contrast to the pH in the lumen of the Golgi and TGN (approximately 6.5 to 6.7) or the pH of the cytosol (approximately 7.4). Thus, as lysosomal enzymes are delivered from the TGN to the lysosome (by vesicular transport) they become activated because of the lower pH in the lysosome. In fact, lysosomal enzymes are not delivered directly from the TGN into the lysosome, but are delivered to an intermediate compartment, the late endosome. This is an interface between the secretory pathway and the endocytic pathway (a pathway carrying material that has been internalized from the cell surface). A late endosome fuses with a mature lysosome, delivering the contents of the late endosome into the lysosome. Membrane, and integral membrane proteins, that should be in the late endosome are then retrieved from the hybrid organelle to form a new late endosome and an immature lysosome. The lumenal pH of the late endosome is intermediate between that of the TGN and that of the lysosome (i.e. between 5 and 6). The reduced pH is generated by the action of a proton pump (a vacuolar ATPase) in the limiting membrane of the late endosome and lysosome. Many inherited disorders of lysosomal function have been identified. These diseases and their treatments are described in Chapter 12.8. The special capacity of the lysosomal compartment for complementation by internalizing proteins supplied externally has allowed several important therapeutic enzymes to be developed.
The existence of late endosomes implies that there must also be early endosomes. There are. There is clearly a flow of membrane and protein along the secretory pathway culminating in the fusion of vesicles with the plasma membrane. In the absence of any compensatory membrane internalization, the surface area of the cell would therefore continually increase. Such internalization does occur, thereby ensuring that most cells remain relatively constant in size. The internalization of membrane from the cell surface (a process termed endocytosis) occurs via a variety of routes, the best characterized being clathrin-mediated endocytosis. The process involves the assembly of specific protein machinery at the cytosolic face of the plasma membrane, the invagination of the plasma membrane, and the pinching off of membrane-bound vesicles (or possibly tubules) from the plasma membrane. The protein coat that has been instrumental in the formation of these vesicles then generally disassembles and the uncoated vesicles fuse to form early endosomes; the vacuolar ATPase is already active in early endosomes and they have a lumenal pH of approximately 6.5 to 6.8. The endocytic process not only ensures that a balance is maintained between the amount of membrane inserted in the plasma membrane and the amount removed, but also allows the selective recruitment of specific integral membrane proteins (often with extracellularly bound ligand) into the endocytic pathway.
The different endocytic mechanisms selectively recruit different cargo and thereby serve as molecular filters, internalizing certain integral membrane proteins while leaving others at the cell surface. The complexity of the endomembrane system (i.e. the membranes of the endocytic compartments) in mammalian cells has become apparent in recent years. Thus, for example, in addition to early and late endosomes there are also recycling endosomes. These are compartments from which material that is to be returned to the plasma membrane is retrieved. Such material might be receptors that have been internalized along with their ligand, but which need to be returned to the cell surface having released their ligand at the lower pH of the early endosome/recycling endosome. An example of such a receptor would be the transferrin receptor which releases the iron that is bound to transferrin in the early endosome/recycling endosome, leaving the transferrin receptor and apotransferrin to be recycled to the cell surface for reuse. Other receptors that are internalized are destined for degradation in the lysosome and are delivered to the late endosome. An example of such a receptor is the epidermal growth factor receptor. When this receptor binds its ligand at the cell surface it transmits a cascade of signals across the cell which trigger cell growth and cell division. Such signals should only be transient, otherwise unregulated cell growth and cell division occur; the cell ensures that the signals transmitted by the receptor are transient by internalizing the receptor and sending it to the lysosome for degradation.
All this movement of vesicles and tubules between compartments does not occur at random. It is dependent upon motor proteins and the cytoskeleton. The cytoskeleton is the name given to a framework within the cell which gives the cell its shape and which provides a structure to which organelles and proteins can be attached, thus providing an architecture that gives spatial organization to the cell. There are three main components of the cytoskeleton in mammalian cells: microtubules, actin filaments, and intermediate filaments (see Fig. 4.1.2). Each of these components is a polymer of protein subunits and all three are dynamic structures with the potential for assembly and disassembly according to the needs of the cell.
Microtubules are highly dynamic polymers of heterodimers of α- and β-tubulin which assemble to form long hollow tubes approximately 25 nm in diameter. Monomers of globular (G) actin polymerize to form filamentous (F) actin which is a double-stranded helical polymer with a diameter of 5 to 9 nm. Elongated and fibrous subunits assemble to form intermediate filaments with a diameter of approximately 10 nm (e.g. lamins A, B, and C assemble to form the nuclear lamina that provides the inner lining to the nuclear envelope).
