INTRODUCTION TO CELL BIOLOGY

These notes were put together by Dr Ben Dunmore, a Cellular and Molecular Biologist in the Department of Pathology. Some of the images used in these notes are reproduced with permission from the following three web sites/texts, which you may find useful; http://www.cellsalive.com/index.htm http://www.meddean.luc.edu/lumen/index.html http://www.whfreeman.com/lodish/

The six sections will cover the following topics;

(1) The evolution of cells and the molecules of life (2) The structure of cells (3) The flow of genetic information (4) Signal transduction and the control of cell growth, cell function and cell death (5) Building bodies the concepts behind complex organisms (6) Function, development and pathology

Section 1: The evolution of cells and the molecules of life

(1) Design vs. Evolution of cells. What is the definition of life? Probably one of the best definitions is that living things must possess three properties: metabolism, growth and reproduction. Based on this definition, the smallest unit of life is the cell. This series of lectures looks at the most basic aspects of the biology of cells, both how they work internally, and how they co-operate to form multicellular organisms. Cells differ from man-made machines in one fundamental way - they were evolved rather than designed. This implies that all cell types present today gained their current structure by numerous small modifications of pre-existing structures over millions of years. To remind you of the timescale of this process, the earth was formed approximately 4.5 billion years ago, and the first simple cells were thought to have appeared approximately 3.5 billion years ago. Between then and now a great deal of evolution has taken place! Charles Darwin developed the principle that organisms vary randomly (variation), and that the fittest organisms (those best adapted to their environment) are the most successful in surviving and passing their genes to future generations. This is the basis of the theory of natural selection.

Cells are extremely complex so complex that our understanding of their structure and function is very much incomplete. It is interesting to consider that, even with the most advanced technologies of today; engineers are not yet able to design a machine that can mimic or replace a cell. The difficulty lies in the seemingly impossible design requirements. For example, such a machine would need to perform the functions of a specialised cell (eg. secretion, contraction, electrical conduction etc.), be packaged into the small size of a cell, repair and replicate itself, and communicate with and respond to its environment. Cells are remarkable in their adaptiveness and functionality. However, they can sometimes malfunction or become overwhelmed by the demands placed on them. This is the basis of most human and animal disease. (2) The History of Cell Biology. The history of cell biology is fascinating. It involved the scientists of the day heroically proposing and testing hypotheses that went against the dogma of their time. The discoveries they made are remarkable, given the primitive facilities available to them. Since the study of cells requires techniques to image them, the history of cell biology is intimately tied up with imaging technology. One of the earliest cell biologists was Robert Hooke, who in 1655 used a primitive microscope to observe the cells of a cork tree. In 1683 Leewenhoek discovered bacteria. In 1838, modern cell biology was born when two scientists Scheiden and Schwann proposed the cell theory. Among other things, their theory suggested that all life forms were made of one or more cells, and that cells only arise from pre-existing cells. (This theory still holds true, with the minor caveat that viruses are only alive while infecting a cell). Following the cell theory, small structural units known as organelles were discovered within cells - Kolliker described mitochondria in 1857 and Golgi described the Golgi apparatus in 1898. (3) The size of cells. In order to understand cell biology, it helps to have a concept of the sizes of the cells and the structures they contain. It also helps to know a little about the techniques used to observe cells. For an idea of relative sizes, please see the diagram below. (If included on this

diagram, bacteria would be often approximately 1m in size; viruses are often <100nm in size).

Many of the structures within an animal or plant cell can be observed with a light microscope, which has a limit of resolution of about 200nm (0.2 m). This limit is due to the wavelength of light (0.4-0.7m). For finer detail or to observe smaller structures electron microscopes are used. (4) Cells as part of a hierarchy of life. Cells can be viewed as part of a hierarchy of life. At the base of the hierarchy are the molecules that make up the cells. Above this are structures found within some cells known as organelles. In multi-cellular organisms the hierarchy has further layers above the cells themselves, including tissues, organs and the entire organism. Over and above individual organisms, there are yet further layers of the hierarchy that will not be considered in this course, consisting of; populations, communities, ecosystems and the biosphere. (5) The molecular building blocks of life.

To understand cell biology you first need to begin at the bottom of the hierarchy described above the molecules which are the most basic building blocks of life. Cells are 90% water. Of the remaining molecules present, cells are made up of approximately; 50% protein, 15% carbohydrate, 15% DNA and RNA and 10% lipid. These compounds are described below. A) Deoxyribonucleic acid (DNA) DNA belongs to the chemical class of nucleic acids. The science of DNA goes back further than you may think. DNA was discovered in 1868 by a Swiss biologist called Friedrich

Miescher. In 1952, Alfred Hershey and Martha Chase used viruses that contained radioactively labelled DNA to show that DNA is the genetic material (this famous experiment is known as the Hershey-Chase experiment). In late 1953, James Watson and Francis Crick at Cambridge proposed a model of the structure of DNA. For this work they shared the 1962 Nobel prize for Physiology and Medicine with Maurice Wilkins. (Another scientist, Rosalind Franklin who contributed to this discovery died before the she could receive a part of this Nobel prize). DNA is a molecule made up of a series of building blocks known as nucleotides. Each position in a DNA molecule can use any one of four nucleotides. The nucleotides are held together by a phosphate backbone. DNA forms a two-stranded helix, with each strand complimentary to the other. The sequence of the nucleotides along the two strands represents the genetic code (see diagram below). The role played by DNA, how it is organised into genes and how it is passed down through generations will be discussed in Lecture 3.

B) Proteins Proteins are the workhorses of the cell. They are assembled according to information contained in the DNA genetic code. Proteins are manufactured by joining together small building blocks are known as amino acids. The chemical bonds that join the amino acids are known as peptide bonds, hence protein chains are also known as polypeptides. Each position in a protein molecule can use any one of 20 different amino acids, therefore, an almost infinite range of different protein structures can be made. Localised regions of proteins can fold into diverse shapes (known as secondary structure). Two examples of secondary structure you may come across are -helices and -sheets (see diagram below).

