Jig's Tissues

Lecture 1: Epithelial cells 1) Demonstrate the following on a suitable transmission electron micrograph: nucleus; nucleolus; nuclear envelope; mitochondrion; rough endoplasmic reticulum; smooth endoplasmic reticulum; ribosomes; Golgi apparatus; secretory granules; plasma membrane; cytoskeletal components.

2) Describe the three main components which constitute the cytoskeleton.

• The cytoskeleton is a group of polymers which form structurally important cytoplasmic components

Microtubules – made from tubulin (polymers of α and β tubulin) , ~20nm diameter; involved in cell shape and act as ‘tracks’ for the movement of other organelles and cytoplasmic components within the cell. It is also the major component of cilia and flagella.

Intermediate filaments – made from filamentous proteins which from rope-like filaments. The type of IF a cell has is characteristic of cell type e.g. epithlia have cytokeratins, mesenchymal cells have vimentin, neurones have neurofilaments protein. The IF gives mechanical strength to the cell, links desmosomes (cytokeratin), and stabilising the nuclear envelope (nuclear lamins).

Microfilaments – made from actin; involved in cell shape and movement (e.g. muscle contractility) and associates with adhesion belts and other plasma membrane proteins. Accessory proteins, e.g. myosin, act with actin to control actin organisation and cell movement.

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Cytoskeleton organisation is highly labile, and is controlled by many factors. Re-organisation occurs during cell locomotion, cell division etc 3) Explain the following terms used to describe features of epithelial cells: apical surface; basolateral surface; brush border; microvilli; cilia; cell junction; basal lamina (basement membrane). Demonstrate them on suitable transmission electron micrographs.

• An epithelial sheet has two faces; the apical surface is free and exposed to the air or to a watery fluid; the basolateral surface rests on some other tissue – usually a connective tissue – to which it is attached.

• Supporting the basal surface of the epithelium there lies a thin tough sheet of extracellular matrix, called a basal lamina, composed of a specialised type of collagen (Type IV collagen) and various other molecules. The basal lamina acts to isolate the epithlial cells from the mesenchyme /connective tissue, it also acts for cell adhesion.

• The apical and basal faces of an epithelium cell are as a rule chemically different, reflecting a polarised internal organisation of the individual epithelial cells: each one has a top and bottom with different properties.

• The baso lateral surface is the basal and lateral surface (often grouped together as they have similar properties.

• Intestinal epithelial cells have microvilli which are projections from the apical surface of an epithelial cell that is supported by a central core of microfilaments associated with bundling proteins such as villin and fimbrin. The brush border is the densely packed microvilli on the apical surface of, for example, intestinal epithelial cells.

• Cilia are motile appendages of eukaryotic cells that contain an axoneme, a bundle of microtubules arranged in a characteristic fashion with nine outer doublets and a central pair (‘9+2’ arrangement).

• Cell junctions are specialised junctions between cells which give epihelia mechanical integrity and act to seal the intercellular pathway, allowing layers of epithelia to form.

4) Describe the major types of cell-cell junction in an epithelium.

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Epithelial cells make organised, stable cell-cell junctions to form continuous cohesive layers; have a distinct apical surface at the luminal surface, and a basal surface in contact with the ECM; the lateral membrane is the membrane running between.

Cell-cell junctions give the epithelium mechanical integrity and seal the intracellular pathways There are two forms of junction, zonulae (belts) or maculae (spots) Tight junctions (zonulae occludens) – near the apical membranes, seals paracellular pathways and segregates apical and basolateral membrane polarity Adhesion belt (zonulae adherens) – situated just basal to the tight junction; composed of a trans-membrane adhesion molecule called cadherin, which associates with the microfilament (actin) cytoskeleton; this controls the stability of the other junctions (the master junction) Desmosome (macula adherens) – found at multiple sites between adjacent cells, composed of a cadherin like trans-membrane molecule linked to the intermediate filament cytoskeleton, providing good mechanical strength between cells Gap junction (macula communicans) – consists of clusters of pores formed from 6 identical subunits in the membrane that are continuous with membrane pores, allowing passage of ions and small molecules between cells; passage is dependant on pH, [Ca2+], voltage etc, by opening/closing pores Synapse – mainly in neural tissue; information is passed one-way via neurotransmitter signalling system Explain the meaning of the term “extracellular matrix”.

