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BIOL 1501

BIOL 1501: Cell Biology Dr. Ireland – Winter 2014 Two Types of Microscopy • Fluorescence microscopy, scanning electron microscopy Stem Cells • Gene delivery • Marrow stroma • Bone • Adipose • Tendon and ligament • Cardiac muscle • Cartilage • Biopharmaceuticals Viruses • A lot of controversy whether or not viruses are considered as living species. Cell Composition • A bacterial cell is composed of… o 70% water o 30 % chemicals  15% proteins  6% RNA  4% ions, small molecules  2% phospholipids  2% polysaccharides  1% DNA Molecules in Cells • Water is the most common compound in cells. • Cellular chemistry based on the carbon atom. • Biomolecules are mostly comprised of chemical units called functional groups. • Lipids, sugars, amino acids, and nucleic acids are the most common biomolecules in cells. Bonds • Weak chemical bonds, e.g. ionic and hydrogen bonds, and very important in biomolecules. o They govern the shape of large molecules, and help molecules adhere to each other. • Water is held together with hydrogen bonds. Polarity of Water Molecules- Hydrogen Bonding • Water molecules are polar- the opposite ends have opposite charges. • Polarity allows water molecules to form hydrogen bonds with each other.Hydrogen Bonds • Hydrogen bonds are found between a hydrogen atom covalently bonded to one electronegative atom and another electronegative atom. • In cells, the electronegative atoms are usually oxygen or nitrogen atoms. Water • The properties of water dictate most cellular processes. • Hydrophilic things: compatible with water… o Ions o Other polar molecules o A hydrophilic substance has an affinity for water. • Hydrophobic things: incompatible with water… o Non-charged, non-polar molecules. o A hydrophobic substance does not have an affinity for water. Important Functional Groups • Functional groups: components of organic molecules involved in chemical reactions and interactions. • The number and distribution of functional groups give each molecule its specific properties. • The most important in the chemistry of life: o Hydroxyl o Carbonyl o Carboxyl: carboxylic acids, or organic acids; has acidic properties because the covalent bond between oxygen and hydrogen is so polar. Found in cells in the ionized form with a charge of 1- and called a carboxylate ion. Example: acetic acid. o Amino: amines; acts as a base; can pick up an H from the surrounding solution (water, in living organisms). Ionizes with a change of 1+, under cellular conditions. Example: Glycine. o Sulfhydryl o Phosphate: organic phosphates; contributes negative charge to the molecule of which it is a part (2- when at the end of a molecule, 1- when located internally in a chain of phosphates. Has the potential to react with water, releasing energy. o Methyl Carbon: Organic Molecules • Cells are 70-95% water. • The rest is mostly comprised of carbon compounds. • Carbon can form a huge number of large, complex molecules, such as… o Proteins o RNA o DNA o Carbohydrates Macromolecules • Four classes of large biological molecules: o Carbohydrates o Proteins o Lipids o Nucleic Acids • Small organic molecules are joined together to form these larger molecules. Biomolecules • Sugars form polysaccharides, fatty acids form fats/lipids/membranes, amino acids form proteins, and nucleotides form nucleic acids. • Carbohydrates: sugars and polymers of sugars. o Simplest carbohydrates are monosaccharides, or single sugars. o Carbohydrate molecules are polysaccharides, which are polymers composed of many sugar building blocks. Storage Polysaccharides • Starch, a storage polysaccharide of plants, is made up of glucose monomers. • Plants store starch as granules inside chloroplasts and other plastids. • Glycogen is a storage polysaccharide in animals, made up of glucose monomers. • Humans and other vertebrates store glycogen mainly in liver and muscle cells. Lipids • Lipids are the one class of large biological molecules that do not form polymers. • Lipids are hydrophobic o They consist mostly of hydrocarbons, which form non-polar covalent bonds. • Most biologically important lipids are fats, phospholipids, and steroids. • Fats are lipids: fats are constructed from two types of smaller molecules- glycerol and fatty acids. • Glycerol is a three carbon alcohol with a hydroxyl group attached to each carbon. • A fatty acid consists of a carboxyl ground attached to a long carbon skeleton. • Fatty acids are composed of a hydrophilic carboxylic acid head and a hydrophobic