The entire length of a protein may contain many regions of different secondary structure, which stabilise one another to form a three-dimensional shape known as tertiary structure. For example, a protein that allows lymphocytes to recognise invading cells (known as class I MHC) is made up of both -helices and -sheets that stabilise one another (see below).

A single protein chain is known as a monomer. In order to carry out complex functions, many proteins exist as multimers that consist of two or more linked protein monomers (each of the linked monomers is sometimes called a subunit). The linking of two or more protein monomers to form multimers is known as quaternary structure. One example of a protein multimer is an antibody. Antibodies are defence molecules of the immune system produced by cells known as B lymphocytes. They bind to uniquely shaped regions of proteins known as epitopes. For example, antibodies may bind to epitopes within proteins on the surface of an invading bacterium. Once bound to an epitope, antibodies can inform the immune system of the presence of the invading organism and cause the protein that carries the epitope to be destroyed. In the diagram below, two of the protein subunits that make up the antibody are seen on the right, and the antigen to which the antibody is bound is seen on the left. The antibody subunits together form a precisely shaped pocket that will only bind to a very particular structure of antigen.

The seemingly endless structural possibilities possessed by proteins allow them to perform a wide range of functions. Some of these are listed below;

Function Type of protein

Synthesis / Degradation = Enzymes Structure = Cytoskeleton, Matrix Communication = Hormones, Growth Factors Defence = Antibodies Control of gene expression = Transcription factors
Of these, enzymes are the most important protein-based machines that operate in cells. Enzymes function by bringing the starting molecules of chemical reactions (called substrates) together and by controlling the subsequent chemical reactions. When enzymes speed up chemical reactions they function as catalysts. Enzymes often have complex quaternary structures. Most cellular processes require enzymes. The part of an enzyme that binds to the substrates is known as the active site. Enzymes are usually very specific for the substrates they bind to. This specificity is based on a precise fit between the enzymes active site and the substrate. Many medicines act by mimicking the substrate of a particular enzyme and inhibiting the enzyme either by competing with the substrate or by binding to the enzymes active site irreversibly. C) Triglycerides and Membranes Triglycerides are produced by combining one glycerol molecule with three fatty acid molecules. Triglycerides are useful energy stores. Phospholipids are produced by combining one glycerol molecule with two fatty acid molecules and one phosphate molecule. Phospholipids are essential for the structure of membranes (see below).

Membranes are usually made of two layers of phospholipids which form what is known as a lipid bilayer (see above). This lipid bilayer aligns itself spontaneously with the waterrepellent (hydrophobic) tails of the triglycerides in the centre of the membrane and the waterloving (hydrophilic) glycerol and phosphate molecules are on either surface of the membrane. Cholesterol molecules and protein molecules (such as enzymes and receptors) are integrated into the lipid bilayer. Some proteins (known as peripheral proteins) reside on one surface of the membrane, while others (known as transmembrane proteins) run right through it. Both the proteins and the phospholipids are believed to be relatively free to move laterally in the membrane (as if they were in a fluid). Cholesterol is essential for this membrane fluidity.

This description of membrane structure is known as the fluid mosaic model. Membranes with their embedded proteins carry out many important functions in the cell including; compartmentalisation, catalysis of chemical reactions (via enzymes embedded in the lipid bilayer), selective transport across the membrane (via specialised enzymes that act as channels and pumps), and recognition (via specialised enzymes known as receptors). Often these receptors activate signals inside the cell (described in Lecture 4).

D) Sugars. These contain the 3 atoms Carbon, Hydrogen and Oxygen, therefore they are usually known as carbohydrates (abbreviated to CHO). Monosaccharides (eg. glucose) are single carbohydrate units. Monosaccharides polymerise into disaccharides (eg. sucrose is the combination of the glucose and fructose monosaccharides). Oligosaccharides are formed by the linkage of monosaccharides to produce short chains. Oligosaccharides are sometimes linked to lipids to form glycolipids and to proteins to form glycoproteins. Polysaccharides are formed by the linkage of monosaccharides to produce long chains. Polysaccharides are used for structural strength and the storage of energy (eg. glycogen, starch, cellulose).

E) Small molecules. In addition to the molecules described above, cells also contain a large number of small molecules in solution in water. Many of these small molecules play essential roles in respiration. Respiration is the usual manner in which cells convert food (glucose) into a small energy-containing molecule known as adenosine triphosphate (abbreviated usually to ATP). ATP can be used to power

energy-requiring events in the cell such as; the synthesis of the macromolecules described above, the transport of molecules across membranes, cell movement, cell division and cell suicide.

The form of respiration usually employed by cells is aerobic respiration. Aerobic respiration requires oxygen. There are three steps to aerobic respiration; (i) Glycolysis (where glucose is converted into a molecule known as pyruvate). This produces a small amount of energy which is converted into ATP. (ii) The Krebs cycle converts pyruvate into citrate, producing more ATP. (iii) Oxidative phosphorylation is a chain reaction where energy stored in chemical intermediates produced in the Krebs cycle are converted into heat and yet more ATP. When cells lack oxygen, they produce ATP by anaerobic respiration. This is much less efficient than aerobic respiration at producing ATP, and has the added disadvantage that it produces toxic by-products such as lactic acid.

Section 2: The structure of cells

(1) The two types of cells. Cells can be divided into two types based on their complexity; prokaryotic and eukaryotic. Prokaryotic cells are in general simpler than eukaryotic cells, and evolved earlier. The first prokaryotic cells appeared approximately 3.5 billion years ago, while eukaryotic cells did not appear until approximately 1.5 billion years ago. Multi-cellular eukaryotic organisms then evolved over the last 0.5 billion years.

Prokaryotic cells (eg. the bacteria shown above) have a very simple structure. Only a little internal structure is visible in prokaryotic cells through an electron microscope.

