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Cell Biology

by: Kyler Ondricka

Cell Biology BIOL 3340

Kyler Ondricka

GPA 3.84

Alfred Merrill

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Alfred Merrill
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This 0 page Study Guide was uploaded by Kyler Ondricka on Monday November 2, 2015. The Study Guide belongs to BIOL 3340 at Georgia Institute of Technology - Main Campus taught by Alfred Merrill in Fall. Since its upload, it has received 22 views. For similar materials see /class/233990/biol-3340-georgia-institute-of-technology-main-campus in Biology at Georgia Institute of Technology - Main Campus.


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Date Created: 11/02/15
CELL BIOLOGY TEST 1 OUTLINE CHAPTER 2 21 Covalent Bonds amp Noncovalent Interactions O O O 22 O O O O O O O 0 Many molecules in cells contain at least one asymmetrical carbon atom which is bonded to 4 dissimilar atoms Such molecules exists at optical isomers mirror images designated D or L In biological systems nearly all sugars are D isomers and all amino acids are L isomers Noncovalent bonds are considerable weaker than covalent bonds The 4 types of non covalent bonds in biological systems are ionic bonds hydrogen bonds strongest van der Waals weakest and hydrophobic effects The high degree of binding specificity that results from molecular complementarity is one of the features that underlies intermolecular interactions and thus is essential for many processes critical to life Chemical Building Blocks of Cells Three major biopolymers formed by polymerization rxns net dehydration of basic chemical building blocks are present in cells Proteins composed of amino acids linked by peptide bonds Nucleic Acids made of nucleotides linked by phosphodiester bonds Polysaccharides composed of monosaccharides sugars linked by glycosidic bonds Phospholipids the fourth major chemical building block assemble noncovalently into biomembranes Differences in the size shape charge hydrophobicity and reacticity of the side chains of the 20 AAs determine the chemical and structural properties of proteins The bases in the nucleotides composing DNA and RNA are carbon and nitrogen containing rings attached to a pentose sugar They form 2 groups Purines Adenine and Guanine Pyrimidines Cytosine and ThymineUracil Glucose and other hexoses can exist in 3 forms Open chain linear structure Six member pyranose ring D glucose predominates in biosystems Five member furanose ring Glycosidic bonds are formed btwn either the d or the B anomer of one sugar and a hydroxyl group on another sugar leading to formation of disaccharides Phospholipids are amphipathic possessing both hyd rophilic and hydrophobic properties with a hydrophobic tail 2 fatty acyl chains connected by a small organic molecule often glycerol to a hyd rophilic head Long hydrocarbon chain of a fatty acid may contain no C C double bonds saturated or 1 double bonds unsaturated A cis double bond bends the chain The hydrophobic effect is entropy driven When aggregated there is less order and thus more energetically favorable Triacylglycerols and most other lipids will aggregate to reduce the surface area that s in contact w water via van der Waals and hydrophobic interactions The shift follows 3 patterns Bilayer sheet heads pointing away and tails inside together O I 23 O O O I 24 O O O O O CHAPTER3 Micelle spherical arrangement with heads pointing out Liposome Sphere with smaller sphere in middle donut shaped The stability of the phospholipid bilayer is due to its favorable enthalpy and entropy Chemical Equilibrium The Keq is the measure of the extent of a reaction and depends on the temp pressure and chemical props of the reactants and products but is independent of rxn rate and of the initial concentrations of react amp prod Acids release protons while bases bind them In biosystems most common acidic groups are the carboxyl and phosphate groups while the most common base is the amino group Buffers are mixtures of a weak acid HA and its corresponding base A Biosystems use buffers to maintain their pH within a narrow range Biochemical Energetics Change in free energy AG is the most useful measure for predicting the direction of chemical rxns in biosystems Rxns tend to proceed spontaneously when AG is negative and vice versa The magnitude of AG is independent of rxn rate Many otherwise energetically unfavorable cellular processes are driven by the hydrolysis of phosphoanhyd ride bonds in ATP Directly or indirectly light nrg captured by photosynthesis is the ultimate source of chemical nrg for almost all cells Oxidation rxns are always coupled with reduction rxns OILRIG Biological redox rxns often are coupled by e39 carrying coenzymes such as NAD amp FAD Redox rxns with a positive AE have a negative AG and thus proceed spont 31 Hierarchical Structure of Proteins O O 0 Protein is a linear polymer of AAs linked together by peptide bonds Various mostly noncovalent interactions btwn AAs in the sequence stabilize a protein s specific folded 3D structure or conformation Protein Secondary Structure the a helix B strand and sheet amp B turn are most prevalent elements stabilized by H bonds btwn atoms of the peptide backbone Protein Tertiary Structure results from hydrophobic interactions btwn nonpolar side groups amp H bonds btwn polar side groups and the polypeptide backbone These stabilize the folding of the secondary structure Different combos of secondary structures translate into different specific fxns Incorporation of distinct independently folded regions of 2quotd amp 3rd structures in the course of evolution has generated diversity in protein structure ampfxn Protein Quaternary Structure defined by the number amp organization of individual polypeptide subunits in multimeric structure composed of several identical or different subunits held together by weak bonds proteins Cells contain large macromolecular assemblies in which all necessary participants in complex cellular processes DNA RNA amp protein synthesis photosynthesis etc are integrated to form molecular machines O Homologous proteins which have similar sequences structures amp fxns evolved from a common ancestor 32 Protein Folding O 0 Sequence of a protein determines its 3D structure which in turn determines its fxn ie fxn derives from structure structure derives from sequence Because of this principle newly synthesized proteins must fold into correct shape to fxn properly The planar structure of the peptide bond limits the number of conformations a polypeptide can have The AA sequence dictates its folding structure its native state Proteins will unfold or denature if treated ender conditions that disrupt the noncovalent interactions stabilizing the 3D structure Protein folding in vivo occurs with assistance from chaperones which bind to new polypeptides emerging from ribosomes and prevent their misfolding 33 Protein Function 0 O O Fxns of nearly all proteins depend on ability to bind other molecules ligands The affinity of a protein for a specific ligand refers to the strength of binding usually expressed as dissociation constant Kd Ligand binding sites on proteins are chemically and spatially complementary to the ligands themselves Enzymes accelerate chemical reactions by bringing the reactants together and lowering the energy of activation for the transition state of the reaction progress curve An enzyme active site usually only a small part of the protein comprises 2 functional parts Substrate binding site responsible for extreme specificity of enzymes Catalytic site Initial binding of subtrates S to enzymes E results in the formation of an enzyme substrate complex ES which undergoes 1 rxns catalyzed by the catalytic groups in the active site until the products P are formed and diffuse away from the enzyme E 5 ES ES ES E P Enzyme reactions often also involve conformation changes 0 34 Regulating Protein Function 1 Protein Degradation O O 0 Proteins can be regulated at the level of synthesis degradation or the intrinsic activity of proteins through noncovalent or covalent rxns Life span of intracellular proteins largely depends on their susceptibility to proteolytic degradation Many proteins are marked for destruction with a polyubiquitin tab and then degraded within proteasomes large cylindrical complexes 36 Purifying Detecting and Characterizing Proteins O 0 Various assays are used to detect and quantify proteins Some use light producing rxns to generate a readily detected signal Others produce an amplified colored signal w enzymes Centrifugation separates proteins on the basis of their rates of sedimentation which is influenced by their masses and shapes 0 CHAPTER 4 41 Structure of Nucleic Acids O O OO O O O 0 To conduct a proteomic analysis partial separation and sometimes identification of proteins by means such as gradient centrifugation electrophoresis immunoblotting etc followed by mass spectrometry DNA contains info to specify the AA sequences of proteins It is transcribed into several forms of RNA mRNA tRNA amp rRNA Both RNA and DNA are long unbranched polymers of nucleotides which consist of a phosphorylated pentose linked to an organic base purinepyr Adjacent nucleos are attached via phosphodiester bonds w a 5 93 direction Natural DNA contains 2 complementary anti parallel polynucleo strands wound together in a regular right hand double helix with bases on inside and the sugar phosphate backbones on the outside Base pairing btwn strands and hydrophobic interactions btwn adjacent base pairs stacked perpendicular to the helix axis stabilize the native structure Binding of protein to DNA can deform its helical structure causing local bending or unwinding of DNA Heat causes the DNA strands to denature separate The melting temp Tm of DNA increases w the percentage of G C base pairs Circular DNA can twist onto themselves forming supercoils Enzymes called topoisomerase can relieve torsional stress and remove supercoils from DNA Cellular RNAs are single stranded polynucleos some which form well defined 3rd pseudoknot and 2quotd hairpin amp stem loop structures Some RNA called ribozymes have catalytic activity 42 Transcription O O O O O O 43 O OO 0 Central Dogma DNA 9 RNA 9 Protein Transcription of DNA is carried out by RNA polymerase which adds 1 ribonucleotide at a time to the 3 end of a growing RNA chain The sequence of the template DNA determines the order in which the ribonucleos are polymerized to form an RNA chain Initiation RNA polymerase RNAP binds to a promoter in DNA melts the DNA to reveal the unpaired template strand and polymerizes the first 2 nucleos complementary to the template strand This melted region of 12 14 bps is known as transcription bubble Elongation RNAP moves down the DNA 3 95 melting ahead of the polymerase so that the template strand can enter the active site of the enzyme and allowing complementary DNA stands to reanneal after being transcribed rNTPs are added to growing RNA strand Termination When RNAP reaches termination sequence completed RNA strand is released and RNAP dissociates from DNA Addition of 5 cap and 3 poly A tail occurs during RNA processing where introns are spliced out and exons are combined Decoding of mRNA by tRNAs Genetic information is