hydrocarbon tail. • Proteins • At least 50% of the dry mass of most cells are proteins. • Protein functions include: o Structural support o Storage o Transport o Communications o Movement o Defense against foreign substances • Polypeptides are polymers built from the same set of 20 amino acids. • A protein consists of one or more polypeptides. • Amino acids are organic molecules with carboxyl and amino groups. • Amino acids differ in their properties due to differing side chains. Peptides • Peptides are bonded by peptide bonds. • There is an N terminus and C terminus of a polypeptide chain. • Amino acids are linked by single bonds called peptide bonds, formed by the carboxylic acid group of one amino acid and the amino group of a second amino acid. Protein Structure and Function • A functional protein consists of: o One or more polypeptides o Twisted, folded, and coiled into a unique shape. • The sequence of amino acids determines a protein’s three-dimensional structure. • A protein’s structure determines its function. Levels of Protein Structure • Primary Structure: unique sequence of amino acids. • Secondary Structure: coils and folds in the polypeptide chain. • Tertiary structure: arises from interactions among side chains. • Quaternary structure: found in multimeric proteins. • Polypeptide folds at secondary structure in an aqueous environment, with hydrophobic region at the core containing non-polar side chains, and hydrogen bonds may then be formed to the polar side chains on the outside of the molecule. • Polypeptides may be proteins or protein subunits. • Types of secondary structure: o Alpha Helix o Parallel Beta Pleated Sheet o Anti-parallel beta pleated sheet • Five classes of bonds that stabilize protein structure: o Disulfide bond o Hydrogen bond o Van der Waals and hydrophobic interactions o Ionic bond • Alpha helices are represented by coiled ribbons, strands of beta sheets are represented by arrows pointed from N terminus to C terminus. Sickle Cell Disease • A slight change in primary structure can affect a protein’s structure and ability to function. • Sickle Cell disease: o Inherited blood disorder o Results from a single amino acid substitution in the protein hemoglobin. The Structure of Nucleic Acids • Nucleic acids are polymers called polynucleotides or oligonucleotides. • Each polynucleotide is made of monomers called nucleotides. • Each nucleotide consists of a nitrogenous base, a pentose sugar, and a phosphate group. • The portion of a nucleotide without the phosphate group is called nucleoside. DNA • Level one of DNA organization is a double stranded anti-parallel double helix held together by hydrogen bonds between base pairs. Cell Biology Techniques • Microscopy o Light Microscopy o Electron Microscopy • Biochemical Techniques o Cell Fractionation o Chromatography o Electrophoresis o Molecular Technique • Molecular Techniques Microscopy • Microscopes are used to visualize cells too small to see with the naked eye. • The microscope has much better resolution than the human eye. • In a light microscope (LM) visible light passes through a specimen and then through glass lenses, which magnify the image. • The electron microscope (EM) uses a beam of electrons which is either passed through or bounced off the surface of the specimen. • We can manipulate the following to get a better image: o Magnification: ratio of object’s image size to its real life. o Resolution: the minimum distance between two distinguishable points = clarity. o Contrast: visible difference between regions of the image. • LM can magnify to about 1000x o Can enhance contrast and emphasize specific cell components: staining and labeling o Sub-cellular structures, e.g. organelles too small to be resolved by LM- need EM • Techniques include: o Brightfield unstained o Brightfield stained o Phase-Contrast o Differential interference contrast Microscope Resolution • Resolution in an optical system can be described mathematically by Abbe’s equation: • d = 0.612 * l n sin a o d = resolution o l = wavelength of radiation o N = refractive index of medium between light source and lens o a = refraction angle o 0.612 = Abbe’s Constant Electron Microscopy • Two types of electron microscope: o Scanning electron microscope (SEM) focuses a beam of electrons onto the surface of a specimen: provide images that look 3-D (surface) o Transmission electron microscope (TEM) passes a beam of electrons through a specimen (interior) Cell Fractionation • Breaks cells apart and separates organelles from one another. • Ultracentrifuges