Eukaryotic cells (eg. protozoa, plant cells, animal cells) tend to be larger than prokaryotic cells, and contain specialised organelles and packaging/transport mechanisms that are required to support their larger size. (2) The structure of prokaryotic cells. Nucleoid. The DNA of a bacterium is often visible by electron microscopy as a loose collection in the centre of the cell. Unlike eukaryotic cells, it is not held in place in a nucleus by a nuclear membrane. Ribosomes. These are small structures involved in translating genetic information into proteins (see later for how this is done). They are smaller than the ribosomes found in eukaryotic cells, and appear as granules or specs through an electron microscope. Storage granules. As their name suggests, they store reserves of nutrients and

macromolecules. Plasma membrane (sometimes informally known as the cell membrane). This is a lipid bilayer with proteins inserted into it (see above). Some of these proteins function as ion channels or pumps that can be carefully regulated by control mechanisms within the cell. Other proteins in the plasma membrane are receptors that receive signals from outside the cell. The plasma membrane is selectively permeable. It maintains the correct concentrations of ions and proteins inside the cell, and controls the transport of nutrients and wastes into and out of the cell. Cell wall. This is a semi-rigid wall around the cell composed of peptidoglycans. The cell wall helps to maintain cell shape. Outer membrane. Some bacteria (known as gram ve bacteria) have a second membrane outside the cell wall. Capsule. Some prokaryotes have an outermost layer known as a capsule. This may help them evade the immune system. For this reason, capsules are found particularly on diseasecausing bacteria. Pili. These are hair-like appendages that stick out of bacteria to allow them to attach to other bacteria. A specialised type of pili called sex pili allow bacteria to exchange genetic information with one another. Flagella. Many bacteria have one or more extending flagella to assist their movement. They are turned like propellers by motors located inside the plasma membrane. (3) The structure of eukaryotic cells. Cytosol. This is the soup-like contents of the cell. It is mainly water, but contains many of the essential molecules of life, which will be discussed later in these lectures. These include; ions, proteins, signalling machinery, and energy utilisation machinery. Nucleus. This is a double membrane bag that contains the DNA of the cell. It is easily visible when cells are viewed through a light microscope. The contents of the nucleus are connected with the cytoplasm via nuclear pores. The nuclei of many cells contain a prominent structure known as the nucleolus, which is responsible for the production of ribosomes (see above). Plasma membrane. See above. Endoplasmic reticulum (ER). This structure is a folded membrane continuous with the nuclear membrane. Some regions of the ER have ribosomes embedded in the membrane. Because the ribosomes give them a rough appearance, these regions are called rough ER. Regions of the ER without ribosomes are called smooth ER. The function of the ER is the synthesis of proteins. Golgi apparatus. This structure is similar to a stack of pancakes. Its function is to package proteins and other macromolecules into membrane-bound organelles known as lysosomes, peroxisomes and secretory vesicles (see below) for transport to other regions of the cell. Vacuoles are membrane-bound sacs used for storage or to contain items imported into the

cell. Lysosomes. These contain enzymes used for digestion / disassembly within the cell. For example, in white blood cells that ingest bacteria (the technical name for this ingestion is phagocytosis), the ingested bacteria are contained in a membrane-bound organelle called a phagocytic vacuole. To kill and break down the bacteria, lysosomes containing enzymes fuse with and release their contents into the phagocytic vacuole. Peroxisomes. Some functions of a cell require the production of dangerous chemical intermediates similar to hydrogen peroxide. The enzymes in peroxisomes protect cells from these intermediates. Secretory vesicles. These are used to transport newly made macromolecules such as hormones from the Golgi apparatus to the plasma membrane for release outside the cell. Mitochondria are the power stations of the cell. They consist of a flat outer membrane and a highly convoluted inner membrane. On the inner membrane food and oxygen are used to produce ATP (during aerobic respiration, described above) which then leaves the mitochondria to power many processes within the cell. The mitochondria have an additional function - they acts as a control centre to decide whether cells commit suicide by a process known as apoptosis (discussed in Lecture 4). The existence of a double mitochondrial membrane suggests that mitochondria may possibly be descendants of bacteria that were ingested by an ancestor cell billions of years ago. In support of this theory, mitochondria have their own DNA and their own ribosomes. Mitochondrial ribosomes are more similar to bacterial ribosomes than they are to eukaryotic ribosomes. Cytoskeleton. The function of the cytoskeleton is to maintain cell shape and to assist cell movement. The cytoskeleton is a network of scaffolding spanning the cell. It is made up of three types of protein; microtubules, microfilaments, and intermediate filaments. Microtubules function primarily to maintain cell shape, and are built up of tubulin subunits. Microfilaments function both to maintain shape and to promote movement and transport, and are made up of actin subunits. Proteins associated with the cytoskeleton known as motors do the work of moving organelles around the cell, and of moving the cell itself. Examples include: myosin (an motor that moves along actin filaments) and kinesin (a motor that moves along microtubules). (4) The structure of viruses. Viruses are the simplest and the smallest of organisms. They have a stripped-down minimal structure consisting of a protein capsule containing DNA or RNA (their genetic code). Viruses survive and reproduce by infecting a cell and commandeering the cells machinery to produce more viruses. A class of viruses known as bacteriophage infect bacteria.

Section 3: The flow of genetic information

(1) DNA is an information-containing molecule. As discussed in Lecture 1, DNA is a molecule made up of a series of building blocks known

as nucleotides arranged as two complimentary strands in a double helix. Each position in a DNA molecule can use any one of four nucleotides. The nucleotides are held together by a phosphate backbone and phosphodiester bonds. The sequence of the nucleotides along a DNA molecule represents the genetic code (see diagram below). By convention, one end of a DNA molecule is known as the 5-end and the other as the 3-end. A DNA sequence is read from the 5-end.

