SSN SBPM Workshop~ Exam 2 Handouts **Last year’s histology handouts are available at http://cpmcnet.columbia.edu/student/ssn/histology/block2.htm Function A. Cell Cell Adhesion Molecules Cadherins Ig family Selectins Integrins cell-cell interactions cell-cell interactions cell-cell cell-ECM interactions cell-cell interactions temporary attachment and anchoring Structure 1-pass transmembrane glycoprotein 1-pass tm protein with repetitive extracellular Ig-like domains 1-pass tm protein with several repeated domains, and a lectin (carbobinding) domain - 1-pass alpha beta heterodimers - 10 different alpha and 9 beta chains combine to form many different integrins - each chain binds divalent cations - beta chain has cys repeat structure - Mg++ binds the 2 chains together Integrins Carbohydrates on other cells (surface glycolipids or glycoproteins) RGD sequence on ECM proteins RGD=ArgGlyAsp Large # of integrins bind weakly to lots of matrix molecules (low affinity, high avidity) Adherence of leukocytes to endothelium Interaction between endothelium and leukocytes Hemidesmosome: IF-ac-INT-Matrix 5 extracellular domains, 4 of which are homologous and bind Ca++ Bind to: Cadherin on neighboring cell. Intracellularly cadherin’s interaction with the cytoskeleton is mediated by catenin Example Desmosomes: IF-Cat-CAD-CAD-Cat-IF Adherens Junction Actin-Cat-CAD-CAD-CatActin IF=intermediate filament Cat=catenin CAD=cadherin cell migration Focal Contact Actin-ac-INT-Matrix Ac=accessory protein INT=Integrin 2 B. Structure of Epithelia Classification Simple squamous cuboidal columnar Pseudostratified Stratified squamous cuboidal columnar Transitional Typical Location Function Endothelium, respiratory system Exocrine gland ducts, kidney tubules Lining of intestine Lining of trachea and bronchi Epidermis Sweat glands, ducts Largest ducts of exocrine glands Renal calyces, ureter, bladder Exchange Absorption Absorption and secretion Secretion, conduit Barrier, protection Barrier, conduit Barrier, conduit Barrier, distensible property C. Intercellular Contacts Terminology of Intercellular Contacts Terminal bar = Junctional complex = Zonula occludens = tight junction Zonula adherens = anchoring junction Macula adherens Desmosome = Macula adherens D. Epithelial Transport Apical (lumen) Basolateral (blood) -60 Epithelial Transport Short Tutorial Na+ Na+ -80 -60 K K+ 0 + Na+ movement results in a transepithelial potential difference. Within the cell there is macroscopic electroneutrality; the electrical potential is the same throughout. Cl- wants to go from apical to basolateral, given that the lumen is negative with respect to the blood. Net result: Na+Cl- are absorbed together across the epithelium. This is known as secondary passive absorption. This serves as an osmotic force for H2O transport from apical to basolateral side. Also, the Na+/K+ pump dumps solute in the basolateral space, this raises the osmotic pressure and in turn drives the transport of water across the epithelia. 3 Connective Tissue SBPM SSN Exam 2 October 20, 2004 [email protected] Connective Tissue (CT) Structure = cells + extracellular matrix (ECM) ECM = fibers, ground substance, tissue fluid. Boundaries = basal laminae of epithelia, muscle, nerve. Types and their Functions Fibroblast – makes extracellular fibers & ground substance, give structure Lymphocytes, plasma cells, macrophages, eosinophils – immune system Bone – bone Tendons and ligaments – joint strength and stability Classification of CT Embryonic Connective Tissue Mesenchyme Mucous CT Connective Tissue Proper Loose CT Dense CT Irregular Regular Specialized CT Adipose Blood Bone Cartilage Hemopoietic tissue Lymphatic tissue Embryonic In the beginning… Embryonic mesenchyme becomes all the various CT’s of the adult body. CT Proper -Loose = Areolar = lots of cells and only thin, sparse collagen fibers, abundant ground substance - forms the lamina propria in respiratory and alimentary systems. -Dense = abundant fibers, few cells -in skin, it’s called “reticular” or “deep layer” of the dermis. -in hollow organs, it forms the submucosa. -Dense CT resists stretching and distention. -forms: - Tendons - Ligaments - Aponeuroses 4 Tendons - parallel bundles of collagen with interspersed rows of fibroblasts called tendinocytes. - Muscle-to-bone Ligaments - fibers and fibroblasts in parallel - the fibers in ligaments are less regularly arranged than those of tendons. - Bone-to-bone - Elastic ligaments – subset, has elastic fibers – e.g. ligamenta flava of spinal column yellow Aponeuroses - instead of parallel arrays, fibers are in multiple layers - orthogonal array – cornea – allows for transparency CT Fibers ** Collagen - most abundant - flexible - high tensile strength - appear wavy under