transcribed from DNA into mRNA in the form of an overlapping degenerate triplet code codon AUG is most common start codon UAG UGA amp UAA are stop codons A reading frame the sets of codons is then translated into the linear sequence of AAs in a polypeptide chain tRNA have specific 3D shape including an arm for attachment of an AA and a stem loop with a 3 base anti codon to pair with the its codon from the mRNA O 44 O O O 0 Each of the 20 aminoacyl tRNA synthetases recognizes a single AA and covalently links it to a tRNA forming an aminoacyl tRNA The rxn activates the AA so it can participate in peptide bond formation Stepwise Synthesis of Proteins on Ribosomes Translation RNA 9 Proteins Ribosomes where translation occurs contains a large and small subunit The large subunit contains a 23 S amp SS rRNA in bacteria and 285 SS amp 585 rRNA in eukes The small subunit contains 165 and 18S rRNA respectively as well as E Exit P polypeptide and A Aminoacyl tRNA sites for tRNA matching Initiation ribo subunits assemble near translation start site in an mRNA molecule with the initiating tRNAMet base paired with start codon Elongation continuous cycle of 4 step process involving Loose Binding of aminoacyl tRNA to A site on ribosome Tight Binding of the correct tRNA with proper anticodon to the A site accompanied by release of previously used tRNA from E site Transfer of the growing peptidyl chain to the incoming AA catalyzed by the large rRNA Translocation of the ribosome to the next codon thereby moving the peptidyl tRNA from the A site to the P site and the now unacylated tRNA in the P site to the E site In each cycle the ribosome undergoes 2 conformational changes monitored by GTP Binding proteins Wobble position is 3rd base of codon 1St base of anticodon Termination carried out by 2 factors Those that recognize stop codons Those that promote hydrolysis of GTP followed by cleavage of peptide chain from tRNA in P site Also release of tRNAs and the ribo subunits 45 DNA Replication O 46 0 Each strand of DNA acts as a template for synthesis of a daughter strand and remains base paired to the new strand forming a daughter complex using a semi conservative mechanism discovered by Meselson Stahl New strands are formed in 5 93 direction Replication begins at sequence called origin ori of which there are multiple sites most are A T rich DNA helicases unwind the strand in both directions At fork one strand leading is elongated continuously while the other lagging strand is formed by a series of Okazaki frags formed every few hundred nucleos Frags joined by DNA Ligase Helicases use energy from ATP hydrolysis to separate the strands Primase synthesizes a short RNA primer for the 3 end forming a short 5 RNA 3 DNA daughter strand DNA Repair Mechanisms for DNA Repair include Base Excision Repai fix point mutations DNA glycosylase removes unwanted nucleotide APEI endonuclease breaks backbone where mutation occurred AP lyase removes entire backbone where error was DNA Pol B amp DNA Ligase add correct nucleo Mismatch Repair repairs mutations incorporated during DNA replication similar to Base Excision except it occurs after DNA Replication Nucleotide Excision Repair repairs T T dimer caused by UV irradiation and other chemically modified bases that distort the shape CELL BIOLOGY TEST GUIDE 3 CHAPTER 13 Moving Proteins into Membranes and Organelles Major ProteinSorting Pathways FIG 131 0 Non Secretory Synthesis of proteins lacking an ER signal done on free ribosomes Proteins wo target sequence are released into cytosol proteins w organelle specific targeting sequence are first released into cytosol and then imported to mitochondria chloroplasts peroxisomes or nucleus 0 Secretory Ribosomes synthesizing nascent proteins are directed to rough ER by ER signal sequence After translation is done on ER proteins move via transport vesicles to Golgi complex Further sorting delivers proteins to plasma membrane or lysosomes Protein Sorting delivery of newly synthesized proteins to their proper cellular destinations encompasses 2 different processes 0 1 Non Secretopy Pathway Targeting a protein to membrane of an intracellular organelle can occur during translation or soon after synthesis is complete 0 2 Secretory Pathway process begins in ER thus all proteins are targeted to ER membrane first involves nascent proteins incompletely synthesized once translocated across ER membrane proteins assembled into native conformation by protein folding catalysts present in lumen of ER done in 3 Steps Protein synthesis and translocation across the ER membrane Protein folding and modification inside ER lumen Protein transport to the Golgi lysosomes or cell surface via budding fuse vesicles 0 All proteins except few mito amp chloroplast proteins are encoded by nuclear genes and start their lives in the cytoplasm 0 Proteins are delivered to specific destinations based on address tags or signal sequence SigSeq signal encoded win AA sequence amp receptors can only bind to specific SigSeq Cotranslational Translocation CTT translocation and translation occur simultaneously cells are homogenized breaking apart cells releasing organelles and cytoplasm fracturing the plasma membrane and physically shearing the rough ER into microsomes small closed vesicles 0 Microsomes have similar orientation as rough ER bound ribosomes are on outside bc of this they have much greater buoyant density than other membranous organelles can be separated via combo of differential amp sucrose density g rad ient centrifugation 0 Experiment FIG 134 performed cell free protein synthesis with either microsomes present or absent Absent add mRNA that encodes secretory protein amp ribosomes no microsomes 9 protein is made but will not cross into ER protease sensitive Present add mRNA and microsomes 9 protein is translocated into ER lumen amp signal peptide on N terminus is cleaved protease resistant Conclusion insertion is co translational amp signal peptide is cleaved in ER Steps in Targeting Proteins to ER FIG 136 CTI39 is initiated by 2 GTP hydrolyzing proteins signal sequence recognition mechanism necessary for targeting secretory proteins to ER 2 key components in ER SignalRecognition Particle SRP cytosolic ribonucleoprotein particle that transiently binds to both the ER SigSeq in a nascent protein amp large ribosomal subunit large complex recruited by synthesis of 6 12 signal peptides mostly hydrophobic AAs which halts translation the SRP protein that binds to SigSeq in nascent secretory proteins discovered bc of its ability to chemically cross link to ER SigSeq SRP Receptor complex of ribosome SRP mRNA amp nascent protein is targeted to receptor on ER membrane once bound proteins cotranslationally inserted across ER 0 Steps 1 2 ER signal emerges from ribosome amp bound by SRP 0 Step 3 SRP delivers complex to SRP Receptor in ER membrane interaction is strengthened by binding of GTP to SRP and its Receptor 0 Step 4 Attachment of ribosomenascent polypep to translocon complex on ER membrane associated with the translocation of nascent polypeptides creates opening of translocation channel and insertion of SigSeq amp growing polypep into central pore once translocon opens SRPReceptor dissociate from complex amp hydrolyze their bound GTP GTP GDP Pi allowin ins tion of another ol e chain ampallowin translation to resume Both SRP amp Receptor have GTP attached once translocon opens SRPReceptor complex splits away and GTP GDP GDP attaches to another ribo complex GDP is phosphorylated to become GTP Repeat 0 Step 5 As polypep chain elongates it passes thru translocon channel into ER lumen where SigSeq is cleaved by signal peptidase amp rapidly degraded 0 Step 6 Polypep chain continues to elongate as mRNA is translated toward 3 end bc ribosome is attached to translocon growing chain is passed thru translocon into lumen 0 Steps 7 8 Once translation ends ribosome is released remainder of protein is drawn into ER lumen translocon closes amp protein assumes native folded conformation 0 Step 9 Glycosylation enzymatic process that links saccharides to produce glycans polyoligosaccharide of protein by oligosaccharidal transferase OST which transfers sugars to nascent chain process makes sure proteins fold correctly amp confers stability 0 Summam SRP amp Receptor not only help mediate interaction of nascent secretory protein w ER membrane but also permit elongation amp synthesis of complete proteins only when ER membranes are present bc SRP slows translation when attached to ribosome but not ER membrane ie translocon Translocon FIGS 137 amp 138 0 Translocon must allow passage of variety of polypep seqs while remaining sealed to small molecules ie ATP AAs 0 Must be regulated somehow to keep channel closed in its default stage amp open when ribonascent chain complex binds to it Short helical peptide acts as plug to closeopen channel 0 Sec61a cross linking experiments show that it is a translocon component that covalently binds with nascent secretory proteins as they pass into ER lumen Hourglass shaped channel thru center of pore is lined by ring of hydrophobic isoleucine residues at constricted waist of hourglass forms gasket keeping channel sealed to small molecules even when ol e 39 39 thru l L W lquot NeIther can hydrolyze ATP or otherWIse provide energy to drive translocatIon Energy derived from chain elongation at ribosome translation is sufficient to push polypep chain across membrane in one direction 0 Signal Peptidase transmembrane ER protein that cleaves SigSeq of elongating chain it recognizes seq on C terminal side of hydrophobic core of signal peptide amp cleaves N term o Translocon stays open until translation is complete and entire polypep chain is inside lumen Topological Classes of Integral Membrane Proteins FIG 1310 refers to number of times that its polypep chain spans the membrane amp the orientation of these membrane spanning segments key elements distinguishing different classes of proteins is based on a helices containing 20 25 hydrophobic AAs that contribute to energetically favorable interactions win hydrophobic interior of phosphor bilayer o Classified by their orientation in membrane amp types of signals they contain to guide them 0 SinglePass Proteins includes Types I II amp III only have one membrane spanning a helical segment Type I have cleaved N terminal ER SigSeq anchored in membrane w hydrophilic N terminal region on luminal face aka exoplasmic face side facing cell exterior or lumen amp hyd rophilic C terminal region on cytosolic face facing cell interior Glycophorin LDL receptor Influenza HA protein Insulin amp HGH receptor Type II don t contain cleavable SigSeq oriented w hydrohilic N terminal on cytosolic face amp hydrophilic C terminal on exoplasmic face Transferrin receptor Influenza HN protein Type III same orientation as Type I but lack cleavable signal sequence similar to Type II EX cytochrome P450 0 MultiPass Proteins includes Type IV contains 2 segments Type IV topology corresponds to that of G protein coupled receptors w 7 a helices N terminal on exoplasmic side amp C terminal on cytosolic side this topology can differ from other Type IVs Membrane Protein Synthesis there are 3 types of topogenic sequences direct membrane insertion and orientation of diff classes of integral proteins 