fractionate cells into their component parts- organelles, etc. • Biochemistry and cytology help correlate cell function with structure. Density Gradient Centrifugation • Gradient of sucrose or some other medium. • Separate organelles based on density. Chromatography • Separation of biomolecules on analytical or prep scale. • Usually in column (glass, plastic, steel) • Maintains biological properties • Can study molecules after separation • Ion exchange chromatography: surface change • Gel filtration chromatography: size • Affinity chromatography: biological properties Electrophoresis • Separation of biomolecules on analytical scale • Often in a gel slab • May damage biological properties • Can sometimes study molecules after separation • Polyacrylamide Electrophoresis: o Native protein electrophoresis: surface charge and size o SDS protein electrophoresis: size • Agarose Electrophoresis: o Sub Gel Electrophoresis: size – DNA Membrane Structure • Lipid bilayer: o Mixture of proteins and lipids o Non-covalent bonds o Continuous double layer o 4-5 nm thick o Various proteins wedged in between the lipids o Relatively impermeable barrier to most hydrophilic molecules o Proteins anchored in a sea of lipid  Various functions: enzymic, transport, structural, receptors. Lipids • Fats • Oils • Some vitamins • Some hormones • Membrane lipids are glycerides Phospholipids • Two fatty acids and a phosphate group are attached to a glycerol o Fatty acid tails are hydrophobic o Phosphate group and its attachments form a hydrophilic head • Amphipathic molecules o Both hydrophilic and hydrophobic • Add phospholipids to water o Self assemble into a bilayer o Hydrophobic tails point toward the interior • Self repair when damaged • Phospholipids are the major component of all cell membranes o The most abundant lipid in the plasma membrane Steroids • Lipids characterized by a carbon skeleton consisting of four fused rings • Cholesterol, an important steroid, is a component in animal cell membranes Plasma Membrane • The boundary that separates the living cell from its surroundings • Fluid mosaic model o A membrane is a fluid structure with a “mosaic” of various proteins embedded in it • Exhibits selective permeability o Allows some substances to cross it more easily that others • Freeze-fracture studies of the plasma membrane supported the fluid mosaic model o A specialize preparation technique o Splits a membrane layer along the middle of the phospholipid bilayer o Electron microscope The Fluidity of Membranes • Phospholipids in the plasma membrane can move within the bilayer • Most of the lipids, and some proteins, drift laterally • Rarely does a molecule flip-flop transversely across the membrane Membrane-Associated Disease • Problems with membrane structure can lead to disease • Hyaline membrane disease (surfactant deficiency disorder) o Preterm infants o Lungs not fully mature at birth o Respiratory distress o Poor O2/CO2exchange • Alzheimer’s disease o Accelerated membrane P-lipids turn over o Abnormal membrane repair o May contribute to amyloid deposition o Ultimately results in synaptic loss • Heart Disease o Faulty membrane repair o Emphasized by vigorous exercise/muscle weakness Membrane Proteins • Membranes contain many different proteins embedded in the fluid lipid bilayer • Proteins determine most of the membrane’s function • Peripheral proteins are attached to the surface of the membrane • Integral proteins penetrate the hydrophobic core • Integral proteins that go all the way across the membrane are called transmembrane proteins • The hydrophobic regions of integral proteins consist of one or more regions of non-polar amino acids, often coiled into alpha helices Membrane Lipids • Fatty acids vary in length and number and locations of double bonds. • Saturated fatty acids have the maximum number of hydrogen atoms possible and no double bonds • Unsaturated fatty acids have one of more double bonds • Lipids made from saturated fatty acids are called fats- solid at room temperature o Most animal fats are saturated • Fats made from unsaturated fatty acids are called unsaturated fats or oils, and are liquid at room temperature. • Membrane lipids have a mixture of the two. Membrane Proteins • Integral proteins that go all the way across the membrane are called transmembrane proteins. • The hydrophobic regions of the integral proteins consist • Six major functions of membrane proteins: o Transport o Enzymic activity o Signal transduction o Cell-cell recognition o Intercellular attachment