A gene is a section of a DNA molecule that contains all the information required to manufacture a protein. Thousands of genes may be contained on a single continuous DNA molecule. In many cells, each DNA molecule (along with some supporting proteins) is coiled up to form a chromosome. Before cells divide by mitosis, each DNA molecule in the cell is duplicated. To achieve this, the DNA is split into its two strands, and new complimentary DNA strands made by a process known as DNA synthesis. DNA synthesis employs a family of enzymes known as DNA polymerases. The end result of DNA synthesis is a complete set of chromosome duplicates, known as sister chromatids. This duplication allows a set of complete doublestranded DNA molecules to be passed to each daughter cell during mitosis. (4) Genomes and Mutations. The sum total of all genes possessed by an organism is known as the organisms genome. 9 The human genome contains approximately 3 x 10 nucleotides and probably consists of about 40,000 genes. Each cell in the same organism has exactly the same genome, unless a mutation has occurred in one or more of the genes in a cell. If a mutation occurs in a cell that will become an egg or sperm, this mutation can be passed on to the offspring of the organism, and is known as a germline mutation. If a mutation occurs in a type of body cell that will not become an egg or sperm, it cannot be passed on to offspring, and is known as a

somatic mutation. Somatic mutations that promote cell growth or survival are one cause of cancer. In humans, the genome is contained on 46 chromosomes. These are made up of two nearly identical (homologous) sets of 23 chromosomes, one set inherited from each parent (ie. 23 maternally-derived and 23 paternally-derived chromosomes). One pair of chromosomes are known as sex chromosomes, because they control whether a fertilised egg will become a male or female. Sex chromosomes are described as X and Y males have one X and one Y chromosome while females have two X chromosomes.

Homologous maternally-derived and paternally-derived chromosomes usually contain the same genes at the same positions. However, the DNA sequence of a gene on the maternallyderived chromosome may differ slightly from the sequence of the same gene on the paternally-derived chromosome. These two slightly differing genes are known as alleles. The slightly differing information contained in each allele may encode a slightly different characteristic (eg. eye colour). These characteristics are known as a phenotype, and the combination of genes that encode these characteristics is known as a genotype. Note that the two alleles compete to determine what the phenotype will be. Sometimes one allele dominates the other allele in this competition (the allele that wins is called the dominant allele, and the other allele is called the recessive allele). Two types of information are contained in a gene to allow the manufacture of a protein; (i) information about the structure of the protein and (ii) information about when and where the protein will be made. The parts of a gene containing the information about when and where the protein will be made (as well as containing some junk DNA) are known as introns. The part of a gene containing information about the structure of the protein is known as the coding region. The coding region, along with small regions called the 5 and 3 untranslated regions (UTR) are contained in those parts of the gene known as exons. The exons of a gene are usually discontinuous, separated by several introns. (5) Expression and the central dogma. Expression is the manufacture of a protein based on the information contained in a gene. Expression involves a flow of information from DNA RNA Protein and is known as the central dogma (devised by Francis Crick, see below).

The first step of expression is the manufacture of messenger ribonucleic acid (mRNA). This process is known as transcription. During transcription, an mRNA molecule is copied from the DNA sequence. Transcription is the most important control step during expression. A particular gene may only be transcribed in a small proportion of an organisms cells, and even then only at particular times. The timing and abundance of transcription in any cell is regulated by proteins known as transcription factors which bind to sequences within the introns of a gene that are known as promoters and enhancers. The activity of transcription factors, and their localisation to the nucleus, is regulated by networks of molecular signals operating within the cell (described below). During transcription the introns are removed from the mRNA by a process known as splicing, which produces an mRNA consisting of only the exons joined together. This mRNA consists of a 5-UTR, a continuous coding region, and the 3-UTR. The 5-UTR and 3-UTR contain information specifying, among other things, the stability of the mRNA. After it is made, the mRNA exits the nucleus through the nuclear pores to enter the cytoplasm. The mRNA then functions as a set of plans (a template) to guide the manufacture of a protein. The sequence of nucleotides in the mRNA specifies the sequence of building blocks that will make up the protein. (These protein building blocks are known as amino acids). It takes 3 nucleotides to contain enough information to specify which amino acid will be used at each position in the protein. There are twenty possible amino acids for each position. The manufacture of a protein using the mRNA template as a guide is known as translation and takes place in ribosomes on the rough ER. The newly manufactured proteins are then polished off in other parts of the cell. This polishing off involves the folding of proteins into their correct shape, the assembly of multiple proteins together into large complexes, and the linking of proteins to lipid and carbohydrate molecules. Many organelles are involved in this polishing process. The newly made proteins are transferred to the Golgi apparatus in transport vesicles where they are further processed and then packaged into lysosomes, peroxisomes, or secretory vesicles that then carry then to their correct destination.

Section 4: Signal transduction and the control of cell growth, cell function and cell death
(1) Signal transduction. Cells must actively respond to their environment and to their internal milieu in order to survive and function. To do this, they have evolved an internal communication network that is loosely referred to as signal transduction. Proteins traversing the plasma membrane (receptors) acquire information from the extracellular space and relay this information into the cytoplasm. Information available in the extracellular space includes; (i) contact signals from neighbouring cells, (ii) chemical signals produced by nearby cells (known as paracrine signals), and (iii) blood-borne chemical signals produced by cells in distant parts of the body (known as endocrine signals). The chemical signals that specifically bind to receptors are known as ligands, and the act of a ligand binding to a receptor is known as ligation. Examples of ligands include; growth factors (polypeptides that promote the growth and survival of particular cell types), cytokines (specialised growth factors that act on cells of the immune system) and hormones (which may regulate particular physiological processes).

Following ligation, regions of a receptor that protrude into the cytoplasm pass information to cytoplasmic molecules and thus initiate signalling cascades. The key concept of a signalling cascade is that the initial signal from a receptor is amplified by the stepwise activation of a number of further signals inside the cell, much like the formation of an avalanche from a small snowball. The cascade of signals may have several effects such as; (i) activation of transcription factors that alter the expression of genes, (ii) control of enzymes that actively produce a molecule for secretion from the cell, (iii) control of cell division, and (iv) activation of cell suicide (described below). Frequently, the signals from several receptors interact to form signalling networks. These networks serve to integrate all the signals impinging on the cell to determine many aspects of cell function. There are several types of receptor. Some of the most important are discussed briefly below; (i) Growth factor receptor kinases. A kinase is a type of enzyme that adds phosphate

groups to a substrate (see diagram below). When the extracellular region (extracellular domain) of this type of receptor is ligated by a chemical signal, the intracellular region (cytoplasmic domain) of the receptor adds phosphate groups to (phosphorylates) specific cytoplasmic proteins. These in turn phosphorylate further proteins to initiate a signalling cascade. Kinases that phosphorylate proteins on tyrosine amino acids are known as tyrosine kinases. Enzymes called phosphatases remove the added phosphates and serve to control the signalling cascades initiated by the receptors (see below). (ii) Growth factor receptors linked to kinases. These do not have any kinase activity of their own, but their cytoplasmic domains bind to and activate kinases in the cytoplasm. Sometimes they do this through a set of intermediate molecules known as adaptor proteins.