light microscope, and eosinophilic, unless you use the trachonmason something something stain…where it appears blue - subunits: collagen fibrils - each collagen molecule is a triple helix composed of 3 intertwined polypeptide chains - every 3rd amino acid in the alpha chains is a glycine—essential for the triple-helix conformation. - Associated with sugar groups—hence called a glycoprotein. If you learn nothing else about CT, learn about the different collagen types. Type I Collagen -loose and dense CT Type II Collagen - hyaline and elastic cartilage - fine fibrils Type IV Collagen - nonfibrillar network - provides structural cohesion to the basal lamina Collagen Formation: 1. polypepetide chains are produced by polyribosomes of rER, then discharged into cisternae of rER. 2. Within the cisternae of rER and golgi, posttranslational modifications occur… 3. Cleavage of the signal peptide 4. Hydroxylation of proline and lysine residues while the polypeptides are still in the nonhelical conformation 5. Addition of O-linked sugar groups to some hydroxylysine residues and N-linked sugars to the two termini 5 6. Formation of a triple helix by three polypeptide chains, except at the terminals where the polypeptide chains remain uncoiled 7. Formation of intrachain and interchain H-bonds that influence shape and stabilize the interactions of the polypeptides 8. Now you have procollagen 9. It moves to the exterior of the cell by means of exocytosis of secretory vesicles (MT’s do this) 10. Then there are extracellular events: 11. Procollagen peptidase associated with cell membrane converts procollagen to collagen as it leaves the cell 12. Aggregated collagen aligns to form final collagen fibrils Reticular Fibers -framework of various organs. -“Reticular” = net - you’ll usually see it in the silver stain. Elastic Fibers -allows tissues to stretch and distend -elastic property is due to its unusual polypeptide backbone that causes random coiling Can you believe it, there are even clinical correlations in the Connective Tissue chapter! *Marfan’s Syndrome – a complex, autosomal dominant, CT disorder, in which expression of the fibrillin gene is abnormal…absence of elastin-associated fibrillin microfibrils. Consequence: abnormal elastic tissue. -elastic tissue important in: -vertebral ligaments - larynx - elastic arteries Ground Substance -filler -occupies space between cells and fibers -made of proteoglycans and hyualuronic acid -proteoglycans = glycosaminoglycans and a core protein Extracellular Matrix -fibrous proteins, proteoglycans, several glycoproteins (fibronectin & laminin) -fibroblasts attach to the ECM Connective Tissue Cells Resident Cell Population - fibroblasts (and myofibroblasts) - macrophages - adipose cells - mast cells - undifferentiated mesenchymal cells 6 Wandering/Transient Cell Population - lymphocytes - plasma cells - neutrophils - eosinophils - basophils - monocytes Fibroblasts and Myofibroblasts -fibroblasts are responsible for the synthesis of collagen, elastic, and reticular fibers, and even the complex carbohydrates of ground substance. -they’re a replicating population of cells. -myofibroblast is a cross between fibroblasts and smooth muscle cells. -implicated in wound contraction Macrophages - “big eaters” - phagocytic cells derived from monocytes - contains: - large golgi - rER - sER - mitochondria - secretory vesicles - lysosomes -secrete mediators of immune respons, anaphylaxis (dying from eating peanuts), inflammation. -interact with MHC II on CD4 T Cells. Mast Cells and Basophils - mast cells – important basophils – poor-man’s mast cell mast cells contain histamine in their granules histamine secretion can result in immediate hypersensitivity reactions, allergy, anaphylaxis also secrete leukotrienes and prostoglandin D mast cells are abundant in CT of skin and mucous membranes. Not present in brain and spinal cord. Basophils are…well, I could say a lot, but long story short, basophils are…unimportant. Adipose Cells - a CT cell specialized to store neutral fat. What’s neutral fat, you ask? Good question. Let’s go ahead and surmise that one can lead a gratifying medical career without knowing what neutral fat is. Educated guess: the fat that’s not brown fat. 7 Undifferentiated Mesenchymal Cells and Pericytes - - - still present in the adult? Who knows? Who cares? Actually, very important. They have the potential to give rise to new differentiated cells, which can serve a function in repair and formation of new tissue, as in wound healing, and development of new blood vessels (neovascularization). Pericytes are found around capillaries and venules – wrapped around – makes a neat little barrier. I want to say they show up in your kidneys, around the glomeruli, but it would be prudent to question my instincts. The fibroblasts and blood vessels within healing wounds develop from undifferentiated mesenchymal cells associated with the tunica adventitia of venules. Lymphocytes, Plasma Cells, and Other Cells of the Immune System - immune responses Lymphocytes = T cells, B cells, and NK cells. Plasma cells are antibody-producing cells derived from B cells. You will have to identify it in histo practical. It has A LOT of rER cause it’s a B-cell “factory.” Five bucks says you’ll hear that term. Eosinophils, Monocytes, and Neutrophils are also observed in CT - their presence generally indicates acute inflammation. 