The different components of the cytoskeleton provide complementary features of the cellular architecture. Microtubules serve to localize organelles within the cell and provide the tracks along which many classes of transport vesicles and tubules move, the movement being powered by motor proteins (notably kinesin and dynein) attached to the membranes of the vesicles or tubules. Microtubules also play a critical role during cell division, since they are pivotal in the physical separation of chromosomes during mitosis. Actin filaments can cross the cell and provide the structure that determines the shape of the cell’s surface. These filaments play major roles in protrusions from the cell surface. For example, they run along the length of the microvilli that extend from the apical surface of polarized epithelial cells, and they are absolutely necessary for cell locomotion since concerted rearrangements of the actin cytoskeleton underlie cell movement. Myosin motor proteins also interact with actin, the best characterized such interaction being between actin and myosin II in skeletal muscle; this interaction is responsible for generating the force that is required for muscle contraction. Both microtubules and actin filaments are highly dynamic structures. Their assembly and disassembly is precisely and finely regulated by a host of cellular proteins in response to a range of extracellular signals. Intermediate filaments are relatively stable by comparison, and provide mechanical strength to the cell.
The dynamic cell
The preceding superficial overview of the secretory and endocytic pathways in mammalian cells highlights their dynamic nature. The elegant cartoons that grace most textbooks in this field indicate the subcellular organelles and other cellular components and their relative positions within the cell, but, because they are two-dimensional static images, they cannot give any indication of the complex dynamics that operate within cells. All the different vesicle budding, vesicle transport, vesicle targeting, and vesicle fusion steps involve the assembly and disassembly of specific and discrete macromolecular complexes. There is exquisite spatiotemporal control of each of these events. The dynamic nature of microtubules and actin filaments adds to the complexity of the interactions that occur within cells.
The dynamic nature of the eukaryotic cell has been made self-evident over the past 10 years or so following the increasingly widespread use of a range of tools and microscopy systems that allow the imaging of specific proteins within live cells. The real breakthrough came with the isolation of the DNA sequence encoding green fluorescent protein (GFP). GFP is encoded by the genome of the jellyfish Aqueoria victoria; it is a protein that is naturally fluorescent, emitting green light when it is illuminated with blue light. The now standard techniques of molecular genetics have allowed researchers to link the DNA sequence encoding GFP to the DNA sequences encoding a range of different proteins. These hybrid DNA sequences can be introduced into eukaryotic cells and the localizations, and intracellular movements, of the hybrid proteins they encode can be monitored by appropriate microscopy techniques. This has allowed us to see the dynamic instability of microtubules within living cells, the movement of proteins along the secretory pathway, the sorting of proteins in the endocytic pathway, and many other cellular processes. Genetic engineering has also provided us with a suite of spectral variants of GFP, each emitting light of a different wavelength (i.e. a different colour), thereby allowing the imaging of two or more different proteins in the same cell at the same time. It is remarkable that, in most cases, the presence of a fluorescent protein attached to a protein of interest has little if any effect on the function of that protein.
It is over 30 years since Singer and Nicholson proposed the ‘fluid mosaic’ model for biological membranes. This proposed that integral membrane proteins could diffuse freely in the sea of the lipid bilayer. The imaging of populations of GFP-tagged proteins has confirmed earlier studies which show that this is essentially the case, but more sophisticated single-particle tracking studies have shown that the plasma membrane is partitioned with regard to molecular diffusion in the plane of the lipid bilayer. This is because of specific interactions with the underlying actin cytoskeleton. These interactions tend to be between actin and the cytosolic domain of specific integral membrane proteins (see Fig. 4.1.3). Such interactions are often indirect, i.e. via one or more intermediate proteins, thereby providing the opportunity for regulation of the interaction. For example the cytosolic domain of the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with a cytosolic protein called EBP50 at the apical surface of polarized human airway epithelial cells. EBP50 in turn binds another protein, ezrin, and ezrin binds the actin cytoskeleton, thereby tethering CFTR to the actin cytoskeleton and keeping it in the right place in the plasma membrane. Similar interactions between the cytosolic domains of specific integral membrane proteins and the actin cytoskeleton most probably occur in the context of organellar membranes.
Differential gene expression
A human body clearly arises from a single cell, the fertilized egg. This single cell eventually gives rise to the multitude of different cell types within the body. For this to occur, cells must grow and divide (and sometimes die) in a highly regulated manner. Not only do the cells need to grow and divide, different populations of cells must differentiate along different lineages in order to generate the different cell types required to populate the different tissues and organs of the body.
Each of the 1013 or so cells in the human body is a phenomenally complex and dynamic entity. Furthermore, different subsets of the approximately 20 000 to 25 000 genes in the human genome are expressed in different cell types, with further variations in gene expression occurring during development and in response to external stimuli. This differential gene expression leads, at least in part, to the diversity of cell types found throughout the body. Thus, for example, a neuron is clearly very different from an epithelial cell lining the gut. However, both have the same fundamental organization described in the preceding paragraphs. They both express a core set of shared genes, providing the fundamental cellular organization, but each expresses a different set of specific genes. The specific genes expressed will help to define the phenotype of the cell. The differences between cells can often be quite subtle, e.g. different cell types have different protein subunits making up their intermediate filaments, different motor proteins are expressed in different cell types, and differential glycosylation of glycoproteins and glycolipids occurs in different cell types.