ECM = The material deposited by cells which forms the insoluble part of the extracellular environment

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It is composed of fibrillar proteins (collagen, elastin) embedded in a hydrated gel (made from proteoglycans); can be poorly organised (loose connective tissue) or highly organised (bone, tendon, cartilage).

Note tissue = cells + ECM

Lecture 2: Epithelial tissues 1) Explain the importance of cell polarity in function of epithelial tissues.

• Epithelial processes i.e. secretion, absorption are usually unidirectional, and polarity is required for this to occur – cell junctions separate the epithelial membrane into apical and basolateral domains, which are biochemically and functionally distinct.

2) Classify individual cell examples within the following categories: epithelial; mesenchymal; haematopoietic; neural.

Recognise the originating cell type of the following types of neoplasm: carcinoma; sarcoma; lymphoma; leukaemia; neuroblastoma; glioma.

• Epithelial cells – cells forming continuous layers, these layers line surfaces and separate tissue compartments; have a variety of functions, for example transport, absorption, secretion and protection.

• Mesenchymal cells – cells of connective tissues e.g. fibroblasts.

• Haematopoietic cells – blood cells and the cells of the bone marrow from which they are derived.

• Neural cells – cells of the nervous system having two main types – neurones (for transmission of nervous impulses) and neuroglia (support cells e.g. astrocytes etc.).

• Carcinoma – epithelial cells; sarcoma – mesenchymal cells; leukaemia – bone marrow cells; lymphoma – lymphocytes; neuroblastoma – neurones; glioma – neuroglia.

3) Discuss the ways in which the cellular organisation of epithelia may be specialised for absorptive, secretory or protective functions.

• Absorptive cells have numerous hair-like projections called microvilli on their free surface to increase the area for absorption.

Often contain many mitochondria at the basal end for active transport.

• Secretory cells are found in most epithelial layers. These specialised cells secrete substances onto the surface of the cell sheet.

Secretory epithelial cells are often collected together to form a gland that specialises in the secretion of a particular substance.

Exocrine glands secrete their products (such as tears, mucus, and gastric juices) into ducts (through the apical layer). Endocrine glands secrete hormones into the blood (through the basal lamina). The secretory cells contain many secretory granules the apical cytoplasm, Golgi apparatus, lots of RER. • Protective cells, such as the skin, are usually stratified-squamous. The upper surface of the skin is a squamous cell surface which is the protective surface. Below this layer is the cuboidal basal layer containing stem cells for the renewal of upper layers.

4) Discuss the pattern of cell division found in different epithelia undergoing normal turnover, growth or regeneration.

• Prophase – chromatin fibres condense and shorten, to form the characteristic chromosome with double-stranded chromatids held together by a centromere; later the nucleolus disappears and the nuclear envelope breaks down.

• Metaphase – the chromosomes align along the metaphase plate.

• Anaphase – the centromeres split, separating the sister chromatids which move to opposite poles becoming daughter chromosomes; beginnings of cytokinesis.

• Telophase – daughter chromosomes uncoil to diffuse chromatin; re-formation of nuclear envelope, nucleolus etc.

• New cells are born from stem cells in the basal layer, they eventually lose contact with the basal lamina and move more outward, differentiating as they go

Lecture 3: Extracellular matrix 1) Give examples of the multifunctional role played by the extracellular matrix (ECM) in maintenance of structural and functional integrity in a wide variety of tissues.

• The ECM is a complex network of proteins and carbohydrates between cells, containing fibrillar and non-fibrillar components and is an essential component of all multi-cellular organisms • it determines mechanical/physiochemical properties of the tissue and is not inert – it reacts with cellular receptors and influences growth, adhesion and differentiation of the cells and tissues with which it interacts • it also provides structural and physical support – in the form of cartilage, bone, tendons and ligaments.

