Fundamentals of Cellular and Membrane Structure Exam Review - PDF

Fundamentals of Cellular and Membrane Structure Monday 8/26/24 Dr. Fliesler Prokaryo?c Cell vs Eukaryo?c Cell Prokaryo'c Cells Eukaryo'c Cells...

Fundamentals of Cellular and Membrane Structure Monday 8/26/24 Dr. Fliesler Prokaryo?c Cell vs Eukaryo?c Cell Prokaryo'c Cells Eukaryo'c Cells No internal organelles Have internal organelles Have cell walls No cell wall Have flagella No flagella Have plasma membrane Have plasma membrane Organelle: specialized structure designed to carry out a specific func?on within cell Cell Membrane Func?ons Separate intracellular vs extracellular Selec?ve permeability barrier Subdivide organelles, regula?ng biochemical processes Residence for receptors, etc. to facilitate signal transduc?on, cellular communica?on Membrane Compartmentaliza'on: spa?al segrega?on of biochem. processes in the cell Increase reac?on efficiency (e.g., [substrate] and spa?al localiza?on) Avoids “fu?le cycles” (e.g., separates anabolic/biosynthe?c from catabolic/degrada?ve rxns.) o E.g., faXy acid synthesis (in cytosol) vs. oxida?on (in mitochondria) Conven?onal Cellular Compartments Organelle Func'on Nucleus Gene?c info. Assembly of ribo. subunits Structural support Ribosomes Protein synthesis RER Protein synthesis and processing SER Lipid synthesis Golgi apparatus Protein processing Lysosomes Diges?on and recycling Peroxisomes Oxida?on of faXy acids, ethanol, etc. Vacuoles Varies- colora?on, storage of oils, carbs, waters, or toxins. Mitochondria ATP produc?on Chloroplasts ATP and sugar produc?on via photosynthesis Cytoskeleton Structural support Movement of materials Plasma Membrane Selec?ve permeability- maintain intracellular environment. Cell Wall Protec?on Structural support RNA RNA is required for the integrity of mul?ple nuclear and cytoplasmic membrane-less RNP granules Ribonucleaoprotein (RNP) granules= RNA + proteins o Considered “organelles” w/o a membrane o Found in nucleus and cytoplasm of euk. cells o Ac?va?on of RNase-L= disintegra'on of many (not all) RNP granules Lipid Droplet: Once not considered an organelle due to their lack of membrane-binding, lipid droplets are now considered an organelle Molecular aggregates exist in: Small, soluble, oligomeric aggregates that develop into… Large insoluble protein aggregate aka inclusion bodies Electron microscopy (EM) reveals cellular ultrastructure Cytosol= largest compartment in cell followed by: Mitochondira RER Membrane composi?on 1. Lipids (3 classes): o Glycerophospholipids o Sterols o Sphingolipids 2. Proteins 3. Carbohydrates o Glycoproteins (> 90%) o Glycolipids (< 10%) 4. Glycosaturated RNAs o AXached to cell surface o RNA molecules bound to sugars General Composi?on of Membranes Lipid class composi?on varies among differ. cell types and subcellular compartments Protein: lipid ra?o o Higher protein: lipid ra?o= higher biological ac?vity Glycerophospholipids (GPLs) “Backbone” glycerol is esterified at C-3 with polar “head group” (phosphoric acid) plus choline, ethanolamine, or inositol. o Ethanolamine as the head group= protonated compared to choline head § There are differences in chemical nature and size of “head groups” C-1 and C-2 hydroxyl groups esterified with nonpolar faXy acid “tails” o Saturated FA at C-1 o Unsaturated FA at C-2 Ether-linked Lipids GPLs with ether (instead of ester) linkage at C-1 Ether (C-O-C) linkage hydrolyzes with acidic pH but is stable to basic pH FA: carboxylic acid deriva?ves of long-chain hydrocarbons Saturated: alipha?c, straight-chain, no dbs Unsaturated: olefinic, bent-chain, cis dbs Polyunsaturated FAs (PUFAs) Examples: EPA, DHA, and arachidonic acid Have more than one db E.g., omega-3s, 6s, etc. o Numbering starts at carboxylic end (COOH) o “Omega-#” starts at the methylated side (CH3) Unsatura?on Effect More dbs= decrease/lower melt. temp. More dbs= decrease effec5ve chain length o Membrane thickness would be reduced § Unsaturated chains (dbs) take up less space than saturated chains (no dbs) Lipid “Rules of Thumb” Increase carbon #= increase melt. temp. Increase unsatura?on (more dbs)= decrease melt. temp. The physical proper'es of phospholipids tend to be conferred by their cons'tuent FAs Increase dbs= increase unsatura?on= decrease melt. temp. Sphingolipids “Backbone” sphingosine is an amino alcohol (rather than glycerol)  Amide bond: sphingosine + FA= ceramide o Prevalent in neural ?ssues Sphingomyelin (SM or Sph) is the only sphingolipid that is a phospholipid o Other sphingolipids are glycolipids Gangliosides are complex sphingolipids that contain N-acetylneuraminic acid Sterols Cholesterol is the predominant sterol NP, planar, 4-ring nucleus Polar, 3-beta-hydroxyl group Dipolar o Cholesterol is dipolar Membrane Lipids are Amphipathic Hydrophilic (polar, charged) head group Hydrophobic (NP, non-charged) tail group Hydrophobic Effect Lipids via thermodynamics spontaneously assemble into self-organized structures in mixed lipid-water systems o Maximizes P-P and NP-NP interac?ons Minimizes overall free energy of system E.g., too much water and not enough air, a micelle is formed as lipid tails avoid water Phospholipids mixed with water spontaneously form