Alternative splicing and post-translational modifications
The fact that there appear to be only 20 000 to 25 000 genes in the human genome does not mean that only this number of proteins can be encoded by the genome. Many genes are subject to the process of alternative splicing, whereby specific exons are included or excluded as the precursor mRNA is processed (spliced) in the nucleus to remove the noncoding intron sequences. Thus one gene can give rise to several related, but different, mRNA transcripts. Furthermore, differential processing and differential post-translational modification of proteins leads to further variety in the range of protein products produced from the genome.
The range of proteins in a cell (the proteome) is therefore potentially considerably larger than the number of genes in its genome. In the case of cytosolic proteins, and the cytosolic domains of integral membrane proteins, many of the post-translational modifications that occur are transient and reversible. Thus, many such proteins are subject to phosphorylation (the addition of a phosphate group to the side chain of a specific amino acid). This process is catalysed by specific enzymes (kinases) and occurs on specific serine, threonine, or tyrosine residues in target proteins. This modification is reversible by the action of members of another family of enzymes (phosphatases) that remove the phosphate. Phosphorylated proteins have different activities, and often interact with a different subset of proteins, from their nonphosphorylated counterparts; thus, reversible phosphorylation is a mechanism whereby the cell can regulate interactions, and thereby processes, occurring within it. It is not uncommon for a kinase to be activated by phosphorylation and it is not uncommon for one kinase to activate another by phosphorylation, thus establishing a signalling cascade that has built-in amplification of the initial signal—amplification because each kinase is an enzyme capable of acting upon multiple substrate molecules while it is in the active state. The initial signal might be the binding of a ligand to its receptor at the cell surface, e.g. the binding of epidermal growth factor to its receptor at the cell surface. As mentioned earlier, this initiates a cascade of signals across the cell which trigger cell growth and cell division; that cascade of signals is essentially a cascade of phosphorylation events. Such a process clearly needs to be transient or it would lead to unregulated cell growth and cell division. The initial signal is removed by the internalization and degradation of the epidermal growth factor receptor, as previously outlined, but this still leaves an activated kinase cascade perpetuating the ‘grow and divide’ message. It is the action of specific phosphatases removing the phosphate groups from the kinases in the cascade that turn off the signalling pathway. Thus, once again, we have a highly dynamic cellular system with exquisite spatio-temporal control.
Reversible phosphorylation is one example of several reversible post-translational modifications that serve to regulate cellular function. The principles relating to phosphorylation as a form of post-translational regulation, i.e. that phosphorylated proteins interact with different proteins compared to their nonphosphorylated counterparts, or that phosphorylated enzymes have different activities from their nonphosphorylated counterparts, and that this plays a role in the regulation of cell function, also applies to other forms of reversible post-translational modification.
Post-transcriptional gene silencing (miRNA)
The 20 000 to 25 000 genes in the human genome account for only about 2% of the total DNA in the genome. So, what is the role of all the other DNA? A significant amount of it serves structural purposes, e.g. the sequences at the centromeres (middles) and telomeres (ends) of chromosomes, but recent evidence shows that much of it plays crucial regulatory roles, working by the process of post-transcriptional gene silencing (PTGS).
The phenomenon of PTGS was first described in plants, but has subsequently been shown to be widespread in eukaryotes. In PTGS a short (19 to 23 nucleotides long) double-stranded RNA molecule associates with a target mRNA (the nucleotide sequence of one of the RNA strands in the double stranded molecule is complementary to the sequence of the target mRNA). This occurs in the context of a multiprotein complex and leads to either a block in translation or the degradation of the target mRNA. This mechanism therefore regulates protein expression post-transcriptionally, hence the designation PTGS. The short double-stranded RNA molecules are produced from slightly larger precursor RNA molecules known as micro RNAs (miRNAs) which are themselves produced by transcription of relevant DNA sequences by DNA polymerase in the cell’s nucleus. The number of DNA sequences within the human genome that encode miRNAs has yet to be finalized, but there appear to be at least as many such sequences as there are protein-encoding DNA sequences (i.e. conventional genes). miRNA sequences have been shown to play critical regulatory roles in a range of processes, e.g. during development and in the immune response to pathogens. They have also been implicated as playing a role in several disease states, such as heart disease and cancer.
The availability of the human genome sequence has given us access to information concerning the basic building blocks of the cell, but it is how those building blocks are modified and used in a multitude of different dynamic interactions that gives organization, function, and life to the cell. A major challenge of the next decade is to integrate the vast amounts of data that are now available, and will continue to become available, on the molecular mechanisms that underlie cellular organization, structure and function. Such a challenge will have to be met if we are to achieve a clearer, and more complete, understanding of the cell and are to develop the capacity to refine our means of modifying cellular functions that are disturbed in disease.
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