2) Explain the relationship between ECM and connective tissues. Describe some different forms of connective tissue, relating their function to the properties of the constituent ECM. • Connective tissue = extracellular matrix + component cells • cartilage is made from Type II collagen, which is made from 3 type II alpha chains which provides good tensile strength and stability • basement membrane is made from Type IV which forms a flat network shape • Type I collagen is the most abundant protein in the body and consists of 2 type I alpha chains and 1 type II alpha chain.

3) List the major components of the ECM and their derivations and properties. Describe the additional components which may be present in an acutely inflamed tissue.

• Collagens (fibrillar and basement membranes that surround the epithelium and capillaries); • Glycoproteins (for example fibronectins and laminins (basement membrane)) which can control cell migration and direction; • Proteoglycans (for example aggrecan, decorin and perlecan (basement membrane)) which are a specialised subset of glycoproteins • Cellular components are for example fibroblasts, which undertake synthesis of most ECM components, (and in cases of inflamed tissues macrophages and mast cells are also present)

4) Using collagen as an example, describe the synthesis of glycoproteins including the role played by the following organelles: endoplasmic reticulum, Golgi, secretory vesicles. Explain collagen fibril assembly and how different collagens assemble into distinct supramolecule networks with specific functions.

• Over 20 collagen types are known, designated by roman numerals. Each collagen molecule comprises three α chains.

• Type I collagen has chains from two different genes - its composition is [α1(I)]2 [α2(I)] • Types II and III collagen have only one chain - their compositions are, therefore, [α1(II)]3 and [α1(III)]3. (There are over 25 genes encoding collagens in mammals) • Transcription creates mRNA from the relevant genes, it reaches the ribosome and tRNA binds, the signal peptide is recognised by the signal recognition peptide and the complex then docks with the ER, translation, which was previosuly halted, now continues, and then the protein passes into the lumen of the ER through the activated protein translocator, where the signal peptide is cleaved.

• Collagen undergoes post translational modification, (hydroxylation of proline and lysine, this is catalysed by prolyl and lysyl hydroxylases which also require Fe2+ and vit. C), hydroxylation of proline and lysine allow for H-bond interactions and covalent crossbridges between the chains giving collagen its high tensile strength. These modifications occur in the golgi and before the 3 chains combine. Also glycosylation of selected hydroxylysines occurs.

• Each α chain is approximately 1000 amino acids in length, forming left handed helix, as mentioned before the collagen structure is actually a triple helix and in within this triple helix of α chains glycine is the only amino acid small enough to occupy the interior. At the stage at which the triple helix is formed, the α chains are actually pro- α chains as they still are yet to be cleaved.

• Once the triple helix is formed it is stored in secretory granules • On release by exocytosis, cleavage of the pro-peptides occurs (cleavage of the pro- α chains) to form tropocollagen • it is then assembled into collagen fibrils ranging from 10-300nm in diameter, arranged in a characteristic ¾:¼ stagger which provides mechanical continuity;

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fibrils then congregate into collagen fibres, of diameters between 0.5 and 3µm.

Not all collagens form fibrils – Type IV is a network forming collagen present in all basement membranes, although its molecular composition varies from tissue to tissue; it still maintains the traditional alpha helix structure, but it is interrupted at points along the chain giving kinks, which leads to a flat polygonal network structure.

5) Explain the structural and functional significance of the modified amino acids hydroxyproline and hydroxylysine within collagen.

• Proline and lysine are hydroxylated in the ER lumen by hydroxylase enzymes, which are dependant on Fe2+ and ascorbate, which occurs after the collagen is secreted; hydroxyl groups contribute inter-chain strength, allowing for hydrogen bonding; as such they increase the strength and stability of the collagen chain • (scurvy is lack of vitamin C leading to lack of hydroxylation of alpha chains, giving rise to weak collagens which are degraded leading to loss of collagen in the ECM).

Lecture 4: Connective tissues 1) Describe the properties of the soluble components of the ECM and summarise the molecular characteristics of glycoproteins, proteoglycans and glycosaminoglycans.

