Exam 1 Study Guide 1. Light microscopy shine a light on a sample and see what is reflected or transmitted a.

Limit of resolution that wavelength of incident light limits resolvability b. Components of the cytoskeleton are too small to see in the light microscope unless they are tagged with fluorescent material 2. Fluorescence microscopy shine a light on a sample and see the light the sample gives off a. No limit of resolution can resolve much smaller objects Types of Membrane Protein a. Peripheral (extrinsic) only on 1 layer (does not have to be in hydrophobic layer) b. Integral (intrinsic) goes through bilayer or lipid-anchored = transmembrane i. Single-pass (channel) ii. Multi-pass 1. Extracellular domain, Intracellular Domain, and Hydrophobic Domain 2. Even number of transmembrane segments = Amino and carboxyl end of peptide chain is on same side 3. Domain in-between two hydrophobic domains could be intracellular or extracellular

Freeze fracture a. Freeze fracture only works with transmembrane proteins (bumps are transmembrane

proteins)
b. c. d. e. f. g. Split bilayer into two leaflets: P (protoplasmic) and E (extracellular) faces Bumps = transmembrane protein inside the cell or in the cytoplasm Proteins that extend across the layer do not break it is stuck to one side The protein that is exposed after splitting the membrane appear as bumps The protein underneath the leaflet is not seen by the microscope Amino acids can have ends on the same size of the membrane

1. Peroxisomes a. Enzyme: catalase i. Made on free ribosomes b. Function: fatty acid metabolism 2. Lysosomes a. Enzyme: acid hydrolases i. Made on membrane bound ribosomes (rough ER ) ii. Acid hydrolases packaged into endosomes at the Golgi iii. Carried to the lysosome b. Break down old/unwanted components
c. Lysosomes originate from endosomes that bud off from the Golgi apparatus

3. EMS Endo-membrane system a. ER, Golgi, lysosomes, endosomes, transfer vesicles ALL don t touch cytoplasm disconnected from the rest of cell (Peroxisomes, nucleus, mitochondria)

b. Describes pathway for exocytosis: rough ER, then Golgi, then transfer vesicles to the outside (don t touch cytoplasm) i. Proteins found in the cytoplasm are made from free ribosomes ii. Proteins not found in cytoplasm are made from bound ribosomes (ER) 1. A protein must be made in ER for it to be inserted into a membrane iii. ER makes proteins, Golgi packages the proteins

c. The newly made membrane protein would be inserted in the ER membrane --> membrane of a vesicle --> Golgi membrane --> membrane of a vesicle --> plasma membrane.
Antibodies a. Bind to a specific antigen b. Antigen can also can something else bound to it c. Two methods i. Direct immunofluorescence 1. Use one antibody with tag a. Rabbit anti-mouse = antibody from rabbit that directs against mouse antigen ii. Indirect immunofluorescence 1. Use two antibodies from different animals a. First antibody acts as antigen for the second b. Goat anti-rabbit = antibody from goat that directs against rabbit antigen (= 1st antibody is from rabbit) c. Allows for greater intensity and more variability in binding d. The antibody needs to be washed off each time to remove unattached e. The fluorescence substrate should be added last (otherwise it glows everywhere) Red blood cells (RBC s) a. Model choice for experiments because they have no internal membrane and little cytoskeleton (Only MF s, no MT s) iii. Only cytoskeleton in RBC is the actin/spectrin/ankryin web, directly underneath the plasma membrane iv. No membrane bound organelles no nucleus b. Lysed hole in membrane, so material can flow in/out c. Disrupted membranes broken into fragments v. After resealing, can be in original orientation or inverted (50% chance of each) Anchorage of proteins proteins do not have to be mobile in the plasma membrane a. Anchored by cytoskeleton inside the membrane i. Peripheral proteins 1. Ankryin anchor protein that supports multi-pass channels (e.g. band 3 protein) a. Ankyrin linked to BOTH an integral membrane protein

(such as the anion exchanger or another transmembrane

protein) and a peripheral membrane protein (such as spectrin)