(iii) Ion channel receptors. These receptors are coupled to an ion channel, so when ligated they allow passage of the ion across the membrane. (iv) G-protein-coupled receptors. These evolutionarily ancient receptors often span the membrane seven times, and so are also called 7-transmbrane spanning receptors. These receptors activate nucleotide-containing molecular complexes called G-proteins, which in turn activate other enzymes (eg. they frequently activate an enzyme known as adenylyl cyclase) that mediate downstream events in the cell. Gprotein-coupled receptors are responsible for smell and taste sensation and for the response of cells to adrenaline. (v) Cytoplasmic receptors. These are receptors for lipid-soluble ligands that can traverse the plasma membrane. Some of these receptors act directly as transcription factors themselves (eg. the steroid hormone receptors). (2) The cell cycle.

All cells have a similar life cycle known as the cell cycle (see the diagram below). Different cell types spend different amounts of time in each phase of the cell cycle.

Non-dividing cells (such as many of the stable long-lived cells in the human body) are sometimes considered to be outside the cell cycle and are known as quiescent. Alternatively, they are sometimes confusingly referred to as being in the Gap-0 [G0] phase of the cell cycle. Some but not all G0 cells are able to re-enter the cell cycle. For example, the G0 cells that line blood vessels are normally quiescent, but can re-enter the cell cycle to allow new vessels to grow, if and when required. The active phases of the cell cycle include the Gap-1 [or G1] phase, the S phase (when DNA synthesis occurs), the Gap-2 [or G2] phase, and the M phase (when mitosis occurs - see below). Cells that are actively moving through the cell cycle are said to be proliferating. The control of the cell cycle is complex, and involves the interaction of a large number of proteins. The major proteins involved in controlling the cell cycle are the cyclins and the cyclin-dependent kinases (Cdks). Together, these regulate the progress of a cell from G1 to S or G2 to M. A protein known as p53 functions to block the cell cycle if the cell is damaged, presumably to allow time for repair. (3) Cell division. During the M phase of the cell cycle cell division occurs. Cell division is made up of two sequential events. First, the nuclei of the cell divides by a process known as mitosis. Then, the cytoplasm of the cell divides by a process know as cytokinesis. The key feature of mitosis is that the two daughter cells are provided with a complete copy of the genetic information of their parent (ie. the two daughter cells inherit copies of both the maternally-derived and the paternally-derived chromosomes possessed by the parent cell). To achieve this, all of the chromosomes of the parent cell are duplicated before cell division takes place to form sister chromatids (as discussed in the previous lecture). During the phase of mitosis called metaphase, the sister chromatids align on a structure called the equatorial plate. During the phase of mitosis called anaphase, sister chromatids separate to form the chromosomes of the two daughter cells. Newly formed cells in G1 and G0 may grow in size

and become specialised to serve a particular function, or may themselves divide to form yet more daughter cells. (4) Meiosis. Meiosis is the process by which a small number of cells, most commonly in the reproductive organs of multi-cellular organisms, divide to form gametes. The key feature of meiosis is that each daughter cell obtains only have half the genetic material of the parent cell (ie. only one copy of each chromosome). In meiosis there are two cell divisions. In the first division, the parent cells homologous chromosome pairs are separated. (To put this in another way, the parent cells maternally-derived and paternally-derived chromosomes are separated from one another). In the second division of meiosis, the sister chromatids are separated, much as they are in mitosis. The end result is a set of four daughter cells, each carrying half of the parent cells genetic material. During meiosis, three processes occur which enhance genetic variability. (i) During the first division of meiosis, there is an opportunity for a few segments of each maternally-derived chromosome to be swapped with the corresponding segments of each paternally-derived chromosome. This swapping promotes genetic variability, and is known as crossing over. Crossing over occurs by a process known as homologous recombination. (ii) When the chromosomes are divided into four parcels by the two meiotic divisions, they are assorted independently into the four daughter cells. (iii) Which of the four daughter cells contributes to a new organism (fertilisation) is, by and large, random.

(5) Cell death. The death of cells in a multi-cellular organism can occur in two main ways. (i) Necrosis. This occurs when cells are killed by overwhelming damage that cannot be repaired. Often this form of death kills large numbers of adjacent cells in a tissue (eg. the death of heart muscle during a heart attack). (ii) Apoptosis. This is the suicide of a cell, and is sometimes given the alternative name of programmed cell death. At first it may seem counter-intuitive that cells commit suicide. However, suicide of cells is essential to; shape tissues during development, to maintain the correct numbers of each cell type, to remove immune cells that incorrectly attack their host organism, and to remove damaged, cancerous (transformed) or virallyinfected cells. Apoptosis is regulated by complex intracellular and extracellular signalling networks. Important apoptosis regulators in extra-cellular signalling networks are Fas and the tumour necrosis factor (TNF) receptors. Important apoptosis regulators in intra-cellular signalling networks are the Bcl2 family, APAF-1 and cytochrome C. Many of the intracellular signalling networks that control apoptosis reside on the mitochondrial membranes. When cells die by apoptosis, they have a characteristic appearance of nuclear and cytoplasmic shrinkage and membrane blebbing (see the time-lapse pictures of cells dying by apoptosis, below). The key enzymes that kill a cell during apoptosis are known as caspases. Once dead, apoptotic cells are removed (phagocytosed) by cells of the immune system known as macrophages.

Section 5: Building bodies the concepts behind complex organisms

The remaining two lectures cover the evolution, structure and development of multicellular eukaryotes (briefly!) The Visible World. The incredible diversity of life as we see it today is a relatively recent development in evolutionary terms. Life emerged around 3.5 billion years ago in the form of prokaryotes. These had the world to themselves for the next 2 billion years. This has been termed The Age of Prokaryotes a little misleading, as prokaryotes are still very much with us. Around 1.5 billion years ago, the first eukaryotes evolved. However, it was only 0.5 billion years ago that multi-cellular eukaryotes arose. The incredible diversity we (literally) see around is a product of the last 500 million years of evolution, less than one-sixth of evolutionary time to date. Cell size.