BILAYERS Lipids must be arranged as bilayer Maximizes P-P and NP-NP interac?ons Maximizes hydrophobic effect Organized Lipid Structures Phospholipids are typically cylindrical, they form bilayers, liposomes/vesicles but NOT micelles FA are wedged, shaped and form micelles Evolving Concepts of Membrane Structure Overton (1895) discovered that a substance ability to pass through membrane was related to its chemical nature NP substances passed through quicker than polar substances o The excep?on being water Danielli-Davson (1930-1940s) “Sandwich” model Globular proteins formed a layer above phospholipid heads Includes protein channels (“pores”) Cons of model: o Energe?cally unfavorable  o Later found to be not true: globular proteins are NOOT found in a layer above LP heads JD Robertson (1957): “Unit Membrane” Hypothesis “Trilaminar unit”: 2 outer dark lines (interpreted as protein monolayer) separated by a lighter “inner core” line (interpreted as lipid bilayer) Proposed ALL cellular membranes are like this Fundamentals of Cellular and Membrane Structure Monday 8/26/24 Dr. Fliesler Lecture Summary Cells are compartmentalized o Anatomically: via membranes o Func?onally: conven?onal vs current defini?ons Membrane compartmentaliza?on: o Increases reac?on efficiency o Avoids “fu?le cycles” Prokaryotes do NOT have internal organelles RNP (RNA + protein)= organelles that lack membranes Membranes are amphipathic o Polar (hydrophilic), charged head group o Non-polar (hydrophobic), non-charged tail FA chains Cytosol is the largest compartment in the cell Cell membranes are made up of: 1. Lipids § Glycerophospholipids (GPLs) C-1: saturated FA C-2: unsaturated FA C-3: esterified glycerol backbone § Sterols Cholesterol is the major sterol o Dipolar: having equal and opposite electric charge § Sphingolipids Sphingomyelin is the major sphingolipid Sphingosine backbone 2. Proteins 3. Carbohydrates § Glycoproteins § Glycolipids 4. RNA In eukaryotes, the major glycosphingolipids (GSLs) are PC, PE, and PS FA o Saturated: alipha?c, straight-chain, no dbs o Unsaturated: olefinic, bent-chain, cis dbs High (protein: lipid) ra?o= high bio. ac?vity Increase FA chain length= increase melt temp. Increase dbs= decrease effec?ve chain length o Membrane thickness would be reduced § Unsaturated chains (dbs) take up less space than saturated chains (no dbs) Increase FA unsatura?on= increase dbs= decrease melt temp. Increase carbon number= increase melt temp Physical proper'es of phospholipids tend to be conferred by their cons'tuent FAs  Hydrophobic effect: lipids in aqueous lipid-water mixtures spontaneously self- assemble into organized structures o Minimizes free energy o Maximizes polar-polar and NP-NP interac?ons Molecular geometry governs the type of organized structures formed o Phospholipids + water= form bilayers o Phospholipids are cylindrical and form bilayers, liposomes/vesicles NOT micelles o FAs are wedge-shaped and form micelles NP pass quicker through membrane than polar molecules Problems with Danielli-Davsion Model o Energe?cally unfavorable o Later found to not be true Most common structural mo?f of ALL biological membranes is the lipid bilayer The “unit membrane” hypothesis (JD Robertson): all bio. membranes share the same func?onal structural mo? o Trilaminar unit: 2 outer dark lines separated by a lighter “inner core” (lipid bilayer) Biophysical methods o Microscopy o Spectroscopy o X-ray diffrac?on Biochemical/Cell biological methods o Immunofluorescence o Chemical modifica?on Historical Perspec?ves: “Fluid Mosaic” Model and Beyond Wednesday 8/28/24 Dr. Fliesler Lecture Summary Singer-Nicolson “fluid mosaic” model (1972) o Proteins float in a “sea” of lipids with rela?vely few constraints to diffusion within the bilayer plane Proteins in fluid mosaic model are categorized based on the strength and nature of their associa?on with the lipid bilayer o Peripheral (extrinsic) § Loosely associated with bilayer § Require mild tx to remove them from membrane o Integral (intrinsic) § Strongly associated with bilayer § Require harsh methods to remove them from membrane The transbilayer distribu5on of proteins, lipids, and carbohydrates is ASYMMETRICAL o Help generate fluidity differences in the 2 halves of bilayer At the cri'cal micelle concentra'on (CMC): detergent molecules self-associate and form micelles Detergents: o SDS: unravel protein and denatures them o Lipopep?de (LPDs): retain nega?ve, 3D structures of protein Choline-PLs (PC, Sphingomyelin) prefer extracellular (luminal) leaflet of bilayer o Glycolipids exclusively on outer leaflet of bilayer o Tend to have more saturated FAs Amino-PLs (PE, PS) prefer inner (cytoplasmic) leaflet of bilayer o Tend to have more PUFAs Physical state of membrane lipids depends on composi?on and temperature o More unsatura?on = more fluid o Higher temperature = more fluid  Cholesterol= “fluidity buffer”: can enhance or restrict fluidity, depending on ambient temperature rela?ve to mel?ng temp of lipids o Below melt temp: increase fluidity o Above melt temp: decrease fluidity Lateral and rota'onal diffusion of lipids and flexing of PL acyl chains are rapid Transverse (“flip-flop”) diffusion of lipids is extremely slow