8 SBPM Muscle Information Structure of the Sarcomere The basic unit of skeletal muscle organization is the long, cylindrical, multinucleated skeletal muscle cell. The cell itself contains a number of cylindrical contractile units called myofibrils. The plasma membrane of the cell (also called the sarcolemma) invaginates along the length of the cell, forming T (transverse) tubules. The myofibrils are composed of thick and thin myofilaments (myosin and actin, respectively). These are organized into discrete, repeating units called sarcomeres. The sarcomere is the functional unit of contractility of the myofibril. The myofibril consists of thin filaments, made of the protein actin, and thick filaments, made of the protein myosin. On the thin filament are four other proteins: tropinin T (binds to tropomyosin), troponin C (binds to calcium), troponin I (binds to actin) and tropomyosin (prevents myosin from binding to actin) The sarcoplasmic reticulum of the muscle cell serves the same function as the endoplasmic reticulum does in other cells. It is principally important because it is a Ca storage site, and at certain points called triads, the terminal cisternae of the SR are juxtaposed with the T tubule, allowing close coordination of signals from outside the cell with signals inside the cell. The organization of the sarcomere is as follows: the thin filaments, composed of actin, are anchored into a structure called the Z disk, which is composed of alpha-actinin, and this is stabilized by the protein nebulin. The part of the sarcomere that is only composed of thin filament is called the I band. The A band is that part that contains the thick filament, made of myosin and stabilized by titin anchoring the thick filaments to the Z disk. The H band is that region in the center of the A band that does not have thin filament juxtaposed with thick filament. It is important to remember: when a muscle cell contracts, the I band and H band shorten, whereas the A band stays the same length. The Z disks move toward each other. The organization of the sarcomere makes sense in the context of length-tension relationships. There is point of maximal overlap between the thick and thin filaments: too much more overlap would leave little distance for shortening, and too little overlap means that the thick filament can not “grab” the thin filament well enough. Z Disk A Band I Band H Band 9 Variations of Muscle Cells Skeletal Cardiac Smooth Cell shape Long, cylindrical Branched Spindle-shaped Number/Location of Nuclei Multiple, peripheral Single, central Single, central and “corkscrewed” Striated? Yes Yes No Sarcomere? Yes Yes No Gap Junctions? No Yes Yes (some) T tubules? Has triads at the A-I junction Has dyads at the Z disks (instead of two SR terminal cisternae, only one) None Voluntary? Yes No No Important Histologic Features Peripheral nuclei Intercalated disks No striations Skeletal muscle is voluntary, striated muscle. It is characterized by invaginations of the cell membrane called T tubules that form triads at the A-I junction of the sarcomere. Skeletal muscle fibers are activated as parts of motor units, receiving peripheral innervation. Smaller motor units, found in places like the extraocular muscles, have a small number of muscle fibers; larger motor units, found in places like the quadriceps or biceps muscles, have a larger number of muscle fibers. As a muscle starts to contract, first smaller motor units contract and then, as the force increases, larger motor units are activated. Cardiac muscle is striated muscle as well, but is made up of individual muscle cells (as opposed to multinucleated syncitia like skeletal muscle). It is characterized by gap junctions that exist at the intercalated disks which serve to spread ionic current throughout the muscle so that contraction occurs uniformly. As opposed to the triads that distinguish skeletal muscle, cardiac muscle has dyads of SR next to T tubules near 10 the Z disk. Cardiac muscle is automatic in its contractility, controlled by the rhythm set by the SA node. However, it is also innervated by autonomic nerves to alter