2. Spectrin forms a web with short microfilaments to support proteins a. Defects cause cell fragility b. Peripheral membrane protein 3. Actin forms long microfilaments 4. Band 4.1 glue protein that organizes spectrin + actin into networks a. Also supports glycophorin protein b. Anchored by extracellular matrix (ECM) i. Fibronectin adhesive protein holds the ECM together ii. Collagen major structural protein of the ECM iii. Only ECM has carbohydrates Bilayer chambers a. Weak acids do not dissociate in acidic environment - unionized form crosses bilayer b. Weak acids do dissociate in basic environment trapped in one chamber c. Same for weak bases d. Note: Lysosomes are an acidic environment traps weak bases inside Nuclear envelope a. Contains TWO lipid bilayers (inner and outer): 4 monolayers b. Space in between is called perinuclear space c. Nuclear pores are gaps in the nuclear envelope d. Nuclear proteins (ribosomes) on the outer bilayer Cytoskeleton 3 components y Part of the cytoplasm: Originates near the nucleus ( - end) and branches out toward the cell membrane ( + end) 1. Microtubules (MT) a. Hollow and largest diameter b. Made of tubulin i. MT = Polymers of globular subunits (alpha and beta tubulin) c. Motor molecules i. Kinesin away from origin (Korner): plus end ii. Dynein towards origin (Draw): minus end 1. Minus end also known as centrosome or MTOC (microtubule organizing center) d. Role i. Spindle fibers to pull apart sister chromatids during mitosis (metaphase) ii. Mt s are also present in interphase e. Drugs i. Colchicine shifts toward monomer 1. Destabilizes MT ii. Taxol shifts toward polymer 1. Taxol binds to MTs (polymerized tubulin) and prevents depolymerization of MT. 2. Stabilizes MT

iii. Both drugs inhibit movement along track 2. Microfilaments (MF) a. Solid and smallest diameter b. Made of actin i. Polymers of globular subunits (G-actin monomer, F-actin polymer) c. Motor molecules i. Myosin 1. using ATP to move along MF track carries with it vesicles and other large structures 2. have two fibers sliding past each other to shorten/lengthen structure d. Role i. Cleavage furrow to separate into two cells during mitosis (telophase) e. Drugs i. Cytochalasin shifts toward monomer (G-actin) 1. Mnemonic: a s in Cytochalasin and actin and microfilament 2. Destabilizes MF ii. Phalloidin shifts toward polymer (F-actin) 1. Stabilizes MF iii. Both drugs inhibit movement along track 3. Intermediate Filaments (IF) a. Solid b. Polymers of fibrous subunits i. Many different kinds of monomers ii. Tetramer = basic subunit iii. Protofilament long structure formed by many tetramers c. Function: Structural support, NOT movement i. No motor molecules d. Types of IF s i. Nuclear IF s 1. Lamins (intermediate filaments) a. Only cytoskeletal structure in the nucleus b. In the inner nuclear membrane ii. Cytoplasmic 1. Type of IF depends on the tissue 2. Note: MF and MT only in the cytoplasm (unlike IF)

ECM 1. Three types of molecules a. Structural Proteins i. Collagen ii. Elastin b. Adhesive Glyco-Proteins (some sugar attached) connect materials in ECM together i. Fibronectin

ii. Laminin c. Proteoglycans (lots of sugar attached) provide gel-like matrix for ECM 2. Glycosylation only in ECM (phosphorylation in cytoplasm)

Junctions ALL made of transmembrane proteins 1. Cell-cell junctions a. Gap junctions i. SIX connexins (transmembrane proteins) form connexons by weak bonds ii. Gap junction is a gated tube formed from two connexons (=transmembrane proteins) 1. allows small materials (ions and water) to travel 2. NOTE: the channel can be closed iii. Small gap between cells 1. Material can pass in-between the cells (around the tube) iv. Cytoplasm of two different cells connected v. Main function: transport diffusion across protein channel b. Tight junction i. Two claudins (transmembrane proteins) connect two cells together ii. No gap between cells cells are touching iii. Multiple claudins arranged to form a tight seal (ridges) material cannot travel through iv. Main function: structure, prevent diffusion across cells c. Adhesive / Anchoring Junction i. Two cadherins connect two cells together 1. Cadherins are transmembrane proteins ii. Cadherins attached to MF s or IF s through linker plaques inside the cell membrane iii. Two types of adhesive junctions 1. IF s with cadherins: desmosomes (spot plaque) a. Intermediate and desmosomes b. There are proteins connecting the two cells at a desmosome, but the IF associated with desmosomes are inside the cells. 2. MF s with cadherins: adherens junctions (belt plaque) a. Microfilament and adherens iv. Main function: strength (cross-linking) 2. Cell-ECM junctions (contains integrins, NOT cadherins)