The evolution of visible organisms has involved limited increase in cell size and is mostly explained by the increase in cell numbers. The needs of cell groups. There are many needs for groups of cell to be able to form a complex organism. These include: Cell-to-cell structural linkages. Cell-to-cell communication over short and long distances.

-A sense of self. -An ability to specialise. The ability to co-ordinate cell division and specialisation during development. The remaining two lectures deal with these aspects. Dictyostelium discoideum. An example of some of the needs of a multi-cellular organisms are given by Dictyostelium discoideum. This remarkable organism spends much of its time as a free-living amoeba. It happily divides and proliferates until put under stress, the most common being starvation. -

When starved, the cells begin to aggregate towards signalling centres. These centres are composed of groups of cells that produce a diffusible signal molecule, cAMP. Initially there are multiple signalling centres. As aggregation continues, smaller signalling centres are turned off by the stronger ones and, eventually, one centre dominates. The amoebae aggregate to this centre to form a slug. The slug migrates, then differentiates into spores, a spore case and the stalk, to lift the spore case above the medium. Released spores can survive for up to several years, before hatching into a free-living amoeba. This organism represents many of the problems which face more complex multicellular organisms; the need for long distance signalling (cAMP in aggregation), cell-to-cell adhesion (after forming a slug), cell-to-cell signalling over short distances (co-ordinating slug movement), cell-to-cell sliding (during slug movement and spore development) and cell specialisation (into stalk, spores and spore-case). Question: How is it possible to explain, in terms of Natural Selection, the altruism of the stalk amoeba, given that they will not pass their genes on in the spores?

Linking Cells Together.

For cells to be able to work together they need strong cell-cell connections to produce groups of cells with similar functions (tissues) and collections of tissues to form organs. These connections must be firm, but flexible and permit short distance cell-to-cell communication and communication between different organs at greater distances, to synchronise their activity.

Cell Adhesion Molecules (CAMs) are responsible for holding the cells of an organism together. These form bonds between adjacent cells and between cells and the extra-cellular matrix that surrounds tissues. Cell-to-cell linkage. There are five main classes of CAMs involved in cell-cell linkages; Cadherins, Igsuperfamily, Selectins, Mucins and Integrins. These can form linkages between similar cells (Homophilic) and dissimilar cells (Heterophilic). Of the above classes, 2+ Cadherins and Selectins require Ca to be functional and the addition of a calcium ion sequestor (such as EDTA) to an 8-cell Xenopus embryo in an isotonic solution (together with a breaker of disulphide bridges, such as beta-mercaptoethanol) will cause the embryo to dissociate into individual cells. Removing the EDTA by addition of excess calcium will permit the cells to reform into an embryo and development to continue. Gap Junctions. Direct cytoplasmic contacts between cells in regions of close membrane association between adjacent cells are called Gap Junctions and allow the free flow of small metabolites.

The 12 connexin subunits form a pore with a diameter of 2-3nM. This potentially allows cells that are mutant for a particular gene to survive if there are wild-type cells surrounding the mutant. This is an example of a Genetic Mosaic. In mammalian females (who carry two copies of the X chromosome) one of the two copies is shut-down early in development. It is random as to which copy is shut down. A female who carries one copy of a mutation on one of the X-chromosomes will become a mosaic for cells expressing the wild-type gene product and cells expressing the mutant form. Tortoise-shell cats (all female) are an example of this. Cell-to-Matrix linkage. In animals, epithelia and most organised tissues are surrounded or underlain by an extracellular matrix of collagen, fibres, proteoglycans and multi-adhesive matrix proteins. The Matrix organises cells into tissues, helps to coordinate their cellular functions and the Matrix also acts as a route to transfer long distance signals to the tissues. Membrane-bound CAMs bind to components of the Matrix. The main type of CAM that binds to the Matrix are Integrins, although Selectins and syndecans are also relevant. Integrins come in two forms, alpha and beta with a functional integrin being a heterodimer of an alpha and beta subunit. In mammals, there are at least 17 alpha and 8 beta subunits, producing at least 22 known heterodimers. The particular combination determines what component of the matrix the Integrin binds to. When necessary, the rigid connections between cells and the matrix can be broken. Anexample of this is the migration of keritinocytes to a wound area to assist in closing it. There are two ways that this can be achieved; for example disintegrins bind to theRGB amino acid motif of integrins and prevent their binding to the Matrix (snakevenom also

includes disintegrins) or proteases can degrade matrix components. The Matrix.The Matrix is formed from a range of components, including lamins, fibronectins,glycoaminoglycans and proteoglycans. These form a network within the matrix. Themain protein component of the Matrix is Collagen.Collagen is an important flexible protein used throughout the body that gives tissues agreat deal of their strength.

Collagen is produced in a precursor form called procollagen and prolyl hydroxylases are needed to convert it to mature collagen. The failure of these enzymes prevents the collagen being sufficiently hydroxylated to form a stable triplex at room temperature. An essential co-factor of these enzymes is ascorbate (Vitamin C) and the lack of it causes a condition called scurvy, a major problem for early sailors. This leads to weakness in the tendons, skin and blood vessels. The solution was to take a stock of oranges on long voyages.

Cell-to-cell communication.
Coordination between the different specialised tissues of a complex organism and also between cells within those tissues is essential for an organism to function. This requires communication between adjacent cells to co-ordinate functions within a tissue and communication between the different organs, to maintain the organism. We have already explored intra-cell and cell-environment signalling (see above) and much of this applies to signalling in a multi-cellular organism. There are four major forms of signalling: -Endocrine. -Paracrine. -Autocrine. -Signalling between plasma membrane attached signals. (a) Endocrine signalling.

Endocrine: Hormones act a distance and are usually carried to their targets by the blood system. An example is the control of blood glucose level by insulin. Insulin stimulates the uptake of glucose from the blood into cells. (b) Paracrine signalling.