• Glycosaminoglycans are unbranched polysaccharide chains consisting of repeating disaccharides • Proteoglycans are core proteins which are covalently linked to one or more glycosaminoglycan chains. There are several proteoglycan families based on structural and functional characteristics: Aggregating (interact with hyaluronic acid) eg aggrecan; Small leucine rich eg decorin; Basement membrane eg perlecan, agrin; Cell surface eg syndecans 1-4 • glycoproteins have multiple domains for example integrin/fibrin receptors.

2) Describe the roles of glycoproteins in matrix assembly, cellular interactions and wound repair.

• Fibronectin derived from 1 gene, with around 20 variations arising due to alternate splicing at mRNA level; Large multidomain molecule (dimer = 500kD), capable of interacting with cell surface receptors and other matrix molecules

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There are no known mutations since it is essential to embryonic life; it is also important in regulating cell adhesion, migration in embryogenesis and tissue repair it forms a mechanical continuum with the actin cytoskeleton of many cell types in wound healing, it leaks from the blood vessels and coats the collagen, creating a temporary contained ECM with the fibrin mesh; this allows for the synthesis of the new membrane, repair via cell division followed by the dissolution of the temporary ECM.

Laminin -

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plays an integral part in the basement membrane and associates with other components such as type IV collagen, entactin and proteoglycans; consists of 3 chains of between 140-400kD – alpha, beta and gamma, assembled into a cross-shaped molecule:

its roles include interaction with cell-surface receptors such as integrins and dystroglycan; regulation of tissue differentiation, formation of cell-matrix junction and cell migration (e.g. neurite outgrowth).

Specific chain mutations associated with inherited diseases such as muscular dystrophy and epidermolysis bullosa

3) Relate proteoglycan structure to the function of specialised matrices and to the regulation of collagen fibril assembly.

• Proteoglycans are divided into several families depending on their structural and functional characteristics.

• Aggregating – e.g. aggrecan found in cartilage.

• Small leucine rich e.g. decorin in the regulation of fibril assembly.

• Basement membrane – e.g. perlecan and agrin.

• Cell surface – e.g. syndecans which act as receptors in growth factor signalling.

GAG is linked to a serine on the core protein via a link tetrasaccharide (xylose, galactose x2, glucuronic acid).

• Cartilage – consists of a hyaluronic acid/aggrecan complex; hyaluronic acid is unique from other GAG in that it has no core protein, is synthesised at the cell surface and is unsulfated; due to the sulphate groups on aggrecan, however, the matrix can retain water which is important in shock absorption – (arthritis results from a degradation of the hyaluronic acid/aggrecan complex).

• Blood vessels – versican is present is the aortic media and helps absorb shockwaves generated by the heartbeat.

Lecture 5: Fluid compartments of the body 1) List the main fluid compartments in the body, and give an estimate of the size of each.

• Intracellular fluid is 55% of body water.(23L) • Extracellular is 45% of body water (19L) and contains: Interstitial fluid is between cells and accounts for 36% of body water. (15L) Blood plasma contains 7% of body water. (3L) Transcellular fluid e.g. cerebrospinal, ocular and synovial fluid accounts for 2% of body water (1L) 2) List the main features of the composition of each compartment.

• Main extracellular cation in Na+. Main intracellular cation is K+.

• An important intracellular signalling ion in Ca2+.

• Main extracellular anion is Cl-. Main intracellular anions are organic phosphates.

• Proteins are intracellular anions in low concentration but have a high charge. _______________________________________________ in plasma*** in muscle ( mmol/l) ( mmol/l) ______________________________________________ Na+ + K 2

C+ aCl Organic phosphates 1Protein 17-

150 5

10 150

2 110 5

1045 130

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2

pH 7.4 7.1 ---------------------------------------------------------------------osmolarity 285 mosm/l 285 mosmol/l

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note also that the overall osmolarity of the two main compartments (intracellular and extracellular fluid) are the same!

3) Define “osmosis”. Explain how tonicity is different from osmolarity.

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Osmosis is the movement of water down its own concentration gradient. Osmosis moves water toward the area of higher osmolarity.

Osmolarity is a measure of the concentration of solute particles in a solution.

Tonicity defines the strength of a solution as it affects the final volume. Tonicity depends on cell permeability whereas osmolarity does not.