o Attachment to the cytoskeleton and extracellular matrix (ECM). Protein Movement in Membranes • Proteins can move around- not a random movement, and have restrictions of where they can move to. • Some proteins bind to other cells, allowing cells to form clusters. • Some proteins bind to molecules in the extracellular space, permitting cells to attach and crawl on these molecules. Membrane Carbohydrates and Cell-Cell Recognition • Cells recognize each other by binding to surface molecules. o Usually carbohydrates, on the plasma membrane. Synthesis and Sidedness of Membranes • Carbohydrates on the external side of the plasma membrane vary among species, individuals, and even cell types in an individual. • Membranes have distinct inside and outside faces. • Proteins, lipids, and associated carbohydrates are distributed asymmetrically in the membrane. o Determined when the membrane is built by the ER and Golgi apparatus. Membrane Selective Permeability • A cell must exchange materials with its surroundings. • This is controlled by the plasma membrane. • Plasma membranes are selectively permeable • While large charged molecules cannot penetrate The Permeability of the Lipid Bilayer • Hydrophobic Channel and Carrier Proteins • Some transport proteins- channel proteins- have a hydrophilic channel through which some molecules or ions can pass. • Other transport proteins- carrier proteins- bind to molecules Passive Transport • Diffusion: tendency for molecules to spread out evenly into the available space. • At dynamic equilibrium, as many molecules cross a membrane one way as cross in the other direction. • Substances diffuse down their concentration gradient. o The difference in concentration of a substance from one area to another. • No work needs to be done to move substances down the concentration gradient. • Diffusion of a substance across a membrane Facilitated Diffusion • Transport proteins speed the massive movement of molecules across the plasma membrane. • Channel proteins provide tunnels that allow a specific molecule or ion to cross the membrane. • Channel proteins include: o Aquaporins: facilitated diffusion of water o Ion channels: may open or close in response to a signal (gated channels) Active Transport • Facilitated diffusion is still passive because the solute moves down its concentration gradient. • Some transport proteins, however, can move solutes against their concentration gradients. • Active transport moves substances against their concentration gradient + + o Na /K ATPase: Potassium is moved in; sodium is moved out via active transport. Creates a voltage due to the charges. ATP is released during this process. The sodium and potassium gradients can be used to be move other ions in an out. Gated Channels • Extracellular ligand-gated • Intracellular ligand-gated • Voltage-gated • These are found in extracellular space. Ion Pumps and Membrane Potential • An electrogenic pump is a transport protein that generates voltage across a membrane. • The sodium-potassium pump is the major electrogenic pump of animal cells. • The main electrogenic pump of plants, fungi, and bacteria is a proton pump. Co-transport • Occurs when active transport of a solute indirectly drives transport of another solute. • Plants use the hydrogen ion gradient generated by proton pumps to drive. Transport Types • Two types of co-transport: o Symport: 2 solutes/ions move in the same direction. o Antiport: 2 solutes/ions move in different directions. • Uniport: one solute/ion in one direction. Membrane-associated disease • Problems with membrane proteins can lead to disease. • Cystic fibrosis: o Defect in Cl channels o Excess fluid production in lungs  Respiratory failure o Pancreatic insufficiency o Range of other defects • Cystinuria o Kidney disease o Inherited, autosomal, recessive o Inadequate re-absorption of cystine o Excessive concentration of cystine in urine o May crystallize o Kidney stones Movement of Large Molecules across Membranes- the Endomembrane System Bulk Transport across the plasma membrane • Small molecules and water enter or leave the cell through the lipid bilayer by transport proteins. 6 + • Large molecules, such as polysaccharides and proteins, 10 times bigger than Na ion o Cannot go through the membrane o Requires structural change in the membrane itself o Cross the membrane in bulk via vesicles o Exocytosis and endocytosis Exocytosis and Endocytosis • Import: Endocytosis • Export: Exocytosis The Endomembrane System
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