0 NTerminal ER SigSeq see above 0 StopTransfer Sequence FIG 1311 found in Type 1 proteins when synthesized and enters into translocon it prevents nascent chain from entering farther into ER lumen it then moves laterally btwn translocon subunits amp becomes anchored in phopho bilayer causing close of channel sequence still connects sequences in NH3 and out COO of lumen bc translocon is closed and chain is still connected elongating chain loops out into cytosol until synthesis is completed wherein the ribo subunits are released this allows protein free to diffuse in the membrane 0 SignalAnchor Sequence FIG 1312 found in Type II amp III proteins nascent chain oriented in open translocon with N terminal toward cytosol bc of positively charged residues on outside of membrane as chain elongates and enters lumen signa anchor seq moves laterally out of translocon amp anchors chain in PLBL once protein synthesis is done C terminus is released into lumen while N terminus remains in cytosol ribosomes released Protein Import into Mitochondrial Matrix FIG 1323 once synthesized on cytosolic ribosomes proteins are sorted to mitochondria chloroplasts peroxisomes and the nucleus 0 Inside mitochondria and chloroplasts are lumen called matrix that contain internal subcompartments surrounded by 2x membrane Peroxisomes have single luminal matrix compartment surrounded by single membrane Mitochondria and chloroplasts utilize similar electron transport proteins and F Class ATPase to synthesize ATP same as Gram NEG bacteria they also contain their own DNA that encodes organelle rRNAs amp tRNAs also same as bacteria growth is achieved via incorporation of cellular proteins amp lipids not like bacteria nuclear division Numerous similarities mito amp chloro arose by incorporation of bacteria into ancestral euk cells forming endosymbiontic organelles 0 Protein import requires energy and occurs when outer amp inner organelle membranes are in close contact 0 Sorting of proteins to specific internal compartment is directed by 2 targeting sequences and 2 membrane bound translocation sequences one to direct protein into organelle and another to direct it into correct compartment or matrix 0 Step 1 Precursor proteins synthesized on cytosolic ribosomes are maintained in an unfolded or partially folded state by bound chaperones proteins that assists the non covalent foldingunfolding of proteins 0 Step 2 Precursor binds to import receptor near site of contact w inner membrane 0 Step 3 After binding protein is transferred into general import pore 0 Steps 4 5 Translocating protein moves thru outer membrane channel thru intermembrane space ampthru an adjacent channel in inner membrane 0 Step 6 Binding of protein by matrix chaperone Hsc70 amp subsequent ATP hydrolysis by Hsc70 helps drive import into matrix Uptake targeting sequence is removed by matrix protease amp Hsc70 is released from newly imported protein 0 Ste 7 Protein folds into mature active conformation within the matrix depends on chaps Protein Modifications Folding amp Quality Control in ER membrane amp soluble secretory proteins synthesized on rough ER undergo 4 principal mods before they reach final destination that promote folding of secretory proteins into native structures and add stability to proteins exposed to extracellular environment 0 Glycosylation covalent addition and processing of carbohydrates in ER amp Golgi allow cell to produce array of chemically distinct molecules at cell surface that are basis of specific molecular interactions used in cell to cell adhesion amp communication Glycoproteins proteins with attached carbohydrates includes majority of proteins synthesized on ER OLinked Oligosaccharides carb chains in glycoproteins are attached to hyd roxyl group in serine or threonine residues found in some cytoplasmic proteins as well contain 1 4 sugar residues which are added to proteins by glycosyltransferases located in lumen of Golgi complex sugar by sugar OO NLinked Oligosaccharides FIG 1316 carb chains are attached to the amide nitrogen of asparagines larger more complex than O Linked block followed by ind iv Glycosylation of oligosaccharide chain begins in ER continues in Golgi Common Precursor 14 residue precursor Glc3Man9GlcNAc2 is added to nascent proteins in rough ER removal and addition of sugar residues occur in ER amp Golgi Core Region composed of 5 Y shaped residues Man Man Man GlcNAc GlcNAc found in all N Linked precursor is linked to asparagines which are separate from serine and threonine by only one AA Addition amp Initial Processing 0 Step 1 precursor is transferred from dolichol carrier to susceptible asparagines residue on a nascent protein as soon as asparagines crosses to luminal side of ER 0 Step 2 One glucose residue is removed 0 Step 3 Then two glucoses are removed Q Re addition of one glucose plays role in correct folding of many proteins in ER 0 Step 4 One mannose Man residue is removed 0 Next Step transfer to cis Golgi complex Differences in glycosylation is basis for ABO blood types cell surface glycoproteins amp glycolipids w diff terminal sugars is what controls immune sensitivities 39 01 Gal A GlcNAc Gal B Gal Gal 0 Formation of Disulfide Bonds in ER 0 Proper Folding of Polypep Chains and Assembly of Multisubunit Proteins in ER Protein foldin is assisted b chaeronins which act as uality control llinii i mow ii 7 I LDWCHl Lid ay pill l39lg maxi Misfolded proteIns are retained In ER and degraded EX Cystic Fibrosis disease where mutation is where CFI39R channel is misfolded amp never reaches plasma membrane where is must be in order to properly fxn 0 Specific Proteolytic Cleavages in ER Golgi and Secretory Vesicles CHAPTER 14 Vesicular Traffic Secretion amp Endocytosis Lysosomes organelle with acidic interior generally used for degradation of unwanted proteins and storage of small molecules such as AAs use V class pump to transport into its lumen Processing of N LCs on Glycoproteins wiin Golgi cisternae cisternal MaturationFIG 1414 0 Step 1 Removal of 3 mannose residues in cis Golgi Step 2 Protein moves by cisternal maturation to med ial Golgi 3 GlcNAc residues added Step 3 2 more mannose removed Step 4 3 more GlcNAc residues added Step 5 1 fucose added Step 6 Processing completed in trans Golgi by addition of 3 galactose residues 0 Step 7 Finally linkage of N acetylneuraminic acid residue to each of glactose residues Protein transport thru secretory pathway can be visualized by fluorescence microscopy GFP Tags Brefeldin A tool commonly used in studies of ER amp Golgi causes Golgi complex collapse into ER Protein Sorting at the trans Golgi Network TGN o TransGolgi Network complex network of membranes amp vesicles in which proteins in secretory pathway that are destined for plasma membrane or lysosomes must pass thru TGN is major branch point in secretory pathway ampthru protein sorting a protein can be loaded into 1 of 3 diff vesicles that bud from TGN Constitutive Secretion first type of vesicle moves to and fuses w plasma membrane release contents to exterior of cell exocytosis while membrane proteins from vesicle become incorporated into plasma membrane no signal Regulated Secretion second type secretory vesicles are stored inside cell until signal for exocytosis causes release of their contents at plasma membrane Lysosome third type first transported by vesicles from TGN to compartment called late endosome proteins are then transferred to lysosomes via direct fusion signal Mannose 6 P residues on oligo chains modified in cis Golgi OOOOO Newly synthed lysosomal enzymes bind to M6P Specific Receptor in TGN for sorting to lysosomes M6P receptors are directed into clathrinAP1 vesicles clathrin coat is rapidly depolymerized uncoated transport vesicles fuse w late endosomes which fuse w lysosomes after dephophorylation Coat proteins to cell su rface and M6P receptors are recycled some receptors are delivered Sometimes lysosomal enzymes are sorted from TGN to cell surface amp secreted the enzymes can be retrieved by receptor mediated endocytosis Proteolytic Processing some proteins undergo this after leaving trans Golgi some membrane and many secretory proteins start as long lived inactive precursors called proproteins that require further 0 processing to generate the mature active proteins 2 pathways exist Regulated Secretory Proteins follows pathway of processing proinsulin occurs in secretory vesicles after having budded from TGN two endoproteases PC2 PC3 act on precursors of RSPs to splice polypeptide requires additional cleavages to leave just N terminal B chain amp C terminal A chain linked by disulfide bond mature insulin 0 Constitutive Secretory Proteins follows pathway of processing proalbumin the endoprotease furin acts on precursors of CSPs to splice polypeptide end w mature albumin PC23 vs Furin furin is used for continuously secreted proteins like albumin 0 Endocytosis 0 Examples ReceptorMediated Endocytosis specific receptor on cell surface binds tightly to an extracellular macromolecular ligand that it recognizes plasma membrane region containing R L complex then buds inward amp pinches off becoming transport vesicle occurs similarly to packaging of lysosomal enzymes by M6P in the TGN LDL Receptor Endocytosis Goldstein amp Brown 19705 o Ligand LDL low density lipoprotein bad cholesterol o Lipoprotein facilitates in the mass transfer of lipids btwn cells has shell composed of apolipoproteins amp cholesterol containing phospholipid monolayer shell is amphipathic bc outer surface is hyd rophilic amp inner surface is hydrophobic 0 Steps LDL binds to LDL R in coated pits Pits invaginate to form coated vesicles Clathrin coated pits are pinched off by dynamin lose coats LDL dissociates in low pH of early endosome release of LDL Late endosome fuses w lysosomes proteins amp lipids of free LDL are broken down to constituent parts by enzymes in lysosomes LDL goes to lysosomes LDL R recycles to PM neutral pH allows conformational change allowing binding of another LDL particle Transferrin Receptor Endocytosis o Ligand Transferrin Tfn iron 0 Fxns to deliver iron to cells Iron release amp recycling of Receptor ampTfn om Tfn binds iron in circulation of pH 7 Ferrotransferrin transferrin dimer w 2 atoms of Fe binds to transferrin receptor at cell surface Tfn and receptor internalize Low pH 6 causes release of Fe from ligand ligand remains bound to receptor Fe exits to cytosol Apotransferrin Tfn wo Fe and receptor recycle to PM At pH 7 neutral pH causes apotransferrin release from the receptor allowing it to find more iron Difference btwn 2 R L complex doesn t dissociate in late endosome for Tfn bt fi39 6 o Receptor Ligand Binding 239 cha sin Hmediate sor n of rece tors amp liands o Curvature amp Imagination o Pinch Off to Release Coated Vesicle o Vesicle loses clathrin coat very soon after being formed 0 Functions Nutrient acquisition from circulation cholesterol iron IgG Mediated by specific cell surface receptors Regulation of Signal Transduction Receptor down regulation addiction Targeting activated receptor to other compartments Destruction of Foreign Antigens Immunity phagocytosis Re Uptake of NTs Turnover of Cell Surface Proteins 0 Key Steps Ligand binds to cell surface receptor These receptors or other cell surface proteins associate with