4) Define haemolysis. Describe the composition of solution that would cause haemolysis of red blood cells.

• The lysis of red blood cells with the release of their contents • Suitable haemolytic solution: low pH, low osmotic pressure (100 mOsm/kg or less) • Temperature variations (hot or cold) 5) Briefly describe the main mechanisms by which solutes exchange across cell membranes. For each mechanism give an example solute and state whether this is an active or passive process.

• Passive – down an electrochemical gradient (charge + conc.) through lipid: lipids, oxygen, carbon dioxide, steroid hormones. through pores: water, ions, urea. Some are gated and have open and closed states. Gated by chemical ligands and voltage. on carriers: binding of carrier to solute and then a conformational change. (Specific).e.g. facilitated diffusion – transport of lactic acid out of skeletal muscle cells into interstitial fluid.

• Active – can transport up an electrochemical gradient. on carriers: primary active transport. Uses ATP → ADP + Pi + energy e.g. Na/K pump. on carriers: secondary active transport. Uses “downhill” movement of one solute coupled to “uphill” movement of a different solute. Several examples of Na moving into cell and something else out.

• Endocytosis and exocytosis – encapsulation in membrane as solute enters or before it leaves the cell.It is a method used generally for larger molecules e.g. endocytosis of nerve growth factors (proteins) entering, e.g. exocytosis of peptide hormones from endocrine glands.

6) Describe the main types of exchange across the capillary wall, including exchange through endothelial cells and through the pores between endothelial cells.

• Transport can either occur through the endothelial cells themselves or through the pores between the cells.

• Through the endothelial cells: Lipid soluble substances pass through the endothelial cells; exchangeable proteins are moved across by vesicular transport. (note plasma proteins generally cannot cross the capillary wall.

• Through the pores: small water soluble substances pass through the pores between the cells.

7) Define oedema, and outline its causes.

• Oedema – swelling of a tissue because of excess interstitial fluid.

• Causes: 1. Imbalance of forces causing fluid to move between the a. blood plasma b. interstitium, and

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c. lymphatic vessels Increased permeability of capillary walls to plasma proteins.

Lecture 6: Nerve 1) Describe the structural components of a “nerve” and the function of each component.

• Divided into three distinct regions: • Cell body (soma) - Bound by a plasma membrane.

- Contains a nucleus defining the location of the soma.

- Contains the normal complement of organelles.

• Axon (nerve fibre) - Arises from the soma at a region called the axon hillock or initial segment. This is the region where the plasma membrane generates nerve impulses. The axon conducts these impulses away from the soma towards other neurons.

• Dendrites (receiving processes) - Shorter than axons and unmyelinated. Form receiving surfaces for synaptic input from other neurons.

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The neurons transmit signals, the neuroglia (glia-support cells), aid in metabolism and ionic balance, and these are nine times more numerous than the neurons themselves 2) Rapid signalling over long distances is the major function of neurons. Give some examples.

• Myelin is composed of 80% protein and 20% lipid giving high resistance and low capacitance acting as electrical insulators, the myelin is interrupted at intervals by nodes of Ranvier, where the nerve membrane is exposed to the external environment.

• Myelin – since ions cannot cross the lipid content of the myelin sheath, they spread passively down the nerve fibre until reaching the unmyelinated nodes of Ranvier. Nodes of Ranvier are packed with a high concentration of ion channels which, upon stimulation, propagate the nerve impulse to the next node. Therefore, the nerve impulse jumps from node to node along the fibre in a process called saltatory conduction.

• The diameter of the axon also plays a part in the velocity of the electrical impulse. The greater the diameter the faster the axonal conduction.

• The fastest conduction velocity occurs in the largest diameter nerve fibres, which are also myelinated ~ 120m/s, compared to Small diameter, non-myelinated axons ~ 1 m/s • In the CNS it is the oligodendrocyte that forms the mylein sheath and not the schwann cell of the peripheral system 3) Rapid, reliable signalling involves the following: resting potential, action potential, non-decremental spread. Define each of these terms and explain how each is involved in rapid signalling.

