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BIO1350 FALL 2013 PRELIM I MATERIAL: I. MICROSCOPY A. Light Microscopes -magnifies cells up to x1000, details as small as 200nm (=.2 microns) -resolving power depends on wavelength, but light microscope can't resolve more than organelles -cells = mostly transparent bc they are mostly water, so dyes can be used (ex: use a dye that binds to acidic things to highlight the nucleus) 1. Fluorescence Microscopy -two filters: light before it reaches specimen, passing only the wavelengths that excite the particular fluorescent dye; and a filter that blocks out this light and passes only the wavelengths emitted when the dye fluoresces (dyed objects show up bright) *slices/sections, stained → cells 2. Confocal Microscopy: specialized fluorescence microscope that scans specimen with a laser beam, beam is focused onto a single point at a specific depth in the specimen B. Transmission Electron Microscopy -tissue is chemically fixed, embedded in plastic, cut into thin sectioned and stained with uranium and lead (Fix → Dehydrate → Section → Stain → Vacuum) -uses beam of electrons and magnetic coils to focus the beam -CONS: cells dead, not a lot of specificity -HIGH resolving power (2.5 pm) -creates 3D images (Scanning electron microscopy) C. Immunofluorescence: Making and Using Antibodies 1. The Antibody Molecule: proteins, bind to antigens, produced as a defense against infection 2. Antibody Specificity: different antibody molecules, each with a distinct antigen binding site -foreign molecules, viruses, bacteria (Antibodies for aggregates, antibody and antigen aggregates are ingested by phagocytic cells, special proteins in the blood kill antibody coated bacteria/viruses) 3. Raising antibodies in animals a. Inject an animal with antigen A b. Stimulate beta cells to secrete large amounts of anti-A antibodies (antigen binds to a receptor, B cell is stimulated to divide and secrete a lot of the same antibodies in a soluble form) 4. Indirect Immunofluorescence (IMF) -permeabilize cell membrane, fix cell (crosslinking → glues everything in place; detergent to allow antigens to be placed in the membrane) -primary antibody against antigen A (which is attached to a certain organelle in the cell) made in a small animal like a rabbit (by injecting antigen A) and injected into the cell, binds to antigen A -secondary antibody made in a larger animal (by injecting primary antibodies into the larger animal), anti-small animal antibodies made and tagged with fluorophore, injected into cell (bind to anti-A antibodies and then can be seen using light microscopy) D. GFP fusion proteins -protein from jellyfish, emits green light, can be attached to any protein you want to look at (make fusion protein with recombinant DNA) -allows you to look at live cells, see motion of cell organelles -could potentially alter structure and function of some proteins * How do we localize proteins with in a cell? * 1. Sensitivity: fluorescence; shine UV light, glows green (HOW IT FLUORESCES) 2. Specificity: antibodies; GFP fusion (DNA) (HOW IT TARGETS A PROTEIN) II. CELL STRUCTURE A. Cell Theory: Living cells formed by the division of existing cells B. Cell Organelles 1. Cytoplasm: ~54% of cell volume; a transparent substance around organelles in the cell and with in the cell walls (plasma membrane); site of many chem reactions, manufacturing of proteins (ribosomes in cytoplasm) 2. Mitochondria: ~22% of the cell, organelles which generate energy for eukaryotic cells; double membrane, own DNA & ribosomes; uses oxidative phosphorylation to harness energy and produce atp/carbon dioxide 3. Nucleus: information store, ~6% of cell; 2 concentric membranes form the nuclear envelope; nuclear pores; contains DNA; chromosomes become compact as cell prepares to divide 4. Chloroplasts: capture energy from the sunlight, large green organelles found only in cells of plants and algae; double membrane, own DNA & ribosomes; perform photosynthesis, release oxygen as a byproduct 5. Endoplasmic Reticulum: maze of interconnected spaces enclosed by membrane, where most cell membrane components and materials designed for export are made; synthesis of proteins and lipids; rough and smooth ER (ribosomes attached to rough; smooth responsible for detoxification and steroid hormone synthesis) 6. Golgi Apparatus: stacks of flattened membrane-enclosed sacks; receives and chemically modifies the proteins and lipids made in ER and then directs them to cell exterior 7. Lysosomes: small, irregularly shaped organelles in which cell digestion occurs (nutrients; excretion); premature lysosomes = endosomes → ON GOLGI 8. Peroxisomes: small, membrane enclosed vesicles for reactions in which hydrogen peroxide is generated and degraded → ON ER 9. Vesicles: involved in transport 10. Cytoskeleton: system of protein filaments with in a cell → Actin filaments: ~9nm in diameter, involved in cell shape and movement; especially present in muscles → Microtubules: ~25 nm in diameter, help in cell division and organization → Intermediate filaments: ~10 nm in diameter, strengthen cell mechanically *endocytosis: cells engulf large particles or foreign cells *exocytosis: internal vesicles fuse with membrane of cell and release content into external medium C. The Procaryotic Cell 1. Bacteria & Archaea: simplest structure, no organelles 2. no nucleus in cell, no membrane bound organelles 3. cell wall surrounding plasma membrane 4. live on inorganic substances or photosynthesis III. PROTEINS A. Amino Acids are the subunits of proteins 1. Carboxylic acid group and amino group, both linked to the alpha carbon, and a chemical variety from the side chain 2. peptide bonds: covalent linkage between amino acids (formed by condensation reactions → DEHYDRATION SYNTHESIS) B. The 20 Basic Amino Acids found in Proteins 1. Basic Side Chains (POLAR) a. Lysine, arginine, histadine (NH/NH2+/NH3+) 2. Acidic Side Chains (POLAR) a. aspartic acid, glutamic acid (COO- makes it acidic) 3. Uncharged Polar Side Chains a. Asparagine, glytamine (C bound to O and NH2) b. serine, threonine, tyrosine (CH2/OH/CH) → can be phosphorylated 4. Non-Polar Side Chains (hydrocarbons) a. alanine, valine, leucine, isoleucine, proline, methionine (CH3/CH2) b. phenylalanine, tryptophan (carbon hexagon) c. glycine (H); cystine (CH2—SH) C. The Principal Types of Weak, Non-Covalent Bonds 1. Van Der Waals attractions: two atoms attracted until distance between their nuclei is equal to sum of vdw radii (close range interactions) 2. Hydrogen Bonds: formed when H is sandwiched between two electron-attracting atoms (usually N/O) 3. Hydrophobic Forces: Not true forces, but if 2 or more hydrophobic groups are in water, the water will force them together to minimize their disruptive effects on hydrogen bonded water. Hydrophobic forces help fold proteins (hydrophobic side chains come inside, hydrophilic forces face outside) 4. Electrostatic Attractions: between fully charged groups (ionic bond) and between partially charged groups on polar molecules; strongest