its rate and contractility. Smooth muscle is found all over the body: in the walls of the gut, lining the bronchioles, in hair follicles, etc. It is not striated and lacks T tubules. Some smooth muscles are linked by gap junctions; others are not. Instead of Z disks, because smooth muscles lack sarcomeres, they have cytoplasmic densities that serve a similar function of anchoring the thin and thick filaments. Smooth muscle is usually innervated by the autonomic nervous system; some smooth muscle also shows auto-contractility response to stretch (e.g. intestinal muscle contracts when stretched). E-C Coupling In skeletal muscle, a nerve impulse at the neuromuscular junction creates a depolarizing ionic current that is spread along the muscle fibers’ sarcolemmas and down the T tubules. The depolarizing Na current eventually reaches the terminal cisternae of the sarcoplasmic reticulum, which is spatially arranged next to the T tubule at the A-I junction. Voltage sensitive dihydropyridine receptors along the T tubule alter their confirmation and in so doing, open Ca channels in the SR. Calcium in turn binds to troponin C which sits on the thin filament. Troponin C and calcium release troponin I from actin, causing tropomyosin to move out of the way of myosin binding. Then, actin-myosin cross-bridge formation occurs. Relaxation of the muscle occurs when the depolarization ends and a Ca pump in the SR removed calcium from the cytosol. In cardiac muscle, the story is similar to skeletal muscle. In normal ventricular muscle, a wave of depolarization from the AV node results in an inward depolarizing Na current carried by the T tubules. The depolarization leads to the opening of voltage-sensitive Ca channels. The influx of Ca opens ryanodine receptors on the SR to release more Ca (so-called “calcium-induced calcium release”). The rest of the cycle is the same as in skeletal muscle. Smooth muscle E-C coupling is very different, because smooth muscle have no sarcomeres or T tubules. In smooth muscle, depolarization leads to Ca influx. Ca binds to the kinase calmodulin and activates it. Calmodulin activates the enzyme myosin light chain kinase which phosphorylates one of the myosin heads, activating it. Eventually, myosin gets dephosphorylated and this leads to relaxation. Knowing this, one can explain why smooth muscle can contract without external depolarization in response to hormones/neurohormones that induce Ca influx (e.g. oxytocin in uterine smooth muscle; acetylcholine at muscarinic parasympathetic receptors). Cross-Bridge Cycling Once tropomyosin has moved out of the way, actin and myosin are free to interact. The sequence of events that follows allows myosin to bind to and walk along the actin filament, shortening the sarcomere. Step 1: Myosin initially is bound to actin filament once tropomyosin is out of the way. Step 2: ATP binds to myosin, releasing it from the actin filament. Step 3: Myosin hydrolyzes ATP, causing a conformational change that shifts the position of the myosin head. Step 4: Myosin binds to actin again. Step 5: Myosin releases ADP. In doing so, it reverts to its original confirmational state, and propels forward along actin. ATP binds to myosin to repeat the cycle. The cycle continues as long as sufficient Ca and ATP are in the cytosol. 11 Step 2 ATP+ Step 1 Step 4 ADP+ ADP+ Step 3 Physiologic Characteristics of Muscle Activity Isometric contraction refers to contraction at a fixed muscle length (imagine pushing an immobile object). Isotonic contraction is contraction of fixed force over a range of muscle length (like lifting a dumbbell). Preload is a term used to describe the amount of tension a muscle develops at a given muscle length (for instance, if you stretch a muscle to a given length before you allow it to contract, the tension that develops is the preload). Afterload refers to the force against which a muscle contracts. Passive tension refers to the tension that is developed by stretching a muscle to various lengths, and depends upon the elastic properties of the muscle itself. Active tension is the tension that is developed by contraction of the muscle. Total tension is the sum of these two forces. Active tension is directly proportional to the number of actin-myosin cross-bridges that can be formed by a contracting muscle; hence, the spatial characteristics of the sarcomere discussed earlier come into play. The point of maximal cross-bridge overlap is the point of maximal active tension. As the muscle is stretched beyond that point, passive tension increases and active tension decreases. The speed of cross-bridge cycling is dependant on the afterload. As the weight against which a muscle is contracting increases, the velocity of muscle contraction decreases.