clathrin coated pits Coated pits 9 Coated vesicles In early endosomes low pH causes ligands to dissociate from receptors Ligands and receptors can then be sorted to different destinations Autophagy FIG 1435 pathway for delivery of cytosolic proteins and organelles to lysosomes for degradation when cell is under stress such as starvation means eat oneself 0 Step 1 Cup shaped structure surrounds portion of cytosol or an organelle continued addition of membrane leads to formation of autophagosome vesicle which envelops contents via 2 complete membranes 0 Step 2 Fusion of outer 39 w 39 of 39 releases single layer vesicle amp its contents into lysosomes interior 0 Step 3 Lysosome degrades single layer and contents via hyd rolases released AAs are transported across lysosomal membrane into cytosol Key Questions on Regulation of Vesicular Transport 0 How many types of vesicles are there Many 4 Best clathrin vesicles transport proteins from plasma membrane ampTGN to late endosomes Endocytosis at PM TGN lysosome COP I vesicles transport proteins in retrograde direction btwn Golgi cisternae amp from the cis Golgi back to the rough ER Golgi ER COP II FIG 148 vesicles transport from rough ER to Golgi ER ER Golgi Intermediate Compartment CaveolaeEndocytosis o How are vesicles formed Receptors have M5 in tail that allow them to bind to coat and adapter proteins 0 How do receptors associate with coated pits Coat protein binding is regulated by a small GTPase different one for different coats coats deform membrane and shape it into a vesicle o How do vesicles get to the correct destination Once there how do vesicles fuse with the correct compartment SNARE Hypothesis FIG 1410 when vesicles lose coat v SNARE proteins are exposed which join with their specific target cell membrane which has matching t SNARE proteins causes cell fusion on cell surface Summary 0 Receptors have specific sequences in cytoplasmic tail that target R L complex to coated pit o GTPases regulate the assembly of coat proteins on membranes and vesicle formation ARF COP I Dynamin vesicle pinchoff Vesicles transport along cytoskeletal elements to get to correct destination NARE interactions determine if correct destination is reached 0 Vesicle fuses with acceptor compartment 0 Coat proteins and GTPases recycle for next round of transport 0 CHAPTER 15 Cell Signaling I Signal Transduction amp ShortTerm Cellular Responses Goal To understand mechanisms by which cells communicate amp respond to extracellular stimuli Cell Signaling mechanisms by which cells bring about change in response to something could be internal or external EX Fight or Flight Response 00 External Cues hormones light etc Internal Cues cell growthdeath specialized fxns Signaling Molecules hydrophobic molecules ie steroids retinoids can spont diffuse thru PM amp bind to intracellular receptors most signals are too large amp too hydrophilic to penetrate PM and thus bind to cell surface receptors CellSurface Receptors CSR consist of 3 segments segment on extracellular surface one that spans PM and one facing cytosol Signal Transduction Signal molecule SigMol acts like ligand binding to structurally complementary site on extracellular or membrane spanning segments this binding induces conformational change in receptor change in shape is passed down to cytosol segment which recruits other proteins for activationinhibition Important Themes Involves protein to protein communication receptor to enzyme kinase to target Often involves additional secondary messengers cAMP etc Tightly regulated employs On and Off mechanisms Cellular responses usually result from integration of signals from many diff pathways Types of Intracellular Signaling O O Endocrine Signaling SigMols are synthed and secreted by signaling cells endocrine cells transported thru circulatory system amp finally act on target cells distant from their site of synthesis EX commonly used by hormones Paracrine Signaling Sig Mols released by cell only affects those target cells in close proximity EX neurotransmitters growth factors Autocrine Signaling cells respond to substances that they themselves release EX growth factors are secreted to promote own growth amp proliferation 9 tumor cells Signaling Pathway Sumary Hormone H or Ligand L binds to Receptor R 9 Causes Signal Amplification via Primary Effectors Second Messengers amp Secondary Effectors 9 Cellular Response Specificity of R L Binding 0 Purifying cell surface receptors is difficult because of their low abundance 0 5 of all proteins on PM amp these integral membrane proteins first must be solubilized from the membrane w a nonionic detergent then separated from other cellular proteins Use cloning amp expression to identify amp study receptors 9 eliminates need to isolate amp purify CSRs perform functional expression assay see below Each receptor generally binds only a single Sig Mol Ligand this specificity refers to its ability to distinguish closely related substances Ligand binding depends on weak noncovalent forces and molecular complementarity btwn interacting surfaces of a ligand and receptor Dissociation Constant KD measure of affinity of receptor for its li and at equilibrium rate of formation of R L complex equals rate of its dissociation 9 am Low values of K more stable RL complex amp tighter bonding amp higher affinity Binding Assays FIG 154 used to detect receptors amp determine their affinities for ligands for hig h affinity ligands binding assays can determine the Kd amp of receptors per cell done in low temperatures to prevent Endocytosis of CSRs Total Binding Curve TBC represents ligands specifically and nonspecifically bound to high affinity receptors add SBC and NBC nonspecific binding contribution is determined by repeating assay in 100x excess ligand saturates all specific H A sites Specific Binding Curve SBC difference btwn TBC and NBC maximum of the curve indicates the number of total receptors per cell Nonspecific Binding Curve NBC depicts binding ofall labeled ligands to nonspecific sites after all specific sites are saturated by unlabeled ligand 2 conc of ligand required to bind to 50 of surface receptors correlates w 12 of total receptors determined by SBC 9 draw vertical line to find conc on x axis Competition Assays FIG 155 way to detect weak binding of a ligand to its receptor put weak ligand w another ligand that binds to same receptor w H A LOW K measure K for binding of competitor to receptor by determining conc of competitor required to inhibit binding of half the radioactive ligand On graph competitor s concentration K 9 find half point of curve 50 inhibition on y axis ampfind corresponding x value conc often used to determine specific binding o ReceptorLigand Interactions FIG 156 maximal physiological response to an external signal occurs when only a fraction of receptors are occupied by ligand in general the ligand conc required to induce 50 of max cellular response is LOWER than Kd value 39 RL1Rtotal 11KdL 0 Functional Expression Assay FIG 157 can identify a cDNA encoding a CSR in low abundance genes encoding low abundance receptors for specific ligands often can be isolated from cDNA libraries target cells lacking receptors for part ligand are transfected w cDNA expression vector encoding receptor transfected cells exhibit normal cellular response to ligand if cDNA does indeed encode for the functional receptor Sensitivity of Cell Signaling cell sensitivity to external signals is determined by number of CSRs and their affinity for ligand response is NOT proportional to of activated receptors bc most of time you only need fraction of total receptors to be activated to get max response 9 caused by generation of second messenger molecules EX cAMP Ca2 ion Second Messengers carry amp amplify signals for many receptors binding of ligands first messenger leads to short INCDEC of conc of intracellular Sig Mol called second messengers which bind to other proteins modding activity cAMP activates protein kinase A PKA which induces changes in metabolism regulates ion channels Ca2 Ions low conc bc of ATP powered pumps that transfer Ca ions out of cell or into ER increases in conc can cause muscle contraction induce exocytosis of secretory vesicles amp NT containing vesicles change in conc is detected by Ca binding proteins from EF hand family HLH motif binding of Ca amp EF hand family causes shape changes 9 switches activities onoff cGMP activates protein kinase G PKG amp opens cation channels in rods DAG activates protein kinase C PKC IP3 opens Ca channels in ER 0 GProtein Coupled Receptors GPCR most numerous class of receptors all GPCR signaling pathways share these common elements Receptor that contains 7 membrane spanning domains Coupled trimeric G protein which fxns as switch by cycling btwn activeinactive forms Membrane bound effector protein Feedback regulation amp Desensitization of signal pathway Most utilize a second messenger These components are modular can be mixedmatched to create large number of diff pathways GCPR pathways have short term effects quickly modify existing proteins General Structure Contain 7 transmembrane a helical regions H1 H7 4 extracellular segments E1 E4 amp 4 cytosolic segments C1 C4 C4 C3 loop btwn H5 H6 are critical to interactions w G protein AAs that form interior of GCPR are diverse diverse binding capabilities Trimeric GS proteins are made of 3 subunits Ga GB amp Gy w GDP attached to G subunit GB amp Gy subunits stick together Types of GCPRs for Epinephrine Badrenergic Receptor binds on surface of hepatic liver adipose cells cardiac amp smooth muscle cells activates adenylyl cyclase by binding GaS which in turn catalyzes synthesis of second messenger cAMP a adrenergic Receptor binds on smooth muscle cells lining blood vessels cause arteries to constrict by inhibiting adenylyl cyclase via binding Gai o Extracellular Signaling FIG 1513 activation of effector proteins associated with GCPR completed in 6 steps resting state trimeric G protein attached to cytosolic face of PM surrounded by an inactive receptor amp inactive effector on either side Binding of hormone induces conformational change in receptor Activated receptor binds to G subunit Activated receptor causes conformational change in G 9triggers dissociation of GDP Binding of GTP to G triggers dissociation of Ga both from the receptor amp Gay Hormone dissociates from receptor Ga binds to effector activating it Hydrolysis of GTP to GDP causes G to dissociate from effector amp reassociate w Gay Return to resting state conformation O O O O All effector proteins in GCPR pathways are either membrane bound ion channels or enzymes that catalyze the formation of the second messengers Turning Off Signal Signals are transduced by Ga a GTPase switch protein that alternates btwn an active GTP bound and inactive GDP bound state GTP Exchange Factor GEF promotes GTPase activation ON GTPase Activating Protein GAP promotes GTPase inactivation OFF Protein dephosphorylation balances protein phosphorylation Two kinds of Kinases SerineThreonine kinases EX PKA PKC Tyrosine Kinases EX EGF R c src Multiple kinds of Phosphatases PP1 PP2 etc GCPRs That AcitvategInhibit Adenylyl Cyclase GCPR paths that utilize adenylyl cyclase as effector and cAMP as 2quotd messenger are found in most mammalian adipose