• Resting potential: Inside of the membrane is more negative than the outside. There is a membrane potential ranging from -60 to – 75 mV. The resting potential is maintained by the sodium/potassium pump which steadily discharges more positive charge from the cell than it allows in, and by the relatively high permeance of K+, which leaks out of the cell through its membrane channels faster than Na+ leaks in.

• Action potential: A local potential can be of any grade up to the threshold potential. At threshold, voltage-dependent sodium channels become fully activated and Na+ pours into the cell. Almost instantly the membrane actually reverses polarity, the inside acquiring a positive charge relative to the outside. This reverse polarity constitutes the nerve impulse.

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Non-decremental spread: The action potential moves along the axons without decreasing in size.

The positive charge flows through the cytoplasm, activating sodium channels the entire length of the fibre. This series of activations, by propagating the action potential along the fibre with virtually no reduction in amplitude, gives the nerve impulse its regenerative property.

4) Describe the function of myelin and draw a diagram showing its location in the peripheral and central nervous system.

Peripheral Nervous System

Central Nervous System

5) Define “synapse” and “synaptic transmission”. Explain how each is involved in signalling between neurons, or between neurons and another cell type.

• Synapse – specialised structure that forms junctions with other neurons and with muscle cells at the terminal of the axon, and sometimes along its length.

• Synaptic transmission – method of transmitting nerve impulses from one cell to another across a synapse.

• Two methods of synaptic transmission: • Electrical transmission: Far less common than chemical transmission. Transmission takes place through so-called gap junctions, which are protein channels that link cellular contents of adjacent neurons. Direct diffusion of ions through these neurons allows the action potential to be transmitted with little delay or distortion.

• Chemical transmission: Depolarisation of the pre-synaptic membrane terminal and presence of Ca2+ ions in the extracellular fluid is essential for neurotransmitter release from the pre-synaptic terminal.

• The membrane of the pre-synaptic terminal contains voltage dependent Ca2+ channels which open on depolarisation by a nerve impulse allowing Ca2+ entry. In some way, this facilitates fusion of synaptic vesicles containing neurotransmitter substance with the nerve terminal membrane, thus releasing neurotransmitter in the synaptic cleft by exocytosis. The quantal release of neurotransmitter has a critical influence on electrical potential created in the post-synaptic membrane.

• Neurotransmitter binds to receptor proteins on the postsynaptic membrane bringing about a sudden change in permeability to specific ions (Na+ usually). There is therefore a change is electrical potential across the membrane.

• Excitatory post-synaptic potentials (EPSPs – usually due to Na+ influx) bring the membrane potential towards the threshold for an action potential. Inhibitory post-synaptic potentials (IPSPs – influx of Cl-) make the inside more negative and therefore the potential is brought away from the threshold.

Lecture 7: Muscle 1) Draw a sketch of diagrams showing how skeletal muscle shortening can cause joint flexion and joint extension.

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List the main similarities and differences between the following types of muscle: skeletal muscle, cardiac muscle, smooth muscle.

Skeletal 50-150 μm x a few mm up to many mm.

Sarcomeres have banding Fibres electrically independent of each other. Fibres attached at ends to tendons. Multinucleate.

Cardiac Approx. 20 x 100 μm.

Sarcomeres have banding.

Cells mechanically & electrically connected to each other, forming a hollow organ.

Also special cells for conducting impulses.

Smooth Approx. 10 x 100 μm.

No sarcomeres. Smooth, but containing thick & thin filaments. Cells mechanically &, in some tissues, electrically connected.

Innervation

Motor neuron end-plate on every cell. Synapses are excitatory only.

Contraction

Force &/or shortening. Twitch & tetanus.

Force in vivo depends on number of active motor units, freq. of excitation etc.

Neurogenic.

Conducts A.P.s (all-or-nothing) arising only at synapse. Not influenced by circulating hormones. No autonomic innervation.

No motor neurons.

Continuous influence from sympathetic (excitatory) & parasympathetic (inhibitory) neurons.

Twitch only (fairly long duration compared to twitch of skeletal muscle). No tetanus.

Strength of contraction graded by many inotrophic effects & by muscle length. Myogenic.