i. Integrins connected to a protein in the ECM b. Hemidesmosomes i. Half of desmosomes ii. IF s with integrins (spot plaques) iii. Connect to basal lamina 1. Solid layer found in parts of the ECM 2. Mainly laminin and collagen 3. Basement membrane used for support and barrier c. Focal adhesions i. Half adherens junction ii. MF s with integrins (belt plaques) iii. Connect to ECM on outside Epithelial Cells linings of external (skin) and internal surfaces (organs) 1. Two distinct, polarized surfaces separated by a tight junction: prevents passage of ions across cell layer a. Apical Side of membrane facing lumen i. Lumen interior of organ, interior of vesicle = ECM ii. Apical side has microvilli iii. Transport in apical is by active transport iv. Active/passive diffusion present in the apical membrane b. Basolateral (BL) surface side of membrane facing the body i. basal (base) membrane and lateral (side) membrane ii. Hemidesmosomes are on the basal part of the BL 1. Connect to the basal lamina of the ECM iii. Gap, adherens, and desmosomes are on the lateral part of the BL 1. Gap junctions allow passage of ions from cell to cell a. Glucose transported across BL by facilitated diffusion iv. Active/Passive diffusion in the BL membrane 1. Na/K present in BL membrane 2. Epithelial cells contain both MFs and MTs (unlike RBC) 3. There is one belt desmosome that runs around the middle of the epithelial cell, dividing the cell into apical and baso-lateral surfaces. 4. Transport of glucose across intestinal epithelium (IE) a. Glucose in lumen enters apical side and crosses tight junction by Glucose/Na cotransport b. Glucose diffuses through the epithelial cells

c. Glucose leaves BL side by GLUT protein (carrier-mediated protein)

Small-Molecule transport

Two types of curves 1. conc. vs. time curve a. The initial slope of the conc. vs. time curve depends on the amount of transport protein i. Initial Slope = rate of uptake b. Levels off = equilibrium i. concentration of glucose outside = the concentration inside ii. not a measure of protein saturation 2. rate of uptake(flux) vs. concentration a. Level off = saturation of protein b. Directly depends on how much transport protein there is c. Can't tell active transport and facilitated diffusion apart using graphs of influx vs. concentration Types of transport NOTE: ALL transporters are reversible, and direction it 'pushes' depends on the ion gradient 1. Simple Diffusion a. Diffusion of hydrophobic molecules/gases through lipid bilayer b. C(out) = C(in) at equilibrium c. Flux linear with concentration (no limiting factor) 2. Diffusion through a channel a. Diffusion of hydrophilic molecules through a channel protein b. C(out) = C(in) at equilibrium c. Flux linear with concentration, except at very large concentrations 3. Facilitated Diffusion (aka carrier-mediated transport) a. Protein changes conformation to bring molecule inside i. Protein is a carrier or permease (transmembrane) 1. GLUT (Glucose Transporters) a. Glucose requires a transport protein to cross a plasma membrane and enter any cell. b. transports glucose in/out depending on concentration difference i. Orientation/location of GLUT does not matter ii. GLUT transfer glucose out of epithelial cells in the intestine iii. GLUT transfers glucose into epithelial cells in the colon c. GLUT1 plasma membrane of RBC and endothelial cells d. GLUT2 BL surface of epithelial cells i. Ferry glucose out of cells (stored in the lumen) because it is more concentrated there e. GLUT3 similar to GLUT1 and GLUT2 f. GLUT4 muscle and adipose tissue

i. Insulin dependent ii. Protein is inserted into plasma membrane by exocytosis b. C(out) = C(in) at equilibrium c. Flux saturates at high concentrations (all protein occupied), proportional at low conc. 4. Primary Active Transport (2 ions+ATP) a. Use ATP (hydrolyzed inside the cell) to bring molecule up gradient i. E.g. move Na+ outside of cell against gradient b. C(out) < C(in) at equilibrium c. Flux saturates at high concentrations (all protein occupied), proportional at low conc. d. Enzymes i. Kinase 1. Activated by Na+ 2. ATP to ADP, flips pump out 3. Dumps Na+ out of cell, picks up K+ ii. Phosphatase 1. Activated by K+ 2. ADP to ATP, flips pump in 3. Dumps K+ in, picks up Na+ iii. Overall: 3 Na+ out per 2 K+ in per ATP used 1. Na/K pump is antiport 2. an enzyme to add the phosphate at the expense of ATP (a kinase) and a different enzyme to remove the phosphate by hydrolysis (a phosphatase), getting back ATP 5. Secondary Active Transport (2 ions + NO ATP) a. ATP not directly involved b. Movement of one ion down gradient facilitates movement of another up gradient c. Two types: Co-transport and exchangers d. Co-transport (always symport) i. Ex1: Glucose/Na+ co-transport 1. Na comes in down its gradient, allows glucose to come in against gradient 2. Na+/K+ pump needed to establish Na+ gradient 3. Protein: SGLT = sodium-glucose transporter 4. overall concentration of Na+ inside stays the same because of the action of the Na+/K+ pump ii. Ex2: Amino acid/H+ co-transport 1. H+ comes in down its gradient, allows amino acids to come in against gradient e. Exchanger (always antiport, same charged ion) i. Ex1: Ca2+/Na+ exchanger