in absence of water D. Protein Structure 1. Primary (amino acid sequence) 2. Secondary (structure of the alpha carbon backbone) a. alpha helix: amino acid side chains project outwards, H Bonds between carbon group and NH2 group stabilizes b. beta sheet: Carboxyl groups and Amino groups form hydrogen bonds NOT involving side chains; side chains extend above and below the sheet 3. Tertiary: structure of one protein with the positions of all side chains a. backbone, ribbon indicates alpha helices and beta sheets, wire, space fill b. DISULFIDE BONDS! → CYSTINE 4. Quartinary (multiple polypeptide chains) : mediated by non-covalent bonds (hydrogen, hydrophobic, etc. ) *Protein families & Protein Domain* -family = similar ancestors, similar amino acid sequence -domain = any segment of a polypeptide chain that can fold independently into a compact, stable, structure -domains may be recombined to create proteins with different functions IV. CARBOHYDRATES A. Sugar and Polysaccharides B. Structure: (CH2O)n where n = ¾/5/6. usually form rings, with a hydroxyl group C. Roles: Energy source/storage (glycogen/glucose, structural support (cellulose), protein and lipid modification V. NUCLEIC ACIDS A. Structure 1. sugar group, phosphate, base 2. bases: a. PYRMIDINES (1 ring) – T/C/U b. PURINES (2 rings) – A/G 3. identified with three letters – lowercase d = deoxyribose; base, number of phosphates 4. bonds formed between 3' carbon and 5' carbon in two nucleotides through condensation reactions B. DNA: double helix -sugar phosphate backbone, highly charged and polar; strands run ANTIPARALLEL -DNA is replicated from 5' → 3' -DNA wrapped in nucleosomes (~ 11 nm, 147 nucleotides); short double helix is wrapped around histones; histone octomer = 8 histones -during interphase, nucleosomes wrapped tightly into chromatin (~30 nm in diameter); during meiosis, packed at chromosomes so it can be split easily C. RNA -sugar = ribose (two OH groups); uracil instead of thymine -single stranded molecule, folds into 3D shapes (held by hydrogen bonds in base pairing) VI. FROM DNA TO RNA – TRANSCRIPTION -gene expression is regulated, so only certain genes are transcribed A. Transcription produces RNA complementary to 1 strand of DNA (ANTIPARALLEL) 1. RNA polymerases: enzymes that carry out transcription (5' → 3' direction) → catalyze formation of phosphodiester bonds between nucleotides that form the sugar-phosphate backbone -RNA polymerases unwind DNA double helix, use ribonucleotide triphosphates to match with correct base on DNA template at active site, rewinds double helix -phosphorylation of TFIIH tells polymerase when it's over B. Types of RNA 1. messenger RNA: RNA molecules that are copied from genes specifying amino acid sequences 2. ribosomal RNA: forms core of ribosomes on which mRNA is translated into proteins 3. transfer RNA: forms adaptors that select amino acids and hold them in place on the ribosome for incorporation into the protein C. Transcription in Bacteria -promotor: specific sequence of nucleotides indicating the start point for RNA synthesis -terminator: stop site, rna polymerase comes off -sigma factor: on bacterial polymerase, recognizes promotor sequence D. Initiation of Eukaryotic Gene Transcription 1. RNA Polymerase I, II, and III → require accessory proteins to initiate transcription *general transcription factors → assemble on the promotor (TFIID binds first on the TATA box). TFIID causes a local distortion in the DNA; other factors assemble, creating the transcription initiation complex, including RNA polymerase 2. RNA Processing a. RNA capping – modification of the 5' end of the mRNA. RNA capped by an atypical nucleotide (G with methyl group) b. Polyadenylation – newly transcribed mRNAs have a structure at the 3' end. 3' end is trimmed by an enzyme that cuts the RNA chain, repeated A nucleotides to cut the end (poly A tail) → secures the mRNA sequence during transport out of the nucleus into the cytosol E. Introns: Non-Coding Sequences 1. introns not expressed, exons = expressed sequences 2. mRNA spliced, exons sticked together 3. small nuclear RNAs are packaged with additional proteins to form snRNPs, which form the core of the spliceosome 4. lariat structure: G and A form phosphodiester bonds between introns, splicing out exons to be joined together 4. alternative splicing: many different proteins can be made from the same gene (exons = not always exons) 5. why introns? Can use genes differently, can move exons around, allows you to mix and match protein domains F. Export of mRNAs from the nucleus -cell must distinguish between debris and useful mRNA -nuclear pore complex manages this; mRNA molecules eventually degraded by the cell VII. FROM RNA TO PROTEIN – TRANSLATION A. An mRNA sequence is decoded in sets of three nucleotides (64 possibilities for 20 amino acids) called codons 1. translation: conversion of RNA → protein 2. tRNA molecules match amino acids to codons -four short tRNA segments are folded (double helical), clover leaf → L shaped structure (H bonds) -3' CCA end attached to amino acid (buried in enzyme tRNA synthetase) *anticodon: 3 nucleotides that bind the complementary codon in an mRNA molecule B. Specific enzymes couple tRNAs to the correct amino acid *aminoacyl – tRNA syntheses covalently couple each amino acid to its tRNA molecule -in the linkage of amino acid to tRNA, ATP is converted to AMP + 2Pi and a high energy bond forms between the amino acid and tRNA C. The RNA message is decoded on ribosomes 1. ribosome: large complex made from 70+ proteins and four rRNAS; a ribozyme (catalyzed by RNA) 2. three binding sites (E, P, A) on ribosome for tRNA and 1 for mRNA (on small subunit) D. Codons in mRNA signal where to start & stop protein synthesis 1. Initiator tRNA AUG codon carries methionine which is later removed (5' → 3', N terminus → C terminus) 2. translation initiation factors 3. STOP codons (UAA, UAG, UGA) signal to the ribosome to stop translation *release factors: bind to any stop codon that reaches the A-site on the ribosome; water instead of amino acid frees the carboxyl end) E. Proteins are made on polyribosomes 1. polyribosomes: large cytoplasmic assemblies made of several ribosomes → multiple initiations mean many protein molecules can be made in a given time than would be possible if each had to be completed before the next one started VIII. PROTEINS IN ACTION A. Selective binding of proteins to ligands depends on weak, non-covalent bonds 1. protein folding = responsible for creating a binding site B. Proteins as enzymes 1. catalyze covalent bond formation or breakage by lowering the activation energy banner 2. generally, enzymes catalyze in 1 direction 3. EXAMPLE: lysozyme severs bacterial cell wall polysaccharides a. enzyme-substrate complex b. 6 sugar residues, hydrolysis between residues c. enzyme strains (resembles a transition state) a residue, 2 amino acids d. forms temporary covalent bonds *enzyme active site → creates microenvironment C. How do cells carry out unfavorable reactions?? → by coupling them to a very