Spontaneous A.P.s originate in pacemaker cells & are conducted between muscle cells & via specialised conducting cells.

Multi-unit: Autonomic neurons, excitatory & inhibitory. Synapse for each muscle cell.

Single unit: Influenced by neurotransmitters released locally from autonomic neurons, but no real synapses.

Speed of shortening is slow, but can shorten large distance.

Myogenic or neurogenic in origin.

Structure

Membrane properties

Multi-unit: A.P.s conducted only within unit, following nerve A.P. Single-unit: Membrane potential & excitability vary continuously. A.P.s conducted from cell to cell if sufficiently excitable. Modified by autonomic neurone activity, local conditions & circulating hormones etc.

3) Describe the main sub-cellular structures in muscle and outline their functions (include the thick filament, thin filament, sarcoplasmic reticulum, transverse tubules, mitochondria).

• The myofibril within a muscle fibre contains thick and thin filaments which overlap forming bands of darks regions and light regions.

• Two systems separate adjacent myofibrils: • Transverse tubules: Series of channels that open through the sarcolemma to the extra-fibre space. T-tubule system is a network of interconnecting rings, each of which surrounds a myofibril; Provides an important communication pathway between the outside of the fibre and the myofibrils.

• Sarcoplasmic reticulum: Series of closed sac-like membranes. Each segment of the SR forms a cuff-like structure surround a myofibril; Action potential in the sarcolemma and T-tubule causes release of calcium from the terminal cisterns of the SR. Ca binds to filaments and permits cross-bridge attachment.

• Force production and filament sliding by: - cycles of cross-bridge attachment, - conformational change, and - detachment.

- In each cycle, ATP ADP + Pi + energy, with the ATP coming from the mitochondria.

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Explain how the force a muscle produces can be varied to match the task to be performed.

By varying the number of motor units that are active we can vary the force.

The functional unit in skeletal muscle is the “motor unit”. Describe what this term means. What is “recruitment”?

Motor unit – a motor neuron and all the muscle fibres it innervates (has synapses with). The functional unit of normal skeletal muscle.

Recruitment – vary the number of motor units that are active (and thus vary the force).

Lecture 8: Signalling between cells 1) Give examples of why cells in a multicellular organism need to communicate with each other. Provide specific examples of communication between tissues and within a tissue.

• Cells need to communicate, in order to respond to changes in the environment; for example, when you’re watching a scaring film, for example Ghostbusters, the brain detects fear and increases sympathetic nervous activity to the heart; this causes the individual cardiac myocytes to contract faster.

• Essential for coordination of multicellular organism (though even unicellular organisms recognise signals) • Can happen at various levels: - hormone signals to whole organism (e.g. metabolic) - specific signals to targets (e.g. nerve→muscle) - local signalling (e.g. response to infection) - contact signalling (e.g. growth control)

2) Explain with examples the different modes of intercellular signalling: endocrine, paracrine, autocrine, signalling by membrane attached proteins.

• Endocrine – a hormone is released into the bloodstream and acts on a target cell away from it; • Paracrine – involves a localised system that stimulates cells in the immediate microenvironment (signals diffuse locally in the extracellular matrix e.g. inflammatory mediators, growth factors) • Autocrine – is a special case of paracrine signalling, as it has an effect on the microenvironment of its immediate source; • Signalling can be via contact dependant membrane bound signalling proteins. A cell signalling to its immediate neighbour. The signal can pass directly from one cytoplasm to the next via gap junctions, or by the extracellular domain of a membrane protein on one cell binding to another extracellular domain on the second cell e.g. growth control within an epithelium, some developmental signals 3) Explain how an extracellular signal is transmitted intracellularly and can involve a cascade of events.

Direct access to cytoplasm • Hydrophobic (e.g. steroid hormone) • Can cross cell membrane • Intracellular receptors – control DNA expression Indirect access to cytoplasm – internal signal • Either ion flux or enzyme creating second messenger (direct or indirect) • The ligand mediator binds to the receptor, which activates a second messenger; the second messenger is released the cascade follows: signal integration, signal amplification, modulation by other pathways, and regulation of divergent responses such as regulation of a metabolic pathway, gene expression, or promote changes in the cytoskeleton.