muscle amp liver cells Depending on which GPCR is bound b li and aden l lcyclase could be activated Gas OI inhibited G V H39mtilfs 2 quotz GBy subunit is identical in both types of G proteins ho corresponding receptors differ Which pathway is activated depends on type of ligandhormone that binds GCPR Adenylyl Cyclase FIG 1522 membrane bound enzyme contains 2 similar catalytic domains on cytosolic face of membrane amp 2 integral membrane proteins each has 6 transmembrane a helices cAMP activates PKA by releasing its 2 catalytic C subunits when cAMP docks in PKA s 2 regulatory R subunits which contain 2 cAMP binding domains CNB AB when cAMP binds to CNB A it displaces catalytic subunit leading to its activation Signal Pathway L binds R 9 signal cascade via adenylyl cyclase 1St Effector9 cAMP 2quotd Messenger9 PKA glycogen synthase 2quotd Effector9 Mobilizing of ATP G ycogen Pathways Formation Incorporation of glucose from IDP glucose into glycogen is catalyzed by glycogen synthase Degradation Removal of glucose units from glycogen is catalyzed by glycogen phosphorylase Because 2 diff enzymes catalyze formation amp degredation of glycogen the 2 rxns can be independently regulated Regulation of Glycogen Metabolism FIG 1525 An INC in cytosolic cAMP 9 activates PKA 9 inhibits glycogen synthesis directly amp promotes glycogen degradation via protein kinase cascade A DEC in cAMP 9 inactivates PKA 9 release of active form of PP 9 PP promotes glycogen synthesis stops degradation Cellular Responses to Hormone Induced Rise in cAMP Adipose cells 9 phosphorylation amp hydrolyzation of triglycerides yielding free fatty acids and glycerol FAs taken up by kidneys for nrg source DEC AA uptake Liver Cells 9 INC conversion of glycogen to glucose inhibition of glycogen synthesis INC AA uptake INC synthesis of glucose from AA gluconeogenesis Glycogenolysis break down of glycogen to glucose excellent example in which cells respond to complex environment of signals by integrating its responses to gt1 signals epinephrine stimulation of muscle liver cells 9 rise in cAMP 9 promotes glycogen break Muscle Cells stimulation of nerve impulses 9 release of Ca ions 9 INC cystolic Ca conc 9 muscle contraction AND activation of GPK 9 degradation of glycogen to glucose 1 phosphate 9 feuls prolonged contraction Liver Cells FIG 1530 activation of phospholipase C9cleavage of PIP2 by phospholipase C 9 generates two 2quotd messenger DAG IP3 9INC cytosolic Ca conc 9activates GPK in muscles 9glycogen breakdown NC Ca9activates PKC9phosphorylate glycogen synthase9DEC glycogen synthesis Synthesis of IP3 amp DAG Fig 15 29 membrane bound PI kinases place a phosphate on specific hyd roxyl group on I ring producing phosphorylated derivatives PIP amp PIP2 cleavage of PIP2 by phospholipase C yields IP3 amp DAG Down Regulation of GCPR Signaling 1 ever the G subunits amp BARK phosphorylates B adrenergic receptors Barrestin binds to serine and threonine residues in C terminal of GCPR Clathrin ampAP2 promote Endocytosis of receptor Reduction in number of CSRs causes DEC sensitivity to additional hormone o Anchoring Proteins Localization of PKA to specific subcellular regions such as nucleus can be conferred by AKAPS A kinase associated proteins amp restricts effects of cAMP to those regions 0 cGMP Pathway relaxation of arterial smooth muscle Nitric oxide is synthesized in endothelial cells in response to acetylcholine amp subsequent INC in cystolic Ca concentration NO diffuses locally thru tissues amp activates intracellular NO receptor w guanylyl cyclase activity in nearby smooth muscle cells Resulting rise in cGMP activates PKG 9 causes relaxation of muscle amp vasodilation FINAL SUMMARY 0 In many cases the receptors are involved in activation or inhibition of a metabolic pathway secretion or another cell function that does not require changes in gene expression 0 Only a fraction of total surface receptors need to be bound to hormone to induce a maximal cellular response Generation of second messenger molecules cAMP cGMP phosphatidylinositol lipids Cellular responsiveness to hormone is dictated by number of surface receptors 0 GTPases are molecular switches whose activities are regulated by cell surface receptors Protein phosphorylationdephosphorylation is a highly conserved switch mechanism to regulate cell signaling 0 Cellular responses are the result of signal integration from many different pathways 0 CHAPTER 16 Cell Signaling II Signaling Pathways That Control Gene Activity Goal To examine the signaling pathways that cells use to regulate gene expression Receptor Tyrosine Kinases RTKs major class of CSRs most are monomeric single large molecule ligand binding to extracellular domain induces formation of receptor dimers o All have 3 essential components Extracellular domain containing ligand binding site Single hydrophobic transmembrane a helix Cystolic domain that includes region w protein tyrosine kinase activity In resting state intrinsic kinase activity of RTK is very low lacking ligand In Ligand Bound Dimeric Receptor two poorly active kinases are joined amp phosphorylate each other on tyrosine residue in activation lip 0 Phosphorylation leads to conformational change lip moves out of kinase catalytic site that facilitates binding of ATP or protein substrates resulting enhanced kinase activity phosph additional tyrosine residues in cytosolic domain of receptor 0 Resulting phosphotyrosines function as docking sites for signal transduction proteins Multimeric Signaling Complexes assembly is effect of receptor activation 0 Binding of 505 Protein to Inactive Ras Causes Conf Change that Activates Ras FIG 1620 Binding of hormone causes receptor dimerization kinase activation and phosphorylation of cytosolic receptor tyrosine residues Binding of GRB2 amp Sos couples receptor to inactivate Ras Sos promotes dissociation of GDP from Ras GTP binds and activates Ras which dissociates from 505 o Ras MAP Kinase Pathwa FIG 1625 In unstimulated cells most Ras is in inactive form w bound GDP binding of ligand to its RTK leads to formation of active Ras GTP complex Active RasGTP binds to N terminal regulatory domain of Rafserinethreonine kinase thereby activating Raf Hydrolysis of Ras GTP to RasGDP releases active Raf Active Raf phosphorylates activating MEK dual specificity phosph both tyrosine amp serinethreonine residues Active MEK phosphorylates amp activates MAP kinase serinethreonine kinase Dimeric form of active MAPK translocates to nucleus where it activates many TFs which mediate cellular responses 0 Induction of Gene Transcription Tx by MAP Kinase FIG 1627 00 Dimeric MAPK induces TX of c fos gene by direct activation of TCF and indirect of SRF In cytosol MAPK phosphorylates amp activates kinase p90RSK which then moves into nucleus amp phosphorylates serum response factor SRF a TX factor After transocating into nucleus MAPK directly phosphorylates the TX factor Ternary Complex Factor TCF Phosphorylated TCF amp SRF act together to stimulate TX of genes that contains serum response element SRE sequence in their promoter Association of TCF amp 2 molecules of SRF forms active trimeric factor that binds SRE o r 39 39 39 ides PI as SiqnalTran dllcers PI membrane bound phosphorylated inositol lipids many have short term effects cell metabolism and long term effects pattern of gene expression Many RTKs can initiate IP3DAG path by activating phospholipase Cy diff from GCPR RecruitmentActivation of PKB in PI3 Kinase Pathwav FIG 1630 In unstimulated cells PKB is in cytosol w PH domain bound to catalytic kinase domain inhibiting its activity Hormone stimulation activates PI3 kinase amp formation of PI 3 phosphates which serve as docking sites on PM for PH domain of PKB amp PDK1 Full activation of PKB requires phosphorylation both in activation lip by PDK1 amp at C terminus by PDK2 0 Activation of Gene TX by 7 Spanning CSRs FIG 1631 CRE cAMP response element CREB CRE bindng protein only found in nucleus Activation of CREB TF followin li and bindin FIG 1631 Receptor stimulation leads to activation of PKA Catalytic subunits of PKA are released and transocate into nucleus In nucleus subunits of PKA phosphorylate amp activate CREB TX factor Phosphorylated CREB associates w co activator CBPP300 to stimulate TX of various target genes controlled by CRE regulatory element 0 NF kB Signaling Pathway FIG 1635 In resting state dimeric TF NF kB made of p50 amp p65 subunits is sequestered in cytosol bound to inhibitor I kBa Activation of trimeric I kB kinase is stimulated by things like virus infection radiation I kB kinase phosphorylates the inhibitor I kBa which binds E3 ubiquitin ligase Polyubiquitination of I kB targets it for degradation by proteasomes NF kB activates TX of numerous target genes including one that terminates signaling I kBa and ones that promote signaling inflammatory cytokines PRESENTATION 3 RIP3 Switches TNFinduced Cell Death from Apoptosis to Necrosis HYPOTHESIS inferred modulation of energy metabolism by RIP3 in response to cell death stimuli such as TNF can cause cells to switch from apoptosis to necrosis BACKGROUND Necrosis vs Apoptosis 2 types of cell death 0 Apoptosis active self destruction of cells based on their life span affects an isolated cell no release of harmful substances around it eliminates old unhealthy or unnecessary cells Causes stimulus via irradiation or toxic drugs injury removal of repressor agent Triggering of death program 9 intracellular signaling caspases 9 fragmentation of cell into vesicles 9 phagocytosis by neighbor cells 0 Necrosis passive cell death affects a group of cells or tissue stemming from severe injury or disease Causes inadequate blood supply bacterial infection trauma hyperthermia Exhaustion of oxygen or nutriment 9 exhaustion of ATP in the cell 9 impairment of cell membrane 9 enzyme release causes inflammation DEFINITIONS RIP3 receptor interacting protein 3 biological function largely unknown cleaved by caspases after activation of death receptors 0 M tumor necrosis factor 0 zVAD Benzyloxycarbonyl Val Ala Asp fluoromethylketone inhibits TNF induced cell death in A cells enhances it in N cells 0 A Cells NIH 3T3 cells from American Type Culture Collection caspase dep o N Cells NIH 3T3 cells that are caspase independent Caspase inhibition cysteine aspartic proteases distinguishes apoptotic amp nonapoptotic cell death can shift apoptosis towards necrosis or enhance necrosis essential for apoptosis the executors of apoptosis Induce necrosis through kinase activity of RIPl Production of ROC is necessary for this type of necrosis done in mitochondria Want to figure out precise mechanisms underlying necrosis andor apoptosis NIH 3T3 cells undergo apoptosis when stimulated by tumor necrosis factor TNF O 0 One line of cells has been found to not need caspase to do cell death N cells TNF zVAD treated N cells undenNent necrosis METHODS O O 0 Used affymetrix microarray analysis to reveal the genes that regulate cell death they are expressed differently in A cells vs N cells Gene expression could explain switch from apoptosis to necrosis Western Blotting confirmed differing expression of RIP3 in A vs N cells Used Ientiviral vectors to deliver short hairpin RNA shRNA or cDNA into cells lentivirus can infect with 100 accuracy Deletion and point