energetically favorable reaction so net energy = favorable 1. ATP = energy source. Good b/c cleaving phosphate bonds generates a lot of energy; ~50 kg of ATP is synthesized every day 2. coupling reactions to ATP: phosphate transfer (A+B+ATP → AB +ADP + Pi) D. Regulation of Protein Activity 1. transcription control (long term regulation) is the most energy efficient, but has a slow effect 2. control is fastest at the final step (turning proteins on and off) a. phosphorylation – can activate or inhibit protein, phosphate added, done by protein kinases onto serine, threonine, tyrosine. Phosphorylation causes conformational changes → readily reversible by phophatase ~518 kinases (about 2% of proteome) @ any time, about 1/3 of proteins will be phosphorylated → SRC: active SRC increases cell proliferation. To activate, SH2 must be DEPHOSPHORYLATED by phosphotase, and tyrosine on kinase domain must be PHOSPHORYLATED by kinase b. GTP-binding proteins – protein bound to GDP is “off”, GEF comes in and protein loses GDP, picks up GTP and protein is “on.” GAP helps protein go through GTP hydrolysis and P is removed, so the protein is only bound to GDP and is now “off.” -GEF = guanine nucleotide exchange factor: allows protein to lose GDP, will spontaneously pick up GTP -GAP = GTPase Activating Protein removes P ~100 GTP-binding proteins IX. MOLECULAR MOTORS A. Filaments 1. Microfilaments (actin), intermediate filaments, microtubules (alpha beta tubulins) B. Microtubules and Kinesin 1. Microtubules: tubulin heterodimer (subunit) with alpha and beta particles, and GTP binding molecules a. hollow middle, 10 nm diameter, a/bs wrap around microtubule b. POLAR structure → plus end and minus end 2. Microtubules grow from an organizing center (radially away) – minus end in centrosome, plus end outwards 3. Motors in Neuron move down microtubules on axon, carrying organelles and proteins (anterograde = outward movement) 4. most kinesin motors move to the plus end of microtubules; dynein move to the minus end 5. different motor proteins transport different cargos, motor domains = similar; tail domains are different 6. motors arrange organelles → ex: endoplasmic reticulum extends to edges of the cell, although it originates at the nuclear envelope (kinesin motors drag membrane out) → ex: golgi apparatus sits near/on nuclear envelope because dynein brings it towards N.E. C. How does kinesin move along microtubules??? 1. Kinesin structure a. ATP binding domain head with neck linkers in different directions, coiled coil dimerization region, cargo binding tail 2. Couples ATP hydrolysis to conformational change a. leading head binds ATP, trailing has ADP b. binding ATP → conformational change; neck linker swings forward and dock into head (neck linker switches and flips over, pulling head with it) c. new trailing head loses phosphate, new leading head binds to microtubule → head bound to ATP will be tight and docked; head bound to ADP will be weaker and not docked → kinesin has processive movement (step by step, stays on for a long time), events must be coordinated 3. about 14 different kinesins in mammalian cells, different c terminus (motor domain at C terminus) *minus end directed have c terminus then the motor (different orientation) D. Actin Filaments 1. Actin: thin, flexible protein threads, 2 twisted around, + and and – end 2. skeletal muscle cells → muscles, bundle of fibers, multinucleated cells, myofibril (sarcomere) a. sarcomere: thin and thick filaments; thin = actin and thick = myosin b. myosin-II: head, coiled coil, dimerization region. Tail domains bundle around each other, heads stick outwards (staggered) c. during contraction, myosin moves along actin, pulling Z discs together 3. Myosin couples ATP hydrolysis a. binds ATP, head released from actin b. hydrolysis of Atp → ADP + P; mysoin head rotates into cocked state c. myosin head binds actin filament d. “power stroke” release of P and energy; straightens myosin and moves actin *ADP + P → weak affinity e. myosin doesn't work alone; coordination = unnecessary. Myosin is unattached through most of its cycle *rigor mortis → during death, muscles stiffen bc there is no ATP to unbind the myosin from actin 4. some myosin works in muscles; some move like kinesins X. CELL MEMBRANE A. Membranes act as selective barriers -eukaryotes have a plasma membrane and internal membranes surrounding organelles B. Plasma Membranes -receive information, import and export molecules, capacity for movement and expansion, ability to accommodate cell division -composed of lipids and proteins -only 5 nm thick (in comparison to ribosomes, which are about 20 nm thick) C. Membrane Lipids 1. structure: hydrophilic head, hydrophobic tails 2. phospholipids have phosphate in head, glycerol “back bone”, CH2 (hydrocarbon) tails. Generally, 1 chain has a double bond (unsaturated), kinked chain → main phospholipids in choline; phosphatidyl ethanolamine/serine = polar & charged/choline and sphingomyelin → head groups = charged 3. membranes also contain cholesterol and glycolipids, which have no phosphate group 4. cholesterol structure: small, stiff molecule 5. lipid bilayer → flat sheet would be energetically unfavorable, so sheet wrap into a spherical shell so no hydrophobic tails are exposed 6. lipid movement a. lipids can move 2 microns/second (lateral diffusion) b. rotation and flexion 7. saturated lipids are packed really tightly, less fluid, but mixed saturated and unsaturated are more fluid. Cholesterol stiffens membrane making it let fluid and less leaky, animal cells have about 20% cholesterol in their membranes 8. asymmetric membrane: more negatively charged heads inside the cell, glycolipids on the outside. WHY? a. lipids are synthesized in cytosol and go into soft ER. Flippase transfers phospholipids to other half of the bilayer, even # of lipids on both sides of the bilayer but flippases work selectively so type of lipids on either side isn't uniform D. Membrane Proteins 1. Transmembrane, one layer of bilayer (monolayer), lipid linked, protein attached 2. transmembrane proteins have a hydrophobic portion, and hydrophilic regions on either side 3. the peptide chain usually crosses the bilayer in an alpha helix → problem: even though R groups are hydrophobic, the peptide bonds = hydrophilic so to save energy, alpha helices form – tunnel with hydrophilic portions facing in, to be protected from hydrophobic region 4. common structures have multiple domains that cross the membrane → can form a pore with multiple alpha helices (hydrophilic side chains facing aqueous pore) E. FRAP can test protein mobility in membranes 1. FRAP → bleach with laser, gets rid of GFP fluorescence (square, white bleached area). Mobile proteins will move around and the bleached area will once again fluoresce. Some proteins are fluid, but most are anchored. PRELIM 2 MATERIAL I. MEMBRANE TRANSPORT A. most lipid bilayers block passage of most water soluble molecules 1. small hydrophobic molecules and small uncharged polar molecules can go through passively 2. larger uncharged