1. Na comes in down its gradient, allows Ca2+ to flow out against gradient ii. Ex2: H+/Na+ Exchanger 1. Na comes in (down gradient), proton comes out (against gradient) iii. Ex3: Bicarbonate/Cl- Exchanger 1. Bicarbonate flows down gradient, Cl- moves outside against gradient 2. Transporter is Band 3 protein 6. All Na+ transport will indirectly or directly need ATP (to maintain gradient) Big Molecule Transport: Endocytosis vesicle from membrane containing big molecules (antibodies, hormones, channel proteins) go into lysosome to get degraded y Signal is needed for Endocytosis (chemical group or receptor-mediated) y Three types 1. Pinocytosis cell drinking taking a random sip 2. Phagocytosis pseudopods reach out and engulf solids, vesicle formed, actin needed 3. Receptor-mediated endocytosis (RME) a. Receptors (peripheral proteins) bind ligand b. Receptors move to clathrin-coated pits i. Clathrin help the membrane to bend and invaginate to form a vessel ii. Clathrin is a peripheral protein c. Membrane invaginates to form a coated vesicle (pinches off) i. More than one type of receptor (plus or minus ligand) may be included in any one vesicle. The cell does not contain separate vesicles for each type of receptor/ligand combination that is endocytosed d. Uncoating of Clathrin = uncoated endocytic vesicles i. Ligand completely inside the vesicle ii. The receptor has a part sticking out into the cytoplasm e. Vesicle is acidified to become endosome (move H+ into vesicle) or move towards an endosome f. Sorting occurs i. Ligand/Receptor can either be recycled or digested ii. Transferrin 1. Metabolism using iron 2. Ligand = transferrin = apotransferrin + Fe 3. Apotransferrin and receptor do NOT separate + get recycled (to get more iron) a. Separate in the ECM 4. Iron used up in the cytoplasm iii. LDL (low density lipoprotein) 1. Metabolism using cholesterol 2. Ligand = LDL = apoB + cholesterol

3. Ligand is digested (cholesterol used as building block in cells), receptor is recycled (to get more LDL) iv. EGF 1. Signal for growth 2. Ligand = growth factor 3. Ligand is digested, receptor is digested (don t need to keep growing) g. Vesicles buds off and are differentiated i. All different receptors and ligands are separated h. Ligand/Receptor components either degraded in lysosome, used in the cytoplasm, or recycled via exocytosis Types of labeling following material going into a cell 1. Continuous constantly supply radioactive material and follow it a. Multiple parts show radioactivity, though at different levels i. Pattern: Increase, level off 2. Pulse-chase supply a pulse of radioactive material and then ordinary material (chase) a. One part shows radioactivity at a time as pulse passes through b. An entire pulse-chase experiment includes one pulse of labeled material, not many i. Pattern: Increase, reach a peak, and decline 3. GFP fusion protein made from genetic engineering Detection 1. Autoradiography cover cells with photographic emulsion to develop grains on organelle a. More grains = more radioactivity b. Like in situ assay 2. Fractionate break up cells, separate by weight, and measure radioactivity Capillaries 1. Made of endothelial cells 2. Endothelial cells mostly not joined by tight junctions a. Material can pass into capillaries 3. EXCEPTION: brain endothelial cells have tight junctions a. Material cannot pass unless there is transporter 4. Endothelial cells surrounding capillaries are not the same as the epithelial cells surrounding the intestinal lumen a. Epithelial cells surrounding the intestinal lumen take in glucose from the intestine by secondary active transport co-transport with sodium. b. Almost all other body cells, including the endothelial cells surrounding the capillary lumen, take in glucose by passive transport, using a GLUT protein.