mutations revealed that RIPl interacting RHIM RIP homotypic interaction motif domain in RIP3 is required for necrosis MAJOR FINDINGS39 0 0000000000 Focused on RIP3 bc it was only gene expressed differently that inhibited zVAD s enhancement of cell death RIP3 express in A cell switch TNF induced apoptosis caspase independent cell death RIP3 has role in necrosis in the absence of zVAD essential for some forms of necrosis RIPl is required for death receptor agonists to activate necrosis RIPl amp RIP3 are required for TNF induced amp zVAD enhanced necrosis RIP3 may have a role in determining type of RIPl induced cell death RIPl induces apoptosis in absence of RIP3 OR induces necrosis in presence of RIP3 RIP3 is required for zVAD to enhance necrosis PYGL activation by RIP3 contributes to necrosis GLUL and GLUDl mediated use of Glu or Gln as nrg substrates contributes to necrosis ROS production is required for TNF or TNFzVAD induced necrosis in L929 cells MEFs and macrophages Role of RIP3 in apoptosisnecrosis switching should at least partly occur through increasing energy metabolism associated ROS production Energy metabolism affects cell death mechanisms CONCLUSION RIP3 is a potential drug target for necrosis related diseases NEW PROGRESS none really since paper was published 3 months ago FLAWS 0 000 A lot of time they will reveal information or make claims without citing how they got those results very little methodology Not reproducible Basically just listed a bunch of ways that apoptosis or necrosis is caused by Could just read last line of each paragraph to understand article Bunch of mini experiments followed by results amp conclusion PRESENTATION 4 Suppression of Induced Pluripotent Stem Cell Generation by p53p21 Path Background Info O 0000 p53 Tx factor activates Tx of p21 p21 inhibitory protein binds cyclin dependant kinase p53p21 Pathway both proteins monitor cell cycle act as tumor suppressor genes iPS induced Pluripotent Stem cells NanogGFP green fluorescent protein type of reporter system Cell Dividing Under Normal Conditions 0 O 0 p53 works as TF for tumor suppressorpro apoptotic genes MDM2 performs inhibitory binding and ubiquitination of p53 Proteasome degrades marked proteins Cell Dividing with DNA Damage 0 O 0 New surveillance proteins arrive to monitor replication process for DNA damagemutations through pre mRNA regulation ATMATR phosphorylates p53 TWO POSSIBLE PATHWAYS p21 Pathway cell cycle is stopped amp DNA repair goes p21 inhibits Cdc25A which dephosphorylates CDK Cyclin complexes causing activation CDK Cyclin complex phosphorylatesactivates proteins essential for cell cycle progression Hypot Apoptotic Pathway BAX causes release of cytochrome C from mitochondria inducing degradation of cellular components hesis Induced pluripotent stem iPS cells can be generated from somatic cells by the introduction of the transcription factors Oct34 Sox2 Klf4 and c Myc in mice and in humans due to the Why I damag loss of p53 pathway mportant Could enhance stem cell research help advances in medical field repair ed tissues in diseases like ALS Parkinson s Diabetes Muscular Atrophy drug testing p53 deficient iPS cells can grow adult tissues when inside mouse embryos 9 concludes that lack of p53 suppresses p21 0 O O O O O O O I O O O O O O O I O O O O PRESENTATI o Backg O O O O O 0 000000 Results Without c Mvc amp with Oct34 sox2 Klf4 loss of p53 fxn INC iPS cell efficiency wo c Myc retrovirus 10 transduced cells still became iPS cells With all 4 TFs generation of iPS INC further to 20 Do dominant negative mutants of 953 have effect on iPS cell generation substantial INC in of GFP positive colonies present using Pr0275Ser What effect fors cDNA that encodes for wild type 953 have on iPS production using Asp278Asn or Ser58Ala wild type p53 showed DEC in GFP colonies produced Expansions of GFP clones generated by 3 or 4 factor development of teratomas high expression of Oct34 amp Sox2 confirmed pluripotency of iPS cells generated by 3 factors 4 factor cells had low expression of Oct34 amp Sox2 Retroviral expression confirmed by expression level of Sox2 Klf4 ampc Myc in p53 null cells p53 null cells generated by each TF unable to maintain embryonic like morphology Conclusion Loss of p53 is responsible for the observed INC in efficient of direct reprogramming C Myc transgene in p53 null suppresses retroviral silencing and inhibits acquirement amp security of iPS identity for these cells Without p53 present 4 TFs can produce iPS cells from somatic cells p53 DOES suppress iPS cell generation in humans as well as mice Protein p21 was necessary to suppress iPS cell generation p53 amp p21 work together to suppress iPS cell generation However permanent suppression of p53 may affect genomic stability of generated iPS cells Methods microarray analysis amp statistical analysis among others Summary iPS cells generated by regular body cells Oct 31 Sox2 Klf4 cMyc Used nanog GFP reporter system to identify iPS cells specifically amp sensitively p53 null mutants produced the most GFP colonies Mouse embryonic fibroblasts ON 5 An ERMitochondria Tethering Complex Revealed By Synthetic Biol Screen round ER important site for protein amp phospholipid synthesis these proteins amp lipids are then transported to other organelles by vesicular trafficking pathway Mitochondria ATP synthesis cell death signaling cellular differentiation control cell cycle amp growth have been implicated in human diseases can t synth all lipids needed ERMES Model ERM Encounter Structure in order to tether ER amp mitochondria together some proteins must be located in both the OMM amp ER membrane ERMES Model claims all are localized on OMM Previous Studies Shown that transfer of phospholipids into mitochondria does not utilize vesicular trafficking Methods Yeast manipulation Creation of artificial tethering complexes CHIMERA to find mutants w defects in tethering genes Fluorescence microscopy Protein analysis Phospholipid analysis Genetic Selection Genetic Complementation Gene Knockout o Translational Fusion 0 Testing ERMES Model attach GFP amp visualize each protein separately 0 Hypotheses o CHIMERA ra doesn t have enough tethering force 0 CHIMERA ra just labels pre existing ER M interfaces 0 CHIMERA ra is not proved 0 New Hypotheses o Complexes containing SMP containing proteins Mmm1 and Mdm12 could perform similar functions in other eukaryotes o ERMES complexes could provide platforms for specialized molecules to carry out specific roles at the connection 0 Tether predicted to help coordinate mitochondrial growth and influence mitochondrial shape 0 Results 0 Mdm12 amp Mdm34 can be replaced their fxn is partially equal to CHIMERA s o Mdm10 Mmm1 cannot be replaced either have diff fxns or CHIMERA can t tether wo Mdm10 has vital fxns amp Mmm1 o No suppression of anomalous morphology in Mmm1 Mdm10 suggest they re required for tethering by CHIMERA CHIMERA can replace only some proteins For ERMES Model Mdm34 Mdm10 in OMM Mdm12 is on peripheral either Mmm1 in ER Glycosylation suggests Mmm1 is in ER transmembrane Epistatic Mapping combo of deletions in 2 of following genes causes cell death ERMES psd 1 crd 1 9 ERMES genes are all strongly correlated to psd1 amp crd1 o ERMES Deletion reduced rate of lipid exchange impaired cardiolipin production 0 Purpose 0 ERMES acts as a zipper complex that binds ER to mitochondria composed of 4 proteins 2 mito 1 ER 1 peripheral facilitates phospholipid transfer into mitochondria affects cardiolipin production which is necessary for correct mito shape amp allows for respiration O O O O CELL BIOLOGY TEST 2 GUIDE CHAPTERS 712 CHAPTER 7 Transcriptional Control of Gene Expression 71 Control of Gene Expression in Bacteria 0 Regulation of gene expression is fundamental process that controls the development of a multicellular organism from a single fertilized egg cell into the thousands of cell types from which we are made Structure and function of cells determined by proteins it contains The decision to initiate transcription is the major mechanism for controlling production of the encoded protein in a cell Repression mRNA and its encoding protein are synthesized at low rates Repressor proteins that bind to operator sequences which overlap or lie adjacent to promoters binding of a repressor to an operator inhibits transcription initiation by causing the condensation of chromatin around the DNA Activation mRNA and encoding proteins are produced at high rates Activators proteins that bind next to promoters and increase the rate of transcription initiation by RNAP by promoting decondensation of chromatin regulates the lac operon Bacterial cells only synthesize the proteins required for survival under the particular conditions Operons encode enzymes in a particular pathwa Trp encodes 5 enzymes needed in biosynthesis of tryptophan Lac encodes 3 enzymes required for metabolism of lactose sugar in milk All genes within an operon are coordinately regulated all activatedrepressed to same extent In order for transcription to be initiated the RNAP must be associated w one of a small of 0 factors Most common in E coli 07 Works by bringing the RNAP together with a promoter Makes sure transcription starts at 1 site transcription start site 0 Nucleotide sequence of promoter determines its strength frequency of how many diff RNAPs can bind and initiate transcription per minute 72 Overview of Eukaryotic Gene Control and RNA Polymerases 0 Main Purpose of Gene Control execution of genetic program that underlies embryonic development 0 Differences btwn Bacterial amp Euka otic Control Eukaryotic control serves the needs of the whole organism and not survival of one cell Developmental steps cannot be reversed in eukaryotes Eukaryotes have transcription factors TFs that control transcription while bacteria uses RA TFs bind a large distance away from promoter unlike the closeness of prokaryotic RA Eukaryotic RNAPs 3 are more complex than that of bacterial RNAP 1 Similarities btwn Bacterial amp Eukaryotic Control Regulation of initiation is most widespread form of gene control In both the promoter signals the place where transcription starts 00 O O OO 00 O o Activator proteins bind to specific DNA control elements in chromatin and interact with multiprotein co activator machines such as mediator to decondense chromatin and assemble RNAP and general transcription factors GTF on promoters Inactive genes are assembled into regions of condensed chromatin that inhibit RNAP and their associated GTFs from interacting with promoters o Repressor proteins bind to other control elements to inhibit initiation by RNAP and interact w multi protein co repressor complexes to condense chromatin o Promoter a DNA sequence that specifies where RNAP binds and initiates transcription of a gene 0 B bind large distances away from promoter either upstream opposite direction of Tx or downstream same direction as TX bc of the distance multiple TFs can regulate expression 0 3 Types of RNAP differ in sensitivity to q amanitin all contain 2 large subunits and 3 smaller which are analogous to the B 3 q amp 1 subunits of E coli RNAP I transcribes pre rRNA 285 185 585 rRNAs functions in protein synthesis amp ribosome components nonsensitive to q amanitin RNAP II transcribes mRNA snRNAs amp miRNAs