in pure lipid environment but more rapid in bio. membranes o Facilitated by translocases (e.g., scramblases, flippases) Proteins diffuse rela?vely freely within plane of membrane and rotate perpendicular to membrane but transverse (“flip-flop”) diffusion does not occur Lipids in crystal (saturated, straight chain) have liXle movements o Their movement increase as it approaches fluid (increase in chain bends= unsatura?on) Membrane fluidity is influenced by TEMP. and COMPOSITION o Decrease temp= decrease fluidity Carbohydrates are also distributed asymmetrically in bio membranes o Glycosphingolipids (GSLs) and the oligosaccharide chains of glycoproteins are exclusively found on external leaflet of bilayer Lateral phase separa'on: asymmetry of lipids within the plane of membrane Lipid “rats”: transient membrane microdomains enriched in sphingolipids and cholesterol- rela?vely deficient in glycerophospholipids o These regions are thicker than the “bulk phase” of bilayer o Serve as plauorms for signal transduc?on components Membrane thickness varies with faXy acyl chain composi?on of phospholipids Membrane proteins can be classified according to their: o Associa?on with membrane o Topology (e.g., N-terminus out vs in o Number of transbilayer segments (e.g., type I, type II, etc.) Mul?ple ways for aXachment of proteins to membranes: o Acyla'on: involves myristoyl or palmitoyl o Prenyla'on: involves farnesyl or geranylgeranyl covalent modifica?on § Via thioether linkage § CAACX, C-terminal o GPI anchors: occur on Asp residues and involve a membrane phospholipid, a mannose-P-glycan chain and an ethanolamine linkage Membrane Molecular Dynamics Friday 8/30/24 Dr. Fliesler Lecture Summary RBC “ghosts”: prepare by hypotonic lysis of mature RBCs o Offer pure plasma membrane prepara?on- useful for membrane studies Cytoskeleton is o Complex o Triton X-100-insoluble o 3D array of membrane-associated proteins Lipid composi?on in outer and inner leaflets vary o Resul?ng in differences in microdomain physical proper?es § E.g., viscosity affects “fluidity” which affects membrane func?on Polarized epithelial cells have dis?nct, macroscopic membrane domains (e.g., apical, basolateral) o Proteins and lipids may be restricted to apical or basolateral membrane domains o Membrane specializa5ons (e.g., junc?onal complexes, ?ght junc?ons) also represent dis?nct membrane domains Modern biophysical method (e.g., FRAP, FLIP) provide quan?ta?ve means to study protein lateral mobility o FRAP: blast bright light on membrane, measure that spot (?me of recovery) o FLIP: measure distally of bleached area FRAP and FLIP determine lateral diffusion coefficient (D-trans) o Small D-trans= restricted mobility Plasma membrane proteins interact with cytoskeleton (cytoplasmic face) and the extracellular matrix (extracellular face) o Influence degrees of mo?onal freedom of membrane proteins o Restric?ons are due to presence of organized protein domain structure within or associated with membrane Caveolae are morphologically dis?nct from rats by their cave-like architecture and enriched in caveolin o Cavelin: scaffolding proteins that binds cholesterol and signal transduc?on proteins, receptor molecules, etc. clustering them in rats and caveolae o Non-clathrin-mediated endocytosis Rats and caveolae resists solubiliza?on with cold non-ionic detergents (e.g., TX-100) o This property and their low buoyancy allow them to separate from other membrane domains by sucrose density ultracentrifuga?on Subcellular frac?ona?on objec?ves: o Maintain organelle/membrane integrity o Isolate organelle/membrane from other cellular components  o Avoid excessive fric?on to minimize membrane fusion Features in media use to disrupt ?ssues/cells: o Avoid high [salt] (causes aggrega?on) o Avoid Ca2+ (ac?vates proteases) o Osmolarity (iso-osmo?c condi?ons) o pH (7.4-7.5) o Protease inhibi?on Density gradient ultracentrifuga?on o Differen?al rate (large S-value difference= greater separa?on) § Separa?on based on sedimenta?on rate (S) o Equilibrium (isopycnic: buoyant density (p) of par?cle rela?ve to medium) § Buoyancy of par?cle > buoyancy of medium= par?cle sinks § Buoyancy of par?cle < buoyancy of medium= par?cle floats Appropriate density media characteris?cs: o Water soluble o Inert o Low viscosity o Low osmolarity o Membrane-impermeant (otherwise media will penetrate= equal density) o Spectroscopically transparent Subcellular frac?on o Density perturba?on Organelle/membrane purity: o Morphology ultrastructure o Marker molecules (e.g., enzymes, chemical, biochemical, immunological) o Spectrophotometric characteris?cs o Protein composi?ons (SDS-PAGE, WB) Intracellular Compartments and Protein Sor?ng Friday 9/6/24 Dr. Rao Animal Cell A cell is an open thermodynamic system o Can exchange energy and maXer Techniques to Visualize Cells Light microscopy Electron microscopy Staining and labeling 3 Families of Intracellular Compartments Nucleus and cytosol Secretatory and endocy?c organelles Mitochondrial Compartmentaliza?on and Protein Transport Gated transport (con'nuous) o Nucleus and cytosol Transmembrane transport (discon?nuous) o Cytosol and mito/ER Vesicular transport (discon?nuous) o Cytosol and vesicular