Paracrine: Signal molecules transmit across relatively short cell-to-cell distances. An example of this is the transmission of an electric impulse in the nerve axon from the nerve cell to a different cell by release of neurotransmitter across the synaptic gap. (c) Autocrine signalling.

Autocrine: The signal molecule is released from the cell and received by receptors on the surface of the same cell. Many growth factors work in this fashion and unregulated expression of signal or constant activation of the receptor in the absence of signal are often involved in tumour growth. An example is Epidermal Growth Factor. (d) Signalling by plasma membrane-attached proteins.

Plasma membrane attached signals. Very localised signalling between cells where both signal and receptor are bound to their respective cells. An example is in Notch signalling in fruit fly neural development.

A sense of self
Once multiple cells are involved in working together, it becomes important to be able to recognise what is self and what represents a potential invader or pathogen (or even dinner!). Different organisms use different methods or a mixture of methods: Lectins are proteins that recognise cell-surface carbohydrates. Antibodies can also recognise a range of cell-surface and intra-cellular molecules. In humans, the Major Histocompatibility Complex (MHC) is involved in selfrecognition

Antibodies: Antibodies bind to the sugars on the surface of a bacterium and T-cell lymphocytes identify the bound antibodies and destroy the invading cells. Major Histocompatibility Complex.

Class I are the transplantation antigens and are responsible for tissue rejection.Class II are involved in T- and B- cell communication.Class III are known as complement proteins. Intra-cellular invaders.The MHC complex is involved in the presentation of short degradation fragments ofproteins from within the cell. These are 9 13 amino acids and are screened by the T-cell lymphocyte system. Detection of unrecognised fragments by the immune systemleads to the destruction of the cell. In this way, viruses, bacteria and other invaders aredetected and the infected cell destroyed. There are, however, many ingenious waysaround this system.

Stem Cells
These are undifferentiated self-renewing cells. If they can give rise to an entire organism they are called totipotent. If can differentiate into any cell lineage, but they cannot themselves develop into an entire organism, they are called pleuripotent. Embryonic stem cells are an example of totipotent stem cells.

Section 6: Pathology
This lecture covers: Cell specialisation:

Function,

Development

and

Nerve cells Erythrocytes Spermatocytes The problem Aberrant cell division - cancer Xenopus early development Chick limb patterning Drosophila embryo Cell death in development

Development:

Pathology:

When all this goes wrong!

Cell Specialisation. Nerve Cells.

The human brain contains around 10 neurons, which process signals from sensory neurons before responding through motor neurons or through cells such as glands. Between the neurons in the brain are glial cells, which are able to modify the action of neurons. Dendrites connect to other neural or sensory axons and convert the chemical signal they receive into an electrical signal that feeds into the cell body. From there, an electrical signal may or may not be transmitted along the axon to the axon terminals, where a neurotransmitter may be released to pass the signal on to the next neuron. There are two signal types associated with neurons, electrical and chemical. Electrical signals transmit information along the length of the neuron (sometimes metres in length). This involves a rapid depolarisation of localised axonal membrane with a net change of around 110mV. The signal can propagate very rapidly up to 100m/s. These signals can be measured using an electrode.

12

The transmission of the signal from one neuron to another neuron or to another cell is through the release of a neurotransmitter across the synaptic gap. As the depolarisation 2+ reaches the axon terminal, it causes a localised rise in the concentration of Ca . This causes some of the vesicles in the axon terminal to fuse with the plasma membrane, dumping their

neurotransmitter into the synaptic gap. This diffuses across rapidly (around 0.5 milliseconds) and binds to receptors on the post-synaptic plasma membrane. This alters the ion permeability and can lead to a further action potential, a muscle contraction etc., depending upon the cell type.

Erythrocytes.

Erythrocytes (red blood cells) are highly specialised cells and are one of the few cell types to loose genetic information as they develop. In fact, mammalian (although not Avian) red blood cells have no nuclei. The major function of erythrocytes is to carry oxygen to the tissues through the capillaries and to remove waste carbon dioxide. They are packed with Haemoglobin protein, which binds and carries oxygen. The shape of the erythrocytes is also highly specialised.

The shape has two main function: i) ii) High surface area to volume ratio. This makes diffusion as efficient as possible a disc being the shape that has the greatest surface area to volume. The Erythrocyte is also able to deform to fit into capillaries that are even narrower than it is.

In systemic capillaries, where there is high [CO2] and low oxygen, erythrocytes take up CO2 across the plasma membrane and turn it into HCO3 by the action of carbonic anhydrase and the addition of an OH group from water. The proton produced from this reaction binds to haemoglobin, as it releases oxygen. The HCO3 (which is reasonably soluble in the blood plasma, unlike CO2) is exchanged with a Cl ion and leaves the Erythrocyte. This process is reversed when there is high O2 and low CO2, in the pulmonary capillaries. In sickle cell anaemia, the erythrocytes carry an aberrant form of Haemoglobin (S, rather than A). In the homozygous form, this generates spiky erythrocytes and is often fatal, through anaemia. In the heterozygous form, the Erythrocytes only deform under conditions of abnormally low oxygen concentrations and the disease is not fatal. Sickle cell anaemia is more prevalent in some African populations as the heterozygous form also gives resistance to malaria. This positive selection for the heterozygote under malarial conditions may explain why it is more common in these groups of people. Spermatocytes. Spermatocytes in mammals carry half of the genetic information needed to produce a zygote through fertilisation of a haploid egg. Spermatocytes are haploid (through the reductive division of meiosis) and are essentially powerful genetic information delivery machines.

The sperm can swim up to 3mm/minute actually 50 body lengths -and contain a warhead of digestive enzymes in the acrosome at the front of the sperm head. They also contain a battering ram of unpolymerised actin. This forms the acrosomal process that polymerises once the acrosome is activated. The whole is powered by one of the highest concentrations of mitochondria in any human cell type.