4) Define the terms: receptor, ligand, second messenger.

• Receptor – a protein which binds a specific ligand, thus receiving the signal.

• Ligand – a molecule released by one cell which transmits the signal between cells.

• Second messenger – molecules produced intra-cellularly by the activated receptor.

5) Explain the role of receptors in signal transduction: why they are necessary for the relay of specific extracellular signals within the cell and how they define the specificity of the response with respect to cell type and response.

• Signalling molecules are recognised quite specifically by proteins called receptors • These have a ligand binding site which recognises part of the molecular structure of the ligand (signal) • The receptor with bound ligand will then be able to function to cause an effect on the target cell • Ligand-receptor interactions are the targets of many drug actions. Drugs that will bind the receptor and cause the same effect as the natural ligand are called agonists. Drugs that bind the receptor in such a way that they cause no effect but stop the binding of the natural ligand are called antagonists • Receptor proteins allow the transduction of signals carried by hydrophilic messengers into the cell; the receptors are specific to one type of messenger, and are linked to second messengers which can selectively determine the response to the stimulus.

6) Distinguish between direct and indirect activation of second messengers Direct • E.g.growth factor receptors (insulin, epidermal and platelet derived) which act as a tyrosine kinase i.e. add a phosphate to the -OH group of a tyrosine amino acid in a protein • Dimeric ligand causes receptor molecule to dimerise and the two receptor proteins become activated to phosphorylate one another • Other proteins that recognise phospho-Tyr bind the receptor and become phosphorylated themselves

Indirect • The G proteins involved in this pathway are heterotrimeric, with α, β, and γ subunits (there are also other kinds of G proteins in other signalling pathways) • Only the α subunit binds a guanine nucleotide (either GTP or GDP) • The G protein coupled receptors form a molecular family of membrane proteins

7) Provide examples of second messengers. Describe how calcium ions and cyclic AMP act as secondary messengers.

Can be: • Increase in something normally kept at low concentration e.g. Ca2+ • Generation of molecule only used as a signal., e.g. cyclic AMP, IP3 • Modification of a normal molecule, e.g. phosphorylation of a protein • There must be a mechanism that removes the second messenger, so the intracellular signal decays once the extracellular signal stops Ca2+ • ATP-dependent Ca2+ pump in the cell membrane keeps intracellular concentration around 0.1 µM (external about 1 mM) • A rise in [Ca2+] to around 1 µM can be sensed by various cellular processes. Many involve the calcium sensitive protein calmodulin and/or Ca2+-dependent protein kinases.

• After the signal has terminated, [Ca2+] returns to normal levels by the action of the Ca pump cAMP 1. Activation of receptor 2. Activation of G-protein 3. Formation of cAMP 4. Activation of protein kinase A 5. Activation of CREB in the nucleus 6. Transcription of target genes and synthesis of target proteins 8) Describe how protein phosphorylation and dephosphorylation can regulate intracellular signalling cascades. Define the terms protein kinase and protein phosphatase, and describe the 3 main subgroups of each on the basis of the amino acids which are phosphorylated.

• Protein kinase phosphorylates proteins, which activates them • phosphatase de-phosphorylates them, turning them off; however some proteins may be deactivated by phosphorylation • once one protein is activated, it can stimulate another to be phosphorylated, an so on, formed a signalling cascade which can be inhibited at any level and therefore by a variety of factors.

• Protein kinase – enzyme which phosphorylates a protein using ATP • protein phosphatase – protein which de-phosphorylates a protein, yielding one molecule of inorganic phosphate.

• 3 amino acids capable of phosphorylation are serine, tyrosine and threonine 9) Discuss in general terms the globular cellular responses which are regulated by signalling pathways.

• Globular cellular responses keep the cell informed on how it should behave; global responses are either to survive, to divide or to differentiate; without a response, the cell will tend to die, but there possibly exists a positive globular response that induces cell death.

10) Understand the mechanisms of signalling and down regulation.

Amplification

Down Regualtion