lumen Signal sequence directs traffic Found at N-terminus of proteins (3’-end) o Enzymes will remove this sequence to stop instruc?ons Transla?on begins in cytosol Nuclear Pore Complex (NPCs) NPCs regulate nuclear selec?vity Large pores instead of protein transporters Fully-folded proteins CAN be transported into nucleus via NPCs Nuclear Localiza?on Signal (NLS) NLS helps to target nucleus Can be found anywhere in the sequence Point muta?ons can used to test for NLS Nuclear Import Receptors Impor?ns are exclusively found in nucleus Impor?ns bind to NLS of protein and to the NPC of proteins Ras-related nuclear protein (RAN) GTPase Required for nuclear-cytoplasmic transport Gated transport requires energy RAN-GAP= hydrolysis= RAN-GDP= impor?n release in cytosol RAN-GEF= phosphoryla?on= RAN-GTP= expor?n release in nucleus Compartments to Mito. Transloca?on Cytosolic proteins des?ned to be translocated to mito. are: o Unfolded o Coated by HSP70 chaperone protein § HSP70 is a chaperone protein that maintains protein unfolded Mito proteins contain N-terminal localiza?on signal Mito. Protein Translocators Outer o Translocase of Outer Mito. Membrane (TOM): required for import of al nucleus-encoded mito. proteins o Sor?ng and Assembly Machinery (SAM) Inner o Translocase of Inner Mito. Membrane (TIM) § TIM23: transports proteins into matrix § TIM22: mediates inser?on of inner membrane proteins  o Oxidase Assembly Machinery (OXA): helps with inser?on of inner membrane proteins that are synthesize in mito. TOM and TIM usually work together but can work independently Ac?ve Protein Targe?ng to Mito. Inner Matrix HSP70 is stripped in an ATP-dependent manner Transport into the mito requires ENERGY Outer Membrane Inser?on of Porins (w/ SAM) “SAM is helpful with folding protein” Inner Membrane Import N-terminal signal sequence ini?ate import into matrix Hydrophobic sequence stops transloca?on when bound to TIM23 complex Inner Membrane Import- 2nd Route Proteins is delivered completely into matrix space Cleavage of signal sequence= adjacent hydrophobic signal sequence at the new N- terminus Signal directs protein into inner membrane using OXA complex Intracellular Compartments and Protein Sor?ng Pt. 2 Monday 9/9/24 Dr. Rao Protein transla?on begins in the cytosol Uses RNA as template o RNA needs to have expor?n signal to be recognized by expor?n protein= exit nucleus Transcrip?on occurs in nucleus mRNA Export to Cytoplasm Cytosolic ribosomes bind to RNA to prepare for transloca?on ER RER: transmembrane protein produc?on SER: lipid synthesis Signal Hypothesis Helps explain how secretatory, lysosomal, and other vesicular proteins are processed RNA -> nucleus -> cytosol o Factors in cytosol recognize ER and brings them into ER Tes?ng Signal Hypothesis Made microsomes Added high [salt]= naked microsomes Factors bind to microsome Separate factors into frac?ons Recons?tute factor with stripped microsome Monitor transloca?on Signal Recogni'on Par'cle (SRP) Located in cytosol Set the “reading frame”; ini?ates transloca?on SRP uses RNA to draw ribosome into ER SRP scans unfolded chain for hydrophobic segment o Starts at N-term and goes towards C-term SRP + protein= transla?on halted o Buys ?me for ribosome to bind to ER o Prevents protein from folding before being translocated to ER Transla?on ER: co-transla?on (transported into ER as its being synthesized) Mito: post-transla?on (RNA has been synthesized before transported into mito) ER signal sequences and SRP direct ribosomes to ER ER Protein Translocator: Sec61 Soluble Protein Transloca?on (into ER) Channel pore closes but translocator (e.g., Sec61) opens laterally o Allowing hydrophobic signal sequence to diffuse laterally into bilayer where it will rapidly degrade Posi?vely charged AA face cytoplasm ALWAYS Posi?vely is determined upstream in the proteins gene?c sequence Protein Glycosyla?on, Endocytosis, and Vesicular Trafficking Wednesday 9/11/24 Dr. Rao COP2: uses kinesis o ER -> golgi: toward outside COP1: uses dynein o Golgi -> ER: toward inside Material Exchange Endocytosis: membrane fission to internalize material Exocytosis: membrane fusion to dump material ER= manufacturing hub Golgi= sor?ng hub o ER -> golgi (manufactured then transported to ER to be sorted) Receptor-mediated endocytosis Endocytosis selec?vity is due to specific receptor synthesis All vesicles need o Clathrin: forms vesicle skeleton o Dynamin: fission pinch protein § Dynamin requires GTP § Dynasore: dynamin inhibitor o Cholesterol: plasma membrane forma?on § Methyl beta cyclodextrin (MBCD) removes cholesterol LDL receptors are made when cells need lipids Co-transla?onal Inser?on of Proteins to ER Lumen Post-transla?onal Modifica?ons: Glycosyla?on Glycosyla'on: adding sugar o Occurs in ER Glycosyla?on func?on: o Quality control o Facilitate protein delivery o Mediate cell aXachment o Influence protein-protein interac?on o Alter protein solubility 2 Types o N-linked: always occurs on Asp residue § N-X-S/T o O-linked: occurs on Threo or Ser Synthesis of Lipid-linked Precursor Oligosaccharides Oligosaccharide transferase transfers sugar to polypep?de chain o Inhibi?ng transferase= inhibits sugar biding= ER stress Tes?ng Protein Glycosyla?on 