polar molecules and ions can't B. Transmembrane Proteins move water soluble molecules across cell membranes 1. channels vs transporters 2. channels = pores through membrane, can be open or closed, specificity for ions, “doors” 3. transporters have specificity through binding site. Mechanism: transporter undergoes a conformational change, more limited in number of molecules it can transport at a time C. Passive transport – uncharged molecules move DOWN their concentration gradient 1. simple diffusion, channel-mediated, transporter-mediated ex) glucose transporter has glucose binding sites, follows concentration gradient 2. flux of molecules = allowed D. Passive Transport: Charged Molecules 1. influenced by concentration gradient and voltage gradient (“electrochemical gradient”) E. Active Transport → molecules move AGAINST their electrochemical gradient 1. coupled transport to another molecule moving down its electrochemical gradient a. mechanism for glucose – Na+ coupled transporter b. outside cell, transporter is open and Na+ binds bc it is very likely to bind. Bc of Na+, glucose also binds (it will only bind if sodium is bound first). This causes the transporter to change conformation and release Na+ and glucose into the cell, where Na+ is very unlikely to bind bc there is a low concentration. Changes back to open to the outside cell. 2. ATP driven pump a. Sodium Potassium pump = a way to store energy. Keeps [Na+] 20x higher outside and [K+] 20x higher inside cell b. K = lower EC gradient. Three Na+ binding sites, 2 K+ binding sites c. open to cytosol, binding site for sodium. Pump is phosphorylated (ATP donates a phosphate); phosphate →conformational change, so Na+ is ejected and K+ can bind. Potassium causes dephosphorylation of the pump; pump returns to its original state; ejecting K+ and making a binding site for Na+ d. whole cycle happens in ~10 milliseconds 3. light driven pump (bacteria) *channel can't mediate active transport* F. 2 Types of glucose transporters enable glucose uptake 1. intestinal epithelial cells -[glucose] is low in the lumen; high in epithelium; low in extracellular fluid -[Na] is low inside epithelium and high in lumen and outside cell 2. active Na/glucose transport brings glucose from lumen into epithelium, passive transporter takes it out of epithelium and into blood stream *tight junction = no leaking* *Categories (active or passive)* 1. uniport → 1 molecule 2. symport → glucose-Na+ coupled transport 3. antiport → Na+/K+ pump G. Ion Channels (=ion selective) 1. potassium ion channel has carbonyl oxygens (-) a. K+ in water = hydrated (interacting with water molecules) b. K+ has to shed water molecules, so carbonyl groups make similar reactions with K+ → sodium can't make these connections, so it can't go through this channel 2. K+ channels generate the membrane potential (leak channel) (more positive on extracellular side) -Na/K pump causes [K+] to be high inside the cell. K+ channels allow K+ to flow out (concentration gradient), but this changes electrochemical gradient -when K+ channel is open, electrochemical gradient for K+=0 -when K+ channels are closed → leads to membrane potential 3. ion channels can open and close a. stimuli: voltage, ligand-gated (outside or inside cell), stress-gated channels b. ex: hair cells in ear, vibration causes stress on stereocilia, and stress-gated ion channels open → positive ions enter cell 4. diseases caused by ion channel defects – cystic fibrosis 5. each organelle has its own characteristic set of transporters II. PROTEIN SORTING A. How do proteins get into various places in the cell? 1. what determines where the proteins go? → signals in amino acid sequence → sequence = necessary for protein location @ N terminus ex. Import into ER: stretch of hydrophobic residues import into mitochondria: high and charged amino acids import into nucleus: high and charged amino acids B. Transport into Nucleus 1. synthesized in cytosol, can enter as folded, intact proteins, enter through nuclear pores 2. pore surrounded by 8-folds, protein on cytosol face and inner face 3. proteins like fibril, etc. make up the pore. Material in the middle = proteins that line the nuclear pore, unstructured protein, unfolded, form network 4. small molecules can more through by diffusion, but larger (~30 kilodaltons) need active transport *process: nuclear localization signal, nuclear transport receptor can go through the pore (interacts with fibrils, binds to nuclear localization signal and releases cargo when inside nucleus. This is passive → need to concentrate protein into nucleus → need RAN (GTP binding protein) a. RAN GTP is in high concentration in nucleus, binds to receptor so it releases cargo. Receptor and RAN GTP exit, converted to Ran-GDP, so receptor can bind to protein. Ran GDP and Pi immediately dissociate from the receptor. b. how do we get high [Ran GTP] inside cell? High [Ran GEF] inside nucleus, high [Ran GAP] outside nucleus 5. nuclear import vs. export -nuclear export receptor enters nucleus, binds to RAN GTP → allowing receptor to bind to protein with export signal - leaves nucleus, GTP is hydrolized, both GDP and protein fall off, receptor reenters nucleus 6. DIFFERENCES: in import, binding to RAN GTP causes cargo to be released, in export, binding to RAN GTP causes cargo to bind C. Transport into Mitochondria 1. synthesized in cytosol, enter mitochondria through protein channel → must be unfolded 2. signal sequence binds to receptor protein, diffuses through protein translocator, folded in mitochondria 3. transported through both membranes simultaneously D. Transport into ER 1. ER = entry point for proteins destined for a variety of locations (outside cell, membrane, vesicles) 2. proteins not fully synthesized in cytosol 3. an ER signal sequence and an SRP direct a ribosome to ER membrane a. SRP pauses translation, binds signal sequence and slows translation b. SRP binds to SRP receptor on ER membrane, carrying mRNA and ribosome and translated protein onto ER lumen (translocation channel); SRP leaves as ribosome is passed to translation channel, translation resumed transfer of protein but now into lumen of ER c. allows cotranslocation translation (translocation and translation at the same time) 4. soluble protein crosses ER membrane and enters lumen a. signal peptidase clips signal sequence on N-terminus on lumen side b. protein in ER lumen → fully synthesized 5. transmembrane proteins: integrated into ER, has a stop transfer sequence so part of protein stays on cytosolic side (C terminus in cytosol) *multipass protein does this as well, but has multiple “starts” and “stops” START: put material in lumen STOP: put material in cytosol 6. called the rough ER, plug protein closes translocation membrane III. VESICULAR TRANSPORT -proteins are usually synthesized in ER, need to be transported → golgi apparatus → endosomes or transport vesicles to cytosol A. topology of vesicle budding and fusion 1. donor compartment, budding, fusion → bilayer fuses membrane protein join new plasma, secretion 2. topology of lipid bilayer = preserved B. How do vesicles bud and fuse?? 1. when budding happens, vesicles are coated with proteins that help them pinch off (coat proteins responsible for pinching off the vesicle) different coats = different destination 2. clathrin coated vesicle formation a. selection of cargo to be transported is mediated by transport receptors b. adaptins capture cargo receptors by binding their cytoplasmic tails (mediate between cargo receptors and clathrin) c. coat proteins bind adaptins to start formation of vesicle d. dynamin wraps around “bud neck” and cleaves it off e. uncoat → clathrin comes off as soon as vesicle forms so vesicle can bind to the plasma membrane → clathrin assembles into a 3D cage 3. ER → GOLGI protein coating is COPII; golgi to ER protein coating is COPI golgi to endosome protein coating is ADAPTIN endosome to golgi protein coating is retromer cell membrane to endosome protein coating is adaptin II C. How do vesicles know which membrane to go to? 1. protein on vesicle a. RAB (Rab-GTP bound) and SNARES direct transport vesicles b. target membrane contains a tethering protein and t-SNARE c. first Rab GTP is recognized by tethering protein; then v-SNARE and t-SNARE (docking) → fusion, v-snare and t-snare more away d. Rab GAP hydrolizes GTP in RAB, Rab-GDP falls off and goes back to vesicle where GEF rebinds GTP e. vSNARE and tSNARE twist around each other, pulling membranes close (no water between membranes). A second molecule unwinds vSNARE and tSNARE after fusion D. Key Points 1. transport vesicles, receptors, adaptins, coat proteins, rab protein with GTP, rab GTP, tethering factors, RAB GAP/GEF, vSNARE and tSNARE → specific RAB protein and snares for each transport step E. The Secretory Pathway 1. to cell surface a. ER → many proteins are glycosylated in the ER (cotranslational translocation) → 14C sugar (n-linked oligosaccharides) bind to ASN residues on synthesized protein (some not all) b. protein-linked oligosaccharides NEVER reside in the cytosol c. golgi apparatus: oligosaccharides processing as protein is transported through golgi → sorting to plasma membrane or lysosome done by oligosaccharides; signal for targeting to lysosome = a particular sugar residue (soluble and unsoluble) d. exocytosis: trans-golgi network releases cargo through vesicles i. Regulated → wait for a signal before fusion (ex. Insulin) ii. Unregulated (constitutive) 2. Summary: protein synthesis on RER, N-linked glycosylation → tsport to golgi → modification, sorting, transport out of golgi → transport and delivery and fusion in appropriate destination F. Receptor mediated endocytosis 1. LDL enters cell via receptor-mediated endocytosis; LDL receptors a. clathrin coated vesicle carries LDL, LDL transported to lysosome, low internal pH causes release of LDL, lysosome digests particles and frees cholesterol 2. mutations in LDL receptors cause hypercholesterolaemia (too much LDL in blood stream) → receptors can't bind to LDL and take it up, cholesterol increases in blood OR can bind but receptors aren't able to take it up IV. THE CELL CYCLE A. Cell growth and chromosome replication → chromosome segregation → cell division ~ three trillion new cells/day CELL TYPE CELL-CYCLE TIMES Early frog embryo 30 mins Yeast 1.5-3 hr Intestinal epithelial ~12 hrs Fibroblasts ~20 hrs Liver cells ~1 year B. Cell Cycle divided into 4 phases: G1 → S (DNA replication) → G2 → M (mitosis and cytokinesis) 1. timing of cell cycle carried out by cyclin-dependent kinases (CDKs) a. kinases phosphorylate proteins 2. multiple CDKs in vertebrates 3. m-CDK activity = very high during mitosis m-cyclin concentration = highest in M phase, builds up during interphase C. Distinct CDKs associate with different cyclins to trigger the different events in the cell cycle *CDKs are kinases, so they activate cell cycle events through phosphorylation D. Cyclin – CDK complexes are regulated by inhibitory and activating phosphorylations 1. inhibitory kinase (Wee 1) 2. activating kinase (Cak) 3. activating phosphatase (CDC25) remove the inhibitory phosphate 4. structural basis of CDK activity: Inactive CDK → partly active with cyclin → CDK activating kinase → FULLY ACTIVE 5. phosphorylation events responsible for fully activating CDK 6. why is there a sudden rise in CDK activity? Inactive CDC25 phosphatase → activating phosphatase (CDC25) removed inhibitory phosphate put on by Wee 1 → POSITIVE FEEDBACK LOOP! More active m-cdk → more active CDC25 7. at end of mitosis, cyclin levels drop a. ubiquitin = 75 amino acid polypeptide that can be covalently attached to lysine side chain. Ubiquitilize cyclin, leading it to be inactive b. polyubiquitin chain = marks protein for degredation in proteasome E. Checkpoints ensure fidelity of cell cycle → mechanism that puts a brake on the cell cycle to ensure that one process is completed before the next one starts *G2 checkpoint a. is DNA replicated? b. is all DNA damage repaired? * M checkpoint a. are all chromosomes properly attached to the miotic spindle? *G1 checkpoint a. is environment favorable? b. G1 controls cell proliferation → point at which cells decide whether to enter cell cycle based on environmental signals. Generally, mammalian cells will multiply only if they're stimulated by external growth factors. Differences in cell cycle length = dependent on time in G1 (can withdraw to G 0if they won't ever proliferate) EX) DNA damage can arrest cell cycle at G1 checkpoint a. DNA damaged → activated some kinases → p53 is phosphorylated, stable, and active b. p53 = transcription factor, binds to regulatory region of p21 gene c. p21 binds to G1/S-CDK and S-CDK and inactivates them d. p53 is a tumor suppressor, mutated in ~50% of cancers practice questions: Regarding both channels and transporters (True/False): 1. Transporters and channels only catalyze passive transport. false 2. They facilitate transport of equivalent large numbers of molecules. false 3. They have high specificity for the molecules transported. false 4. They both can be involved in active and passive transport. false Consider a positively charged molecule that is at a higher concentration outside a cell. Passive transport of this molecule into the cell is (CHOOSE ONE): 1. Reduced by the membrane potential 2. Enhanced by the membrane potential 3. Unaffected by the membrane potential 4. None of the above Regarding ion channels (True/False): 1. They enable ions to travel up their electrochemical gradient false 2. They enable active transport false 3. They are always open false 4. They may be gated via mechanical stress true 5. They allow more than one type of ion to pass through false 2. inactivation of Ran GEF would... true/false a. inability to transport specific proteins into the nucleus and keep them there TRUE b. diffusion of NTP-cargo protein complex into the nucleus TRUE c. buildup of NTP-cargo protein complexes in the cytosol TRUE d. buildup of NTP-cargo protein complexes in the nucleus TRUE e. release of cargo protein from the NTP-cargo protein complex FALSE 3. if the pH of the early endosome is neutralized, which of the following will happen? a. the lysosomal enzyme will stay in the TGN false b. the lysosomal enzyme receptor will stay in the early endosome true c. newly synthesized lysosomal enzymes will be secreted outside the cell true V. MITOSIS A. How are chromosomes segregated? --microtubules, mitotic spindle B. Required interphase events 1. chromosome duplication (S phase) 2. chromosome cohesion a. need to identify sister strands of DNA b. recognized during S phase c. @ S phase, cohesion molecules (cohesin) form a ring complex (4 proteins) that wrap around sisters and hold them together 3. centrosome duplication C. Prophase 1. chromosome condensation a. DNA (2nm) → beads on a string (11 nm) → chromatin (30 nm) → condensed to mitotic chromosome (~1400 nm) b. condensin – structurally and sequentially related to cohesins, but different roles → link segments of single DNA molecule to compact it (happens in prophase, fall apart during telophase) → can't link sister chromosomes 2. kinetochore assembly a. constriction in DNA b. mediate attachment of microtubules to chromosomes c. each sister has 1 kinetochore, microtubules recognize kinetochores 3. centrosomes separate a. motor proteins between microtubules push them apart b. Kinesin – 5 has four globular heads, so it can bind to 2 microtubules, both + end directed → push centrosomes apart c. Dynein heads move towards centrosome ( – end). Physically attached to cell membrane, reeling in microtubules d. Kinesin – 14 is a dimer, but its heads move to the minus ends of MT – cargo = microtubule; pulls spindles closer as a balancing force to other motors D. Prometaphase 1. nuclear envelope breakdown: double membrane, pores, lamin keeps it intact (intermediate filaments) a. phosphorylation of nuclear pore and lamins → breakdown, chromosomes free in cytosol 2. microtubule capture by kinetochores a. unipolar attachment (both sisters attached to 1 pole); bipolar (both poles attached, kinetochores pulled in opposite directions so it is more stable) b. problems with microtubule capture i. Both sisters attached to 1 pole ii. Both spindles from either pole attach to the same sister → both unstable, enzymes break attachments E. Metaphase 1. 3 microtubules: aster (don't attach), kinetochore (attach to kinetochores), interpolar (connected to both poles) 2. chromosomes line up on metaphase plate F. Anaphase * NECESSARY BEFORE ANAPHASE BEGINS * 1. cohesions degraded by protease called separase a. active separase cleaves cohesins, allowing chromosome separation b. securin = inhibitory protein that binds to separase, making it inactive → APC adds ubiquitin to securin; secrurin is degraded, separase is active → APC also ubiquitinates cyclin @ end of M phase *M-Phase checkpoint* -unattached kinetochore inactivates APC – so, if microtubules aren't properly connected, m-phase checkpoint will be activated and cell won't progress into anaphase 2. anaphase A – chromosomes move to poles -shortening of kinetochore microtubules caused by depolymerization - + ends falling off → kinetochore stays attached through a “sleeve” - collar follows microtubule, moves along and maintains interactions so kinetochore stays on and attached 3. anaphase B – poles move apart (pushed & pulled) a. pushed apart → microtubule growth @ + end of interpolar tubules (Kinesin 5) b. pulled apart → dynein G. Telophase 1. nuclear envelope reassembly → dephosphorylation of nuclear pore proteins and lamins a. pore complexes reassemble around DNA (initially very tight assembly, proteins have to be moved into nucleus again through pores) 2. chromosomes de-condense H. Cytokinesis 1. separation of 2 cells 2. contractile ring of actin and myosin VI. CELL SIGNALING I -in animal cells, all cells need to communicate to know how to act at certain times -example: neutrophils sense certain compounds, polarize, and move towards the source; reorient towards the source -signal in → detected by receiver → transduced → response -about 1/3 of human genome (7,000) are involved in cell signaling A. General principals of signaling 1. signaling molecule, receptor that receives signal, pathway (intacellular) → effector that produces some effect in the cell 1. Signaling molecule: can be peptides, proteins, fatty acids, nucleotides, etc. also light & mechanosensory signals 2. receptor can be membrane bound or inside the cell 3. ISP: relay, amplify, integrate, or distribute 4. effector: can alter gene expression, metabolism, cell shape or movement, etc. B. Signals delivered in various ways 1. endocrine system: molecule (hormone) secreted by endocrine cell, secreted in blood stream, goes to target cells. Long range, non-discriminate (can go to many different cells, as long as they have receptors). Hormone receptors have high affinity b/c they encounter the hormones in a low concentration 2. Paracrine Signaling: signaling cell secretes out of cell but not into blood stream, just around: short range, molecules secreted are local mediators 3. Neuronal Signaling: signal starts in cell body, electrical conduction down axons, releases neurotransmitters (packaged in secretory vesicles); regulated secretion, release contents into synaptic cleft and into target cell. Very specific target cells activated (close proximity to axon terminal); but still long range bc it travels down long axons. Neurons secrete large amounts of neurotransmitters into a small space → high concentration, so receptors don't need a very high affinity 4. Contact dependent signaling: membrane bound molecules are the signaling molecules, only spread signal if they are right next to target cell with receptor (NON soluble signaling molecules) * each cell produces only a limited set of receptors, restricting the types of signals to which it can respond * ability of a cell to respond to a particular signal depends on its: i. Developmental history ii. Current state C. Same signal, different response: Acetylcholine 1. acetylcholine is a neurotransmitter used by a variety of neurons a. heart muscle cells: acetylcholine tells heart to slow down and beat more slowly & with less force b. salivary gland: acetylcholine secreted, activate salivary glands → secretion of saliva * same receptor for both types of cells, but very different responses * c. skeletal muscle cell: different receptor, but skeletal muscle contraction D. Signals can act rapidly or slowly 1. rapid example: seconds to minutes; altered protein function is relatively fast 2. slow example: minutes to hours; altered gene expression takes a long time (requires synthesis of new molecules) E. Types of Receptors 1. Cell Surface Receptors: accept hydrophilic signaling molecules, so need to have receptors open to extracellular space (and then transduce response into cytosol) 2. Intracellular receptors: can accept small hydrophobic signal molecules (i.e. steroid hormones) a. steroid hormones: ring structure, very hydrophobic, testosterone/cortisol, etc; can pass through membrane easily. Receptor inside cell. Steroids enter cytoplasm and bind to an inactive protein, which then is active and enters the nucleus (initiating gene expression) b. each hormone binds to a different protein receptor (activating different genes, determined by type of receptor) c. a given hormone usually regulated different sets of genes d. example: nitric oxide relaxes blood vessels. BV lined by endothelial cells, smooth muscle cells, and nerve cells. Muscle cells expand and contract. Nitric oxide is a response to acetylcholine in the endothelial cells (argenine → NO); NO diffuses across membrane and into a target protein in smooth muscle cells, causing relaxation of smooth muscle cells → nitroglycerine is a drug that treats angina (poor blood flow to heart). Converted to NO to relax blood vessels. i. In the cell, NO binds to guanylyl cyclase (activating it) and producing cyclic GMP from GTP, which increases blood flow. When there is no more NO, phosphodiesterase turns cyclic GMP into GMP (deactivating it). ii. Drug research: inhibit phosphodiesterase, so blood flow is increased. Drug didn't work in treating angina but had an interesting side effect → now used as viagra 3. Cell surface receptors: respond to hydrophilic molecule signals -need a transmembrane receptor. Cascade in signaling (transduction; intracellular signaling molecules). → impact effector proteins and lead to cell response a. ISM can be: kinases, phosphatases, GTP binding proteins, lipids, proteases, enzymes, adaptors, 2 nd messengers (cGMP, cAMP, Ca+2) b. intracellular signaling proteins as switches i. Signal in: phosphorylates protein, makes it active, sends signal out ii. Signal in: GEF removed GDP and adds GTP, protein is on and sends signal out, GTP is phosphorylated by GAP and turned off ii. Simple conformational change (second messenger, protein-protein interaction, etc.) F. What does intracellular signaling accomplish? 1. allows relay, amplification (one extracellular signal molecule can result in the production of a ton of small intracellular messenger molecules); integration (responses from multiple different extracellular signal molecules can come together and be integrated to one response); distribution (pathway diverges into several different pathways → one signal, several different responses) 2. example of integration: two signals, “A” and “B”, both produce activation pathways that can activate two different kinases, and both kinases phosphorylate a protein that requires these two phosphates to be fully active. Or, two different proteins are phosphorylated and then bind together, leading to a response. G. Multiple signals, integrated response (if cells don't get signals, they die → apoptotic pathways, enzymes digest the cell if it doesn't receive “keep living” signals) H. Three main classes of cell-surface receptors 1. Ion channel-coupled receptors a. common in nerve and muscle cells, can change membrane potential, depolarize the cell, calcium 2. G Protein coupled receptors a. bind GTP. Receptor (7 pass transmembrane protein); receive signal, activate g protein, activates an enzyme b. most numerous class (>700 in humans) c. most studied d. most frequently targeted of drugs (~30% of drugs) 3. enzyme coupled receptors a. signal molecule in form of a dimer activates catalytic domain of receptor; or signal molecule activates enzyme on receptor PRELIM 3 MATERIAL: I. CELL SIGNALING II A. G-Protein Coupled Receptors (GPCRs) 1. contain extracellular domain in charge of recognizing signal, transmembrane part (7 domains that cross), and cytoplasmic part that relays the signal 2. largest family of cells surface receptors (>700 in humans), a wide variety of signaling molecules can activate GPCRs, ~50% of all known drugs work through GPCRs (target binding or signaling) 3. receptor receives molecule from outside cell, activating it (sending out signals to activate G protein or allowing it to bind. G protein on cytosol side: alpha, beta gamma subunits. Lipids link alpha and gamma subunits to the membrane. When inactive, G protein is bound to GDP → when active, GDP → GTP, allowing it to bind to the GPCR (GDP leaves, GTP attaches). The complex breaks into activated alpha subunit bound to GTP and activated beta gamma complex (sometimes, not always). These activated subunits activate other proteins which send signals through the cell. RGS inactivates the GTP bound alpha subunit, turning it into GDP so this alpha subunit can bind to other beta and gamma complexes. – receptors can be phosphorylated and bound to arrestins, which end it's ability to bind to G proteins inside the cell 4. most frequent targets: andenylyl cyclase or phospholipase C 5. Adenylyl Cyclase resides on membrane, in close proximity to GPCR. When active alpha subunit binds to it, it is activated. ATP → cyclic AMP. Cyclic AMP is a very small molecule, giving it incredible versatility (can diffuse through cytoplasm). It is a second messenger. This is why GPCRs can act very quickly, because of cyclic AMP. a. cyclic AMP activates PKA → phosphorylates effectors. PKA has two regulatory subunits and two catalytic subunits. The catalytic subunits are freed when phosphorylated, and can phosphorylate other molecules. Ex: can phosphorylate CREB, and the CREB complex binds to target genes. b. cyclic AMP can increase heart rate, breakdown glycogen/fat, can increase cortisol secretion. Many of these are due to adrenaline as a signaling molecule c. example: active PKA phosphorylates kinase phosphorylase, which activates glycogen phosphorylase, which is involved in glycogen breakdown d. example: PKA goes to nucleus and phosphorylates transcription factors, activating transcription and gene expression 6. Phopholipase C (PLC pathway): GPCR activates alpha subunit, activates phospholipase C. catalyzes hydrolysis of inositol phospholipid (membrane bound), consisting of lipid moity and sugar moity that can be hydrolized, and then travel into cytoplasm and be received by ER membrane, releases calcium. The diacylglycerol is recruited to membrane by PKC, and activated by the calcium. a. effects: glycogen breakdown, secretion of amylase, contraction, aggregation B. Enzyme Coupled Receptors 1. inactive catalytic domain on plasma membrane (transmembane) proteins contain catalytic activity by themselves (off when in resting state). When the signal is received, it turns on. ECRs also only crosses the membrane once. Often, signal molecule will form a dimer in the extracellular space, and then attach to two ECRs, both are activated. 