Some of the problems of development: -To create billions of cells from a single fertilized egg cell in a controlled mannersome 500 million million in an adult human -To create asymmetry from symmetry -To create specialised tissues and organs Cell lineage. As the fertilised egg cell develops, individual daughter cells of a single cell division may adopt different fates. Initially, the fertilised egg is pluripotent. This means that it is able to create any tissue in the organism (as it must!). As cell division continues, cell groups begin to loose that ability. There is no loss of genetic information in the majority of cases, just the loss of the ability to access the information Cell lineage is important in development, as certain cell groups are fated to become certain tissues in the adult organism. The strength of this fate depends upon the organism in question. For the worm, C. elegans, cell lineage is of over-riding importance. Individual cells obliterated by laser from embryos will eliminate all of the cell types that are derived from that cell there is no attempt by surrounding cells to compensate for their loss. For most animals, there is more flexibility and for genetic testing of human embryos, it is possible to remove a cell from an early stage (8 16 cell) embryo, without the baby having large parts of their bodies missing, as the surrounding cells compensate for the loss.

The blood cell lineage. The initial cell is able to create all of the final blood cell types shown. At each step downthe lineage, the number of fates open to the cell decreases, until there is finally only onetype of cells each can make. This does not generally involve a loss of geneticinformation (with the exception of the red blood cells), but the loss of the ability to usethat information. The recent cloning of animals has tried to reverse this trend and to resetterminally differentiated cells, to allow them to become pluripotent again.Despite the presence of the genetic information, this process of reprogramming is stillextremely difficult.By the final level, the fates have been decided and the cells are described as terminallydifferentiated. For the erythrocyte precursors cells, where there is only one possiblefate, the cells are called determined although they have not yet terminallydifferentiated. Acute Myeloid Leukemia.AML causes the final steps in the differentiation of the Myeloid precursor to fail. Thismeans that Erythrocytes, Platelets and Granulocytes are not produced, causinganaemia, bleeding and susceptibility to infection, respectively. Acute Lymphoblastic Leukemia.ALL is a failure of the Lymphoid stem cells to differentiate, causing loss of B and T-lymphocyte cells, which are both critical to the immune response and the prevention ofinfection. Unordered growth cancer. Cells sometimes need to be replaced. Intestinal cells have a half-life of a few days and red blood cells of around 100 days, although most cells almost never die or (in the case of brain cells) if they do they are not replaced. So the ability to divide needs to be maintained throughout the life of the adult organism for at least some cell types. Cells have multiple checks and controls on their ability to divide and proliferate. It requires mutations in multiple genes for individual cells to escape control and form a tumour. However, once control is escaped from, proliferation can be very rapid. Some tumours can be benign, but the most

dangerous cancers are those that have the ability to metastasise. Individual cancer cells are then spread through the blood and lymph system and become lodged in other tissue, to form new tumours.

In most cases, the cells maintain their position in the cell lineage at which they becamecancerous and will often express genes appropriate to those tissue types.One tumour type that does not, however, is a teratoma. Here the cells proliferate, butalso undergo differentiation to form final cell types, such as the cell which produce teeth,bone, hair etc.

Cancer emphasises the potentially catastrophic effects of uncontrolled development of cells and make the ability to generate a complete organism from a single fertilised egg all the more remarkable.

Examples of Development
Three examples are given which illustrate some of the principles involved in the patterning of an organism as it goes through development. Many of these principles are conserved across a wide range of organisms. Xenopus laevis is a rather large and unattractive member of the Frog family. It has the advantage of having large egg cells (around the size of small petit pois), which permit the physical manipulation of the resultant embryos. The chick embryo has been used for experimentation, largely because it is possible to physically manipulate the embryo. Drosophila melanogaster (black-bottomed fly.) is often the organism of choice for developmental work, as the entire genetic sequence is available, there are huge numbers of mutant lines available and it has a life cycle taking only 2 weeks. The first two examples

show how induction is central to producing different tissues from the early embryo. The third example is an example of how maternal information (placed into the egg cell during egg development) can effect development. Xenopus laevis.

The figure shows the early stages of Xenopus embryo development.

Initially, an inductive signal from the Vegetal (posterior) pole cells pushes the cells that receive the signal into the next stage of differentiation they have now lost their pluripotency. The second inductive step is from the Spemanns Organiser and further differentiates the Marginal zone into O, M1, M2 and M3. This principle is a critical one in Xenopus and there is evidence that a Window of Opportunity exists. If a signal is received during that window, it will cause the cells to adopt a certain fate. If not, they will adopt a default fate. For Xenopus, this appears to be epidermis. It is possible to show experimentally, that these inductions are actually occurring by transplanting the signal producing cells to a different place or by duplicating the signalling centres.

Chick Wing-Bud.

In the normal chick wing-bud, a region called the Zone of Polarising Activity (ZPA)acts as a signalling centre.This was demonstrated by grafting a second ZPA onto the wing-bud.

From the experiment, it is possible to see that the cells of the wing-bud differentiate according to the position that they find themselves in the signal gradient created by the ZPAs. Thus, a very complex series of developmental events are initiated by a very simple gradient of chemical. For the chick wing-bud, the main signal has been identified as the product of a gene called Hedgehog. Replacing the ZPA will plastic beads impregnated with Hedgehog gives a similar effect as the transplantation, but it is not as complete. Drospophila melanogaster.

Two hours after fertilisation, there are 2,000 nuclei, but only one cell. The advantage of this is the very rapid division of genetic material, without the slower process of producing completed cells. This leads to the nuclei floating in cytoplasm this is called a syncytium. The nuclei finally migrate to the edge of the egg and are enclosed in plasma membrane to form cells at around the 3 hour mark and by 4.5 hours, there are 5,000 cells. Bicoid is a gene that starts the patterning process in Drosophila. The mRNA from this gene, produced by the mother cells, is localised at one end during egg development. When the egg is fertilised, the mRNA is translated and a gradient of the bicoid protein forms. Individual nuclei within the syncytium sense where they are in this gradient and express genes according to their position in that gradient.

Apoptosis - This has been mentioned in earlier lectures as programmed cell death. In
C. elegans, there is a mutation called vulvaless. This involves the worm failing to develop a vulval opening. The consequence of this is that the eggs hatch within the worm and the young eat the adult from the inside. This is an effect of a failed apoptosis, which would normally create the vulva opening.

Pathology this is most broadly defined as the scientific study of disease. It is the
fascinating study of what occurs when the processes you have learnt about in these notes go wrong!