functions to encode protein RNA splicing amp post transcriptional gene control very sensitive to q amanit39n 0 Contains a unique carboxyl terminal repeat in largest subunit RPBl it is crucial for viability in yeast and at least 10 copies must be present to survive o The repeat CTD becomes phosphorylated during initiation and remains as such 0 RNAP II initiates transcription of genes at the nucleotide in the DNA template that corresponds to the 5 nucleotide that is capped in the encoded mRNA RNAP III transcribes tRNA SS rRNA snRNA 7S RNA amp other stable short RNAs functions in protein synthesis RNA splicing signal recognition particle for insertion of polypeptides into ER 73 quot 39 v SPnIIPan in Protein Coding Genes O 0 Types of Alternative DNA Promoters ATA Box 25 35 bp upstream from start site binded by TBP causing the DNA to bend single base changes in the sequence drastically DEC in vitro TX Initiator present in eukaryotic genes lacking TATA Box contain a cytosine C at 1 and an adenine A at 1 start site no consensus sequence around start CpG Island instead of initiator or TATA contain CG rich stretch of 20 50 nucleos win 100 bp upstream of start site not frequent non methylated amp thus not preserved PromoterProximal Elements win 100 200 bp of TX start site contain sequence for binding TFs Enhancers amp Silencers contain multiple short control elements located 200 10K bp upstreamdownstream from promoter win an intron or downstream from final exon of gene P P Elements amp Enhancers are generally cell type specific amp fxn in specific differentiated cells I 74 Activators and Repressors of Transcription TFs which stimulate or repress TX bind to promoter proximal elements and enhancers in euk DNA Structural Motifs in DNA Binding Domains contain 1 q helices that interact w major grooves of DNA 0 O O O O O 2H2 Zinc Finger most common domain in human genome regions fold around central Zn ion producing compact domain from short length of pp chain LeucineZipper has hydrophobic AA leucine at every 7 11 position bind to DNA as dimers HelixLoopHelix similar to zipper except a nonhelical loop of pp chain separates 2 q helix regions in each monomer contains a N terminal middle loop region amp C terminal Cooperative binding of multiple activators to nearby sites in an enhancer forms a multiprotein complex called an enhancesome Assembly of such often requires small proteins that bind to DNA minor groove and bend DNA sharply allowing bound proteins on either side of bend to interact better Multiple TFs Rs amp As act in concert to modulate TX 0 DNA looping explains how enhancer elements can be located at great distances away from promoters I 75 Transcription Initiation by RNAP II GTFs position RNAP II at start sites and assist in initiation Those TFs that assist RNAP 11 w TATA boxes are labeled TFIIA TFIIH largest is TFIID Pre Initiation complex combo of RNAP II and its GTFs bound to a promoter and ready to initiate TX Binding must be done sequentially 1TBP binds to TATA box DNA 2TFIIB 3complex of RNAP II amp TFIIF 4TFIIE 5TFIIH Helicase activity of TFIIH subunit separates template strands at start site requires hydrolysis of ATP I 76 Molecular Mechanisms of Transcription Repression and Activation Eukaryotic TX activators and repressors exert their effects largely by binding to multi subunit co activators or co repressors that influence assemply of pre initiation complex either by O O O O O O O Modulating chromatin structure indirect effect Interacting w RNAP II amp GTFs direct effect The DNA in condensed regions of chromatin heterochromatin is relatively inaccessible to TX factors amp other proteins causing repression of gene expression Repression Domains fxn by interacting with co repressors that are histone deacetylase complexes by deacetylating the N terminal tails in nucleosomes near repressor binding site interactions btwn promoter DNA and GTFs are inhibited 9 repressing TX Activation Domains fxn by binding co activator protein complexes like histone acetylase by hyperacetylating the N terminal tails in nucleosomes near activator binding site interactions btwn promoter DNA and GTFs occur 9 activating TX Activators bound to distant enhancer can interact with TFs bound to promoter bc DNA is flexible and intervening DNA can form a large loop Highly cooperative assembly of the pre initiation complex in vivo requires several activators cell must produce specific set of activators required for TX of a particular gene in order to express that gene I 77 Regulation of Transcription Factor Activity 78 quot 39 39 F lonnation and Termination of Transcription Elongation Termination In euks mechanism for termination of TX differs for each RNAP I RNAP I requires polymerase specific termination factor to bind to specific seq I RNAP II RNA seq must specify site of RNA cleavage amp polyadenylation Poly A tail I RNAP III ends after polymerizing series of U residues easy to melt TX of HIV genome by RNAP II regulated by antitermination mechanism that requires specific binding of TAR sequence near 5 end of HIV RNA Pausing TX causes build up of denatured proteins of which when resuming are transcribed quickly to catch up and reduce accumulation I 79 Other Eukamotic Transcription Systems 0 TX initiation via RNAP I amp III is similar to that of RNAP 11 just use diff GTFs amp doesn t require ATP o Mitochondrial DNA is Txed by nuclear encoded RNAP made of 2 subunits 1 similar to monomeric RNAP from bacteriophage T7 the other resembles bacterial ofactors o Chloroplast DNA is Txed by chloroplast encoded RNAP homologous to bacterial RNAP except it lacks 0 CHAPTER 8 PostTranscriptional Gene Control 81 Processing of Eukaryotic Pre mRNA 0 Events during RNA Processing I 5 Capping I 7 methylguanosine cap is added after RNAP II synthesizes 25 30 bases of new transcript shortly after Initiation Unusual 5 5 phosphodiester linkage since exonucleases only digest 5 3 linkages protects RNA from 5 exonuclease digestion I 3 Cleavage Occurs where AAUAAA amp GU rich regions are I Coupled with polyadenylation occurs early on I Polyadenylation add poly A tail I Signal AAUAAA found in most protein encoding mRNAs also requires downstream GU rich region specific proteins bind to these 2 sites and mediate cleavage I 50 250 A residues added by Poly A Polymerase PAP o Binds before cleava e Ensures rapid polyadenylation as soon as 3 end is cleaved O I RNA splicing I Removal of introns occurs at consensus sequences of 5 amp 3 splice sites I GUAG Rule Splice starts at GU 5 end residue amp ends at AG 3 end residue I Spliceosome mediates pre mRNA splicing formed by 5 snRNPs U1 U2 U4U5U6 0 U1 amp U2 bind pre mRNA followed by U4 5 6 0 U1 amp U4 dissociation activates catalytic activity of spliceosome o TransEsterification 2 5 phosphodie bond forms then standard 3 5 bond Intron released linearized and degraded o Transcript Elongation coupled with RNA processing factors I s RNAP elongates message heterogeneous ribonucleoprotein particles hnRNPs bind to newly synthesized transcript I hnRNPs contain hnRNA which is collection of premRNA and other nuclear RNAs that contribute in splicing polyadenylation amp exportation I premRNA nascent RNA transcripts from protein coding genes amp mRNA processing intermediates 82 Regulation of Pre mRNA Processing 0 Alternative splicing of diff exons is major mechanism for regulation of pre mRNA processing Bc of alt splice of primary transcripts use of alternative promoters amp cleavage at different poly A sites 9 diff mRNAs may be expressed from same gene in diff cell types or stages I Can be regulated by RNA binding proteins that bind to specific sites near regulated splice sites I Splicing Repressors sterically blocks binding of splicing factors or inhibit fxn I Splicing Activators enhance splicing by interacting w splicing factors 83 Transport of mRNA Across the Nuclear Envelope 0 Fully processed mRNAs in nucleus are bound by hnRNPs in complexes known as nuclear mRNPs before translation can occur mRNA must be exported out of nucleus into cytoplasm where translation occurs w ribosomes 0 Nuclear Envelope double membrane that separates nucleus from cytoplasm consists of water resistant phospholipid bilayer amp multiple associated proteins mRNPs tRNAs amp ribo subunits traverse the envelope via nuclear pores 0 Nuclear Pores NP complexes NPCs control import amp export from nucleus I Large complicated structures composed of multiple copies of 30 diff proteins called nucleoporins proteins which are building blocks of NPC allowing transport thru envelope I NPCs are octagonal w 8 filaments extending into nucleoplasm amp another 8 into cytoplasm o mRNPs are exported by a heterodimeric mRNP exporter that interacts w FG repeats of FG nucleoporins direction of transport nuc cyto o Pre mRNAs are not bound by exporter to ensure only fully processed mRNAs reach cyto for TL 84 Cytoplasmic Mechanisms of Post transcriptional Control 0 Regulation of nuclear export is rare transport of pre mRNAs are prevented amp degraded by exosome osome complex capable of degrading RNA in cytoplasm 0 However HIV retrovirus has mech for letting pre mRNA that retain splice site to be exported and TLed 0 Types of Post Transcriptional Control Mechs In Cytoplasm I Work to control stability or localization of mRNA or its TL into protein I Mechs are controlled by short 21 nucleo ssRNA called micro RNA miRNA and short interfering RNA siRNA I miRNA base pairs w target mRNAs inhibiting TL 0 siRNA base pairs w target mRNAs causing degradation miRNA repress TL of specific mRNAs by inn liii r39u ii ITlE i iii and animals39 exression 1 3 of all human genes is regulated by it ii E 39 I m is possible bc sequences don t have to match perfectly RNAI Induces Degredation of mRNA targets that form perfect hybrids w siRNAs evolved as a defence mechanism against viruses Cytoplasmis Polyadenylation promotes TL of some mRNAs it is a process required for TL of mRNAs w short poly A tails phosphorylation of RNA binding proteins from 3 UTR leads to lengthening of 3 poly A tail amp translation occurs in all plants mRNA Degradation occurs as a result of dual shortenin of ol A tail deaden lation followed by exosome mediated i n OR iiv li a quot warm quot Rapid RNA Degradation occurs when euk mRNA proteins are expressed in short bursts leadin to reeated coies of AU rich se in their 3 UTR specific proteins there interact w i 39 Tl 39 39 quot39 39 arr 39 39 promoting rapid degradation For Summary Diagram See Figure 8 1 p 324 CHAPTER 9 Visualizing Fractionating and Culturing Cells 91 Organelles of the Eukaryotic Cell Fig 9 1 0 O 0 00000000000000 92 Li ht Microsco 0 Plasma Membrane controls movement of molecules in amp out of cell amp fxns in cell cell signaling Mitochondria surrounded by double membrane generate ATP via oxidation of glucose amp fatty acids Lysosomes have acidic lumen degrades obsolete materials from cells Lumen aqueous interior containing battery of degradative enzymes Phagocytosis whole cells amp large particles move from cell surface into lyso to be degraded Autophagy old organelles amp cytoplasm degraded by lysosome eating one self Endocytosis soluble macromolecules taken in via invagination of pits in plasma membrane Nuclear Envelope double membrane enclosing contents of nucleus outer membrane cont w RER Nucleolus nuclear subcompartment where most rRNA is synthesized Nucleus filled w chromatin composed of DNA amp proteins site of mRNA amp tRNA synthesis Smooth ER synthesizes lipids amp detoxes hydrophobic compounds Rough ER RER fxns in synthesis processing amp sorting of secreted proteins lyso proteins etc Golgi Complex processes amp sorts those proteins synthesized on RER Secretory Vesicles store secreted proteins amp fuse w plasma membrane to release contents Peroxisomes detox molecules amp break down fatty acids to make acetyl groups for biosynthesis Cytoskeletal Fibers form networks that support cell membranes organize organelles cell moving Microvilli increase surface area for absorption of nutrients from surrounding medium Cell Wall composed of cellulose maintains cell shape provides protection Vacuole stores water ions amp nutrients degrades macromolecules fxns in cell elongation Chloroplasts carry out photosynthesis surround by 2x membrane contain sacs inside Secretion Exocytosis Technigues Fig 9 5 p 376 After synthesis on ribosomes of rough ER proteins are stored in lumen of RER transport vesicles bud off and carry proteins to Golgi In Golgi proteins are concentrated amp packaged into immature secretory vesicles Vesicles coalesce to form larger mature secretory vesicles which become crystallized After vesicles accumulate under apical surface fusion w plasma membrane amp release content Visualizin Cell Structure and Localizin Proteins W In Cells Differential Interference Contrast DIC Microscopy utilize diff in refractive index to enhance contrast allowing for viewing of live cells moving amp unstained tissues PhaseContrast Microscopy does same thing as DIC Bright Id L roscopy contains several lenses to magnify specimen resolution02um Fluorescence MIcroscopy uses epifluorescence light path to localize amp quantify specific molecules in live cells cells are fluorescently stained first could be autofluorescent fluorescent dye or GFP tag limitations blurred image amp thick specimens must be sliced up amp realigned Confocal Microscopy image able to be reconstructed in 2D or 3D space no blurry image bc of pin hole that blocks light from other planes use laser as source of illumination photo scan is timely Deconvolution Microscopy delivers highest possible resolution for light microscopy 0 93 Electron Microscopy Methods and Applications 0 Electron Microscopy provides much better resolution than light microscopy can t use live cells electromagnetic lenses focus high velocity e beam rather than visible light Transmission Electron Microscope e s emitted from filament amp accelerated in electric field condenser lens focuses e beam objective amp projector lenses focus e s passing thru amp project them onto screen entire microscope enclosed in powerful vacuum Cryoelectron Microscopy allows visualization of particles wo fixation or staining freeze sample allowing for live cells hydrated cells amp unstained tissues to be viewed at high resol I 94 Purification of Cell Organelles 0 Cell Centrifugation can separate many types of organelles which differ in size amp density after high speed spinning liquid remaining on top supernatant is removed amp spun at higher speed until separation of organelles occurs at proper density via equilibrium densitygradient centrifugation Lysosomes 112 gcm3 Mitochondria 118 gcm3 Peroxisomes 123 gcm3 o Other techniques used for Cell Fractionation Magnetic Beads ABs attached to magnet captured by magnet field Antibody Pull Down cells join w AB causing complex to be pulled to bottom of jar I 95 Isolation Culture and Differentiation of Metazoan Cells 0 Flow Cytrometry measure of light that cells scatter amp fluorescence they emit while flowing thru laser beam used to identify different types of cells when other methods don t work centrifuge FluorescenceActivated Cell Sorter FACS separates cells based on the cell surface proteins they express can sort thousands of cells amp divide into diff dishes used for purifying white blood cells leukocytes I Labeled cells are passed single file thru laser light beam I Both fluro amp scattered light are emitted scattered light detects shape size of cells I Cells forced thru nozzle which negatively charges each cell proportionally based on fluorescence inside droplet Cells fall btwn electric field each type of cell has specific charge amp according to that charge cells are separated into diff dishes I 96 Mass Spectrometry o Tissues can be imaged via mass spectrometry used to determine cancerous vs noncancerous cells Ionization Electrospray ESI vs Matrix assisted laser dissociation MALDI Ion Analysis use graph to determine chemical make up of cell CHAPTER 10 Biomembrane Structure I 101 Biomembranes Lipid Composition and Structural Organization 0 Plasma Membrane defines cell amp separates inside from outside Membrane Transport Proteins enable particles to cross otherwise impermeable membrane Receptors proteins in plasma membrane that allow cell to recognize chemical signals Lipids affect membrane shape amp fxn anchor proteins to membrane transducer signals to cyto Phosphoglycerides most abundant principal building block of most biomembranes amphipatic made of 2 segments w very diff chemical properties Sphingoloids derivatives of spingosine an amino alcohol w long hydrocarbon chain Steroids in animals cholesterol 4 ring hydrocarbon amphipathic Hydrocarbon tails are hydrophobic heads are polar hydrophilic Both can have different head groups attached to tail with varying net charge Each organelle in euk cells contain unique set of proteins stored in lumen that enable it to do its characteristic cellular fxns Phospholipids Spontaneously Form Bilavers When lipids are dispersed into solution they aggregate to minimize contact btwn hydrophobic surface amp water bc of van der Waals interactions forming either I Micelles spherical formed by single tailed lipids I Liposomes spherical with bilayer hollow center formed by double tailed lipids I Phospholipid Bilayers sheet like Simplest experiment is splitting water in beaker w partition with hole in it add lipids watch as they cover hole with bilayer Lipids are Laterally Mobile in Biomembranes Thermal motion permits lipids to rotate freely around their long axes amp diffuse laterally bc lateral fatty acyl chains stay interior fluidity I Shape amp size of head amp tail influence charge fluidity amp thickness of membrane I 103 Phospholipids Sphingolipids and Cholesterol Synthesis amp Intracellular Moves 0 Cells synthesize new membranes via expansion of existing membranes synthesis of membrane lipids is done in the cytoplasm but final steps are catalyzed by enzymes bound to pre existing cell memb 0 Fatty Acid Synthesis Fig 10 25 p 431 Precursor amp key component to phospholipids amp sphingolipids Major fatty acids in phospholipids contain 14 16 18 or 20 C atoms amp unsaturated chains Fatty acids are synthesized from acetate CH3C00 which is esterized into water soluble coenzyme A CoA of which an example of is acetyl CoA important intermediate 0 Fatty Acid Transport Fattyacidbinding proteins FABPs bind to fatty acids to allow movement thru cytoplasm without esterfying they contain a hydrophobic pocket lined by B sheets of which a long chain fatty acid can fit inside amp interact noncovalently w surrounding protein 000 O O O 0 Cholesterol Synthesis Fig 10 26 p 432 START Acetyl CoA acetoacetyl CoA 9 HMG CoA 9 Mevalonate 9 Isopentenyl Pyrophosphate IPP 9 Farnesyl Pyrophosphate 9 Squalene 9 Cholesterol END 0 Cholesterol amp Phospholipid Transport Fig 10 27 p 433 Vesicles transfer lipids btwn membranes Lipid transfer is consequence of direct contact btwn membranes Transfer mediated by small soluble lipid transfer proteins CHAPTER 11 Transmembrane Transport of Ions and Small Molecules I Transport Proteins embedded in plasma amp other membranes permits controlled amp selective transport of molecules and ions across membrane can be thermodynamically spon or nonspon o Spontaneous molecules move from HI9LOW concentration no nrg required passive o Nonspontaneous molecules move against conc gradient requires nrg ATP Permeability only small uncharged hydrophobic molecules can cross by simple diffusion no need for nrg or transport proteins 0 Diffusion Rate proportional to conc gradient across bilayer amp hydrophobicity and size greater the conc gradient of substance faster its rate of movement across membrane 0 Hydrophobicity measured by partition coefficient K between oil amp water higher K more lipid soluble I Membrane Proteins mediate transport of most molecules amp all ions across membranes 0 TP Powered Pumps pumps utilize nrg released by ATP hydrolysis to power movement of specific ions against their electrochemical gradient P Class Pumps euk plant amp fungi plasma membranes I Ca2 ATPase calcium amp ATP binding9phosphorylation of aspartate9 conformational change9 calcium release9 dephosphorylation9 conf chan e V Class Pumps vacuolar endosmal lysosomal osteoclast plasma membrane I Used to generate pH gradient F Class Proton Pumps bacterial plasma inner mitoch amp thylakoid membrane I Use pH gradient to generate ATP ABC Superfamily bacterial amp mammalian plasma membrane for lipids cancer drugs utilize this bc of its multi drug resistance MDR proteins 0 Ion Channels channels permit movement of specific ions NaKCCa down electrogradient Selective movement of ions creates a transmembrane electric potential difference Inside negative electric potential voltage of 50 70 mV exists across plasma of all cell In animals EPD generated by movement of K ions thru resting K channels stay open I Opening K channels causes hyperpolarization I Opening Na channels causes depolarization I Opening Cl channels causes hyperpolarization Opening KNa nonspecific channels causes depolarization o Transporters fall into 3 categories Uniporters transport single type of molecule down CG EX GLUTl Fig 11 5 Symporters catalyze movement on 1 molecule against CG driven by movement of 1 ions down CG one in some out Cotransport movement of two ions coupled both ions move in same direction Antiporters same as symporters used frequently for intestinal uptake of nutrients I Cotransport movement of two ions coupled one goes in while the other goes out opposite directions NaH Antiporter cation cotransporter exports H from cells in movement coupled to energetically favorable import of Na I AE1 Anion Antiporter anion cotransporter catalyzes 1 for 1 exchange of Cl amp HCO CHAPTER 12 Cellular Energetics 121 Steps of Glucose and Fatty Acid Catabolism Glycolosis amp Citric Acid Cycle 0 ATP adenosine triphosphate found in all life forms even the earliest cells use energy released during hydrolysis of terminal high nrg phosphoanhydride bond in ATP to power many processes energy to drive ATP synthesis comes from 2 sources aerobic oxidation occurs in mitochondria in euks and photosynthesis occurs in chloroplasts in plants amp cyanobacteria Chemiosmosis similar process in plants and animals to generate ATP from ADP amp Pi proton electrochemical gradient is generated across membrane driven by nrg released as e s travel thru electron transport chain nrg stored in gradient is called protonmotive force amp is used directly to power ATP synthesis


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