2. RTKs (receptor tyrosine kinases): tyrosine kinase domain inside cell, phosphorylates tyrosine residues. Signal is bound, catalysic activity activated, each component can transphosphoyrlate other subunit. Phosphates form on the dimer, so it is activated. A variety of proteins are bound to these phosphates on activated receptor a. to inactivate RTKS: protein tyrosine phosphates can remove the phosphates, or endocytois can remove the active receptors from the cell surface 3. signal relayed by activated signaling proteins attached to RTKs into cells interior: a. Phospholipase C (PLC) → IP3 pathway b. inactive Ras Proteins tethered to membranes (inactive when bound to GDP). Adaptor proteins bind to RTKs, and these bind to Ras GEF, which is a Ras activating protein. It switches GDP for GTP to Ras protein, activating it. c. MAP kinase signaling molecule: activate Map Kinase kinase kinase (three kinases). Map KKK activates MAP KK, which activates MAP kinase. Map Kinase mediates activation of mitosis, and also phosphorylates many targets (gene transcription, protein activity, cell proliferation, cell survival, cell differentiation, etc.) d. PI3 Kinase: when cells don't receive any “keep living” signals, they die. Survival Signal activates RTK, activating PI3 kinase which phosphorylates inositol phospholipid. Phosphorylated inositol phospholipid activates protein kinase 1, activating AKT. Protein kinase 2 (activated by another signaling pathways) also activates AKT. Active AKT activates Bad. Unphosphorylated Bad forms a complex, inhibiting an apoptosis inhibitor. When it's phosphorylated, apoptosis inhibitor is active. *SIGNALING PATHWAYS ARE HIGHLY INTERCONNECTED* C. Mutations in cell signaling pathways & their effects: Breast Cancer 1. at least 30% of b. cancer is due to a mutation that causes over expression of the RTK HER2 kinase. It is strongly associated with increased disease recurrence and a worse prognosis. Treatment uses the monoclonal antibody trastuzumab (marketed as Herceptin) that targets HER2 receptor. 2. Her2 is a RTK that responds to a variety of growth factors a. HER receptors receives growth factors. HER2 doesn't need ligand binding to form dimers, and is transactivated by HER1. This mechanism sensitizes cells so they can recognize and respond to growth factors in low concentrations. The more HER2, the more it can respond to low concentrations of growth factors. If HER2 is abnormally overexpressed, cells proliferate more than they should. b. The Herceptin antibody recognizes excess HER2 receptors and prevents it from binding/activating other HER RTKs II. INTRO TO DEVELOPMENT – OVERVIEW A. Intro to Development 1. What is included in the study of developmental biology: embryology, metamorphosis, juvenile growth, adult growth, wound healing, tissue homeostasis, reproduction 2. why study? Intellectual curiosity, medical relevance (in vitro fertilization, cancer biology, birth defects, wound repair, potential for regeneration), agricultural relevance (maximizing yield, in vitro fertilization, cloning of animals) 3. How do we study development? Descriptive embryology, experimental embryology and developmental genetics B. Descriptive embryology 1. put a dye into some cells in development – see what they become (ex. Eye) 2. eggs – watch animals develop (frogs, sea urchins, etc.) C. Experimental embryology 1. get a better understanding of formation mechanisms 2. cut embryos, transplant pieces, put cells in culture and see what they become, etc. D. Developmental Genetics 1. using genetics to get mutations and tell us what genes are important for development 2. normal gene vs. mutated gene 3. human developmental genetics – they come to doctors with abnormalities, try to identify genes based on mapping experiments 4. similar mechanisms promote development of all animals: up to 50% of the genes in worms, flies, or fish are also present in humans and carry out similar functions a. ex: mouse lacking engrailed 1 → put in drosophila engrailed 1; normal development for mouse E. Major Events of Embryogenesis (Embryonic Development) – Fertilization 1. Fertilization, Cleavage, Gastrulation, Neurulation, Organogenesis 2. Fertilization: egg and sperm form zygote. Mother and father: diploid, 2 copies of each chromosome (2n, 46 total chromosomes). 23 chromosomes in egg, 23 in sperm → come together to form diploid (46 chromosomes in zygote) 3. Meiosis: sperm and egg fuse, giving zygote 46 chromosomes. Meiosis = gamete production a. maternal and paternal set, both copied and linked tightly to their twins (sister chromatids). All align on metaphase plate, forming bivalent. Crossing over at chiasmata occurs, exchanging information (leads to genetic variation) b. two copies separate, so each now has 46 again. Then they divide again, giving 4 daughter cells with 23 chromosomes each. Each gamete is different from the others c. meiosis vs. mitosis: Mitosis has DNA replication, no crossing over, each daughter cell has one red and 1 blue chromosome. Meiosis has crossing over/recombination, daughter cells are haploid (23 chromosomes) 4. Fertilization: creates a unique diploid individual, initiates embryogenesis by activating the eggs. Fertilized egg protected by fertilization envelope. Exocytosis releases hydrolitic enzymes stored in vesicles, causing swelling of fertilization envelope F. Cleavage – cell division without cell growth 1. cleaving cells: no G2 phase, no G1 phase. Cells get smaller and smaller. Cleaving cells are called blastomeres, at the end of cleavage, the embryo is called the blastoma G. Gastrulation 1. phase when blastoma rearranges cells 2. cels from outer lager sweep inside, forming a deep cavity. Different cell layers have different fates 3. cells in middle layer (mesoderm) = muscle tissue, inner organs; cells in inner layer (endoderm) = lines digestive tract; cells on outer layer (ectoderm) = skin, surface structures, central nervous system, neural crest 4. germ cells are set aside early in development, do not arise from a particular structure H. Neurulation: first step of developing nervous system 1. part of ectoderm invaginates, pinches off to form neural crest 2. only in vertebrates I. Organogenesis: formation of organs (lungs, stomach, eyes, etc.) J. What Mechanisms drive these events? – cell proliferation, cell specialization, cell interaction, cell movement 1. 411 cell types in humans – what makes them different? Instructional molecules in their cytoplasm, cell-cell interactions, differences in mRNA and differences in proteins (NOT DNA – all cells in an organism has the same DNA) 2. Differential gene expression: all cells have the same DNA, different cells express different subsets of genes, cells “remember” their identity (remember they are skin cells, remember what genes they need to express, etc.). effected by cell signaling, etc. K. Central questions: 1. what makes cell different? Differential gene expression 2. how do cells become different in an organized and reproducible way? Regional specification and axis formation 3. how do cells organize into tissues and organs? Morphogenesis 4. how do similar cells organize into different shapes and patterns? Pattern formation 5. how do cells maintain tissues and organs? Stem cells III. DEVELOPMENT – Part 2 A. How can we know the fate of a cell? 1. cell lineage tracing – looking into the microscope and tracing cells a. C. elegans (worm) – has very few cells, map determined. Only organism for which it is done (few cells, and c. elegans always does this the same way) b. take worms at different stages of development, dissecting them o
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