1. Lec?n: plant proteins that bind to different glycans 2. SDS PAGE: decrease glycosyla?on= decrease sugar= decrease weight 3. Pharmacological agent: glycosyla?on inhibi?on 4. Point muta?on of glycosyla?on sites 2 Classes of Oligosaccharides in N-glycans Complex oligosaccharides High-mannose oligosaccharides Recruitment of COP2 Proteins GTP-bound Sar1 binds to COP2 GTP -> GDP causes Sar1 hydrophobic tails to pop out= coat disassembles Retrieval for ER-resident Proteins (COP1 Vesicles) ER retrieval signals bind to COP1 coats Soluble ER proteins have a KDEL sequence on the C-terminus (carboxy terminus) Ater PTM in golgi is complete, COP1-coated, KDEL-bound KDEL receptors retrieve protein back into ER Vesicular Traffic and Cytoskeletal Structure Friday 9/13/24 Dr. Rao V-SNAREs and T-SNAREs assist with fusion Vesicle membrane (V-SNARE) Target membrane (T-SNARE) Lysosomes Soluble hydroly?c enzymes o Ac?vated by proteoly?c cleavage o Requires low pH Func?ons of Cytoskeleton Cell shape and strength Cellular mo?lity Intracellular mo?lity Microfilaments are smaller than MTs Microfilaments= ac?n Microtubules= tubulin Polarity of Ac?n Filaments Associa?on of ATP-ac?n: (+) end Dissocia?on of ADP-ac?n: (-) end o [Free monomers] influences rate of dissocia?on Rate-limi?ng step= availability of ac?n subunits * hydrolysis occurs AFTER polymeriza?on Ac?n-binding proteins Regulate polymeriza?on Filament length Structure Func?on Ac?n Molecules Ac?n= road Myosin= motor vehicle that uses ATP o ATP hydrolysis drives myosin mo?lity (“power stroke”) o Myosin + ATP= force o Myosin moves towards (+) end of ac?n filaments which is ATP-rich Filament-ac?n (F-ac?n) is required for endocytosis o It drags lysosome carrying cargo into cell MTs Dynein: towards (-) end, retrograde o Endocytosis Kinesin: towards (+) end, anterograde o Exocytosis Centrosomes act as MT organizing centers (MTOCs) o Ini?ate and organize MT growth Gamma-tubulin sits on top of gamma-TuSC (nuclea?on site of MT) o Gamma-TuSC build on each other to form MT MT Treadmilling: (+) grows, (-) shrinks Uses a lot of energy Establishing Cell Polarity and Cell Junc6ons and Adhesions Dr. Luciana Frick Wednesday 10/2/24 Cell Polarity The asymmetric organiza6on of several cellular components Used for specialized func6ons A cell needs to polarize during development, cell division, migra6on Examples of Polarized Cells Epithelial cells Migra6ng cells o Establish a front and rear end within cell to help with guiding miga6on Neurons o One of the most polarized cells in our body (e.g., AP transmission is unidirec6onal) Polarity Establishment and Maintenance Same molecules and mechanisms are used to polarize cells even though they develop completely different cellular func6ons Establishment o Breaking symmetry o Establishing cor6cal landmark o Recruit specific proteins to specific membrane domains Maintenance: reinforce and amplify the polarized state by: o Polariza6on of cytoskeleton o Vesicular trafficking o Build junc6ons and barriers Establishment and maintenance are NOT independent, they are interconnected Establishment: breaking symmetry In germ cells (e.g., egg) Sperm entry o P granules move to posterior end upon sperm entry RNA/proteins of maternal genes In soma6c cells 1) Asymmetric division These 3 events localize PAR 2) Contact with other cells proteins asymmetrically w/in cell 3) Contact with ECM Soma6c cells do NOT have P granules PAR PAR: por6oning defec6ve proteins (and their mRNA) o In both germ and coma6c cells, PAR proteins are distributed asymmetrically PAR genes were discovered in C. elegans Asymmetric cell division: E.g., P granules segregate into one side of cell; when cell divides, P granules end up in 1 daughter cell (aka P cell) PAR Mutants Muta6ons of PAR genes results in P granules localizing asymmetrically PAR Genes PAR-1 and PAR-2 complex= posterior end o The migra6on of this complex to posterior side allows for PAR complex to accumulate in anterior end PAR-3-6-aPKC= PAR complex= anterior end Mlc-4 Gene Mlc-4 gene encodes for a chain of myosin II o Myosin II= motor protein that uses ATP to move along ac6n filaments The silencing of Mlc-4 gene= lack of PAR-6 localiza6on In Germ Cells (e.g., egg) Sperm entry= asymmetrical distribu6on of PAR proteins due to cor6cal flow generated by contrac6on of actomyosin Sperm entry= local weakening of cortex= actomyosin cor6cal flow= PAR complex transported to anterior end o Absence of PAR complex= removal of inhibitory signals= PAR-2 accumula6on in posterior end o Increase PAR-2 prevents back flow of PAR complex back to posterior end ------- Asymmetric Cell Division Produc6on of two daughter cells with different cellular fates 2 mechanisms giving dis6nct proper6es too daughter cells o Intrinsic asymmetry: both cells are different at 6me of division o Extrinsic asymmetry: ini6ally the same but due to external environments, they develop to be different Asymmetrical Cell Division Determines: If a cell differen6ates vs proliferates How cells differen6ate (e.g., what type of cell it will become) Stem Cell Development Mito6c spindles need to be first aligned with the axis of polarity before asymmetrically division can occur a) Extrinsic asym: one daughter cell develops OUTSIDE niche= differen6a6on, allowing for the other daughter cell to self-renew b) Intrinsic asym.: one daughter cell will contain self-renewing factors internally, allowing other cell to differen6ate. Cell with P granules= P cell= will become a germ cell P cells proliferate but do NOT differen6ate into specialized soma6c cells The par66oning of RNA and proteins between sister cells determines different fates o Asymmetric. Cell division= 2 daughter cell contain different RNA and proteins Ul6mate goal: fate diversifica6on -------- Contact with other Cells and ECM Achieved via homophilic molecules such as cadherins Cell junc6ons: Anchoring junc6ons o Cell-cell adhesion o Cell-matrix adhesion Occluding junc6ons (6ght junc6ons) o Seal gaps between cells= impermeable or selec6vely permeable barrier Anchoring Junc6ons Tethered to cytoskeletal filaments (via ac6n intermediate filaments) inside cell Abundant in 6ssues subjected to constant mechanical stress (e.g., skin, heart, GI) Ac6n intermediate filaments are composed of kera6n or desmin Anchoring Junc6ons: Held together via transmembrane proteins o Cell-Cell: cadherins o Cell-ECM: integrins Cadherin Superfamily Includes hundreds of different proteins Structurally, they share cadherin repeats in ECM Cadherins bind via homophilic interac6ons o One type of cadherin only interacts with the same type of cadherin o This selec6vity leads to assembly of organized 6ssues Ca2+ binds to the hinge= prevents it from flexing= rigidifies cadherin in ECM= allowing cadherin to  Cadherin afaches to cytoskeleton with the help of adaptor protein (e.g., p120- caternin, b-catenin, etc.) Desmosomes also use adaptor protein (e.g., desmoplakin, plakoglobin, etc.) o They look like plaques hence “plak” Adherens Junc6ons Band-type of adheren junc6ons= zonula adherens Associated with bundles of ac6n filaments that encircle the cell below the plasma membrane o This network can contract with the help of myosin motor proteins Desmosomes Intermediate filaments are afached to transmembrane proteins that form a dense plaque in the cytoplasm Non-classical cadherins form actual anchor by afaching to cytoplasmic plaque extending through membrane and strongly bind to cadherins coming from the membrane of adjacent cell Basal Lamina Integrins bind to proteins in the basal lamina o Integrins= transmembrane receptors that facilitate cell-ECM adhesion o Integrins are heterodimers and can switch between ac6ve and inac6ve conforma6ons § Ac6ve state: integrins have a higher affinity for basal lamina proteins o Integrin ligands determine the ligand specificity Basal lamina consists of collagen, laminin, perlecan and other macromolecules o These proteins can bind to each other Ac6n-linked Cell-ECM Junc6ons: Focal Adhesions Focal adhesions: integrins that link ECM to ac6n cytoskeleton o ECM side of integrin binds directly to ECM protein (e.g., fibronec6n) while its tail is linked to ac6n cytoskeleton via adaptor proteins (e.g., vinculin talin) Important for cell migraAon Hemidesmosomes Form links between ECM and cytoskeleton (similar to desmosomes except hemidesmosomes have integrins rather than cadherin TJs are cri6cal at determining apical vs basolateral sides of cell TJs are found apically TJ proteins= claudin and occludin o They associate with intracellular proteins called Zonula Occludens (ZO) proteins that anchor strands to ac6n cytoskeleton TJ Vital Func6ons Seal between cells (e.g., prevent entry of substance between cells) Fence between membrane domains (e.g., apical vs basal side) Establishing and Maintaining Cell Polarity in Soma6c Cells Dr. Luciana Frick Friday 10/4/24 Contact w/ Other Cells E-cadherins dimerize at early cell-cell contact o Junc6onal adhesion molecules (JAMs) are localized at TJs o JAM-A is present in early contact points § aPKC helps stabilize the immature junc6on by phosphoryla6ng JAM-A Ac6va6on of Cdc42 and/or Rac1 causes JAM-A to recruit the PAR complex ZO-1 and AF-6 link JAM-A to ac6n cytoskeleton o Promotes forma6on of an ac6myosin ring Contact w/ ECM Accumula6on of ac6n in lamellipodium ini6ates symmetry breakage Lamellipodium afaches to ECM via focal adhesion adaptor proteins Laminin in basal lamina and laminin receptors (e.g., integrins) establish signaling links with actomyosin cytoskeleton= PAR protein localiza6on Cell Migra6on PAR3/6 proteins and aPKC are recruited to the front of migra6ng cells Cdc42 regulates ac6n polymeriza6on PI3Ks- mediated pathways ac6vate integrins that are involved in ini6a6ng the migra6on cycle o Ac6vated integrins= stronger binding affinity In the presence of chemoafractants, PIP3 is produced at the leading edge by PI3K Focal Adhesions (FA)= Molecular Clutches Ac6n polymeriza6on= rapid retrograde cytoskeletal flow FA convert myosin pulling forces into trac6on forces against ECM= promo6ng protrusion and pulls cell body forward Cor6cal Landmark Some proteins and lipids are confined to apical/basal side o This asymmetric distribu6on is maintained by TJs Phospholipids and proteins afach polarity proteins and polarize other proteins to appropriate membranes Establishing Cor6cal Landmark Localiza6on of PIP2 (apical) and PIP3 (basolateral) help establish landmark o PIP2 is phosphorylated into PIP3- there are more PIP3 in cell § PI3k (ac6vated by E-cad.) phosphorylates PIP2 into PIP3 o PAR3 (apical) recruits PTEN (phosphatase) to hydrolyze PIP3 -> PIP2 PAR proteins directly bind to PIP head groups 3 Main Polarity Complexes Crumbs: Crb-Pals-PatJ (apical) Par: Par3-6-aPKC (apical) Scribble: LGL-DLG-Scrib (basal) o Important to maintain E-cad-mediated adhesions Polarity Complexes Explained Crumbs and Scribble are mutually antagonists; their ac6vity is restricted to their respec6ve domains Par complex represses ac6vity of Scribble and promotes Crumbs complex LGL (Scribble) inhibits aPKC (Par) in basolateral aPKC phosphorylates and exclude LGL (Scribble) in apical side How do PAR Proteins Work? (Mul6ple feedback loops) Par 3,6: contain PDZ proteins that are important for scaffolding forma6on Par 1, 4, aPKC: phosphorylate proteins to segregate them in appropriate compartments (e.g., kinases and signaling molecules) P2: E3 ubiqui6n ligase causes protein degrada6on in the incorrect compartment Summary: Par proteins are localized asymmetrically They complex with Rho-GTPase and junc6onal proteins They use various PTMs (e.g., phosphoryla6on, degrada6on), anchoring, and feedback loops to become more asymmetrical and to polarize the proteins and organelles within cell How is Cell Polarity Established? Adhesion with other cells or with ECM ac6vated and relocalize PAR proteins via biochemical forces, interac6on with Rho-GTPases and the actomyosin cytoskeleton PAR proteins then cause polariza6on via mul6ple feedback loops Polarized Cytoskeleton MTs are uniformly polarized o (-) ends toward apical side of cell MTs Reinforce Polarity Par3 afaches to Kif3 and moves towards (-) end (apical) of MT Par3 binds to PIP2/3 which helps retain it in apical side JAM can also help anchor Par3 to membrane In ECs: Nucleus sits on basal side ER and Golgi sit on apical side These posi6ons help guide vesicular transport Golgi Orienta6on Reinforces Polarity In ECs, proteins from ER travel together un6l they reach trans Golgi o They are then separated and transported to appropriate domains 2 ways of sor6ng proteins in polarized EC o Direct o Indirect: inappropriate protein is endocytosed and transported to correct domain (transcytosis) RAB-s and SNARE-s are important for polarized trafficking Rab proteins determine what will be the target membrane Rab protein bind to tethering protein in the membrane Rab11A forms complex with Par3-aPKC complex Nuclear Fallout (Nuf): adaptor of Rab11-GTPase to MT motor proteins Mislocalized PKC is transported via recycled endosome apically o PKC will then be phosphorylated= breaking afachment to transpor6ng protein (e.g., Nuf) When Nuf is phosphorylated, it is pushed out of apical domain If mislocated proteins are unable to relocalize, the protein will be degraded by lysosomes The same molecules and mechanisms used to induce polarity are used to maintain polarity Cell junc6ons are made of transmembrane proteins linked to polarity proteins and the cytoskeleton Junc6ons determine cell domains maintaining polarity Cellular Barriers Spectrin forms netlike meshwork underlining en6re cytosolic surface of membrane o Ankyrins help link the spectrin network Ankyrins also interacts with crumbs, maintaining it in apical domain  Par Func6on is Conserved Regardless of cell type, species; Par-complex is always Par3,6,aPKC and opposite to it is always Par1,2 Par proteins are responsible for polariza6on of all cell types Summary Molecules Mechanism 1. Establishment (Ini6al Cues) Sperm Entry PAR Break symmetry Cell Contact Homophilic adhesion (cadherins, IgCAM, PAR) Break symmetry ECM Contact Laminins, integrins, PAR Orient (up-down, in-out) 2. Establishment/Maintenance Cor6cal landmarks Orient (up-down, in-out) 4. Maintenance Cytoskeleton, - Signaling: Rabs Secretory apparatus localized, polarized trafficking - Flag: phospholipids, PIP2/3, Vamp, SNARE, Rho- sor6ng, delivery of basal/apical GTPase, ac6n, tubulin molecules. - Endosome recycling Forma6on of junc6ons - Junc6onal: cadherins, occludins, JAM Create physical barrier and barriers - Barrier: ac6n, spectrins, ankyrins between cellular domains Planar Cell Polarity (PCP) Dr. Luciana Frick Monday 10/7/24 PCP: polariza6on of 6ssues perpendicular to apical-basal axis Apico-basal polarity: cell polariza6on Planar polarity: 6ssue polariza6on o Apical-basolateral (cell) must be established first then proximal/distal (6ssue) o Unlike establishment and maintenance in cell polarity, 6ssue polarity has independent mechanisms PCP CORE Complex PCP is established and maintained by the CORE complex: PCP is best studied in Drosophila wings Proximal: Van Gogh and Prickle Distal: Frizzled, Dishevelled, Diego Drosophila and Mammals have Orthologous Genes Func6on of PCP proteins is conserved across species Establishment of PCP: Fmi binds Fz preferably ove Vang o Fmi-FZ is more stable than Fmi-Vang Proximal and distal complexes antagonize each other; further amplifying asymmetry Refining Polarity Remove mislocalized proteins Endocytosis Degrada6on Polarized cytoskeleton; direc6ng proteins to proper domains PTM Summary PCP is established using very similar mechanisms to how the aico-basal polarity is established Flamingo is key to breaking symmetry o It binds preferen6ally to Frz over Van Gogh Once in complex, PCP proteins are stabilized and resistant to degrada6on Polarized MT transport helps accumula6on of PCP proteins PTM can also modulate ac6vity and stability of PCP proteins Cells in Tissue Can be Oriented in the Same Direc6on via: 1. Gradients of expression of: Fat, Dachsous, four-jointed a. Influence MT orienta6on= help polarize PCP proteins b. Pk is the key component to read and interpret the Ft-Ds-Fj cues and orient Fmi-Vang-Pk complex and MT properly 2. Gradients of secreted factors: Wnt signaling a. Wnt is more abundant is distal end of wing b. Planar polarity is controlled by non-canonical Wnt pathway c. Orient PCP complexes 3. Mechanical forces a. Contrac6on of hinge causes elonga6on of wing b. This causes PCP complexes to shin orienta6on in order to re-align i. Stabilizes PCP complexes Summary MT of cilia Motor protein: dynein o Dynenin causes cilia to bend= movement (-) end= basal side= MTOCs (+) end= in cilia Types of Polarity PCP is essen6al for morphogenesis Rota6onal Polarity: within a cell Tissue polarity: between cells (across 6ssue) Convergent extension: 6ssue elonga6on in antero-posterior axis Apical constric6on: o Ac6n polymeriza6on is important for this mechanism Cell Polarity and Cancer One theory: once differen6ated, some cells can also develop the ability to proliferate= cancer Orienta6on of mito6c spindle allows asymmetrical cell division o One daughter cell will receive self-renewing factors while the other does not In cancer, both daughter cells develop self-renewing factors Decrease polarity= increase malignancy of tumor Par3= apically localized in cell Loss of Par3= poor differen6a6on= promo6ng prolifera6on Memorize Par proteins Basic components of cell-cell, and cell—ECM CORE complex Know the role of actomyosin C. elegans- figure with steps of fer6liza6on -> last step Signal Transduc.on: Conceptual Framework Wednesday 10/16/2024 Know the following: Signal transduc.on is instrumental in cellular adapta.on and survival Exosomes provide a mechanism for transfer of intracellular components including gene.c exchange between cells Proteins are dynamic molecules that con.nuous structural fluctua.ons between different conforma.ons/states with different energy levels The distribu.on amongst the various conforma.onal states is subject to perturba.on and it is the shiL in the conforma.on popula.on distribu.on of single molecules that governs cellular phenotype (e.g., behavior) All living organisms are controlled by basic drive to survive and propagate. Survival requires coordina.on and thus communica.on Evolu.onary-conserved Mandates Adapt to environment Do not waste energy (i.e., conserve energy) Intercellular Communica.on: between cells Gap-junc.ons: direct connec.on between 2 adjacent cells o 2 types of GJ: § Homotypic: both cells contribute same isoform of connexin § Heterotypic: each cell contributes a different connexin isoform o GP is highly regulated Exosomes: released a lot more during disease o Exosomes provide a mechanism for transfer of intracellular components including gene.c exchange between cells Receptors must be able to recognize input s.muli and must transduce info to machinery o “1st messengers”: intercellular signaling molecules Intracellular Communica.on: within the cell (i.e., organelles) Convey input info from receptor to intracellular targets o “2nd messengers”: intracellular signaling molecules Allows cells to respond and adapt o Allows for signal integra.on and amplifica(on To Change Cataly.c Ac.vity of Protein Alter protein amount o Change transcrip.on and/or degrada.on Alter turnover number o Structure dictate’s func.on Cellular Communica.on Requires:  Specificity and selec.vity Sensi.vity and range of responsiveness Tight regula.on Temporal flexible Adaptability Allow integra.on of mul.ple input s.muli Reversibility Low cost Signal transduc.on: take input s.muli and lead to change in output Signal transduc.on is instrumental in cellular adapta.on and survival Only specific cells express appropriate receptors to sense input s.muli and appropriate end effectors to elicit desired output Ul.mately alters cellular phenotype via sequen.al changes in ac.vity of protein nodes o To change protein func.on, you must change its structure Affinity Affinity= 1/KD (inversed rela.onship) o Higher KD= lower affinity KD= [ligand] resul.ng in half- maximal binding Too high of an affinity= irreversible binding Gibbs Free Energy (DG) Some proteins spontaneously fold into a stable ter.ary conforma.on due to thermodynamics o Spontaneous folding occurs if there is a net decrease in DG § DG in folded < DG in unfolded § More nega.ve DG= more spontaneous o Rate of reac.ons is independent of DG Enthalpy (DH) Measure of total bond energy of the system Enthalpy change (DH)= net change in total bond energy o + DH: product has MORE bond energy than reactants= require energy § Endothermic (heat is absorbed) § Breaking bonds required heat energy o - DH: product has LESS bond energy than reactants= releases energy § Exothermic (heat is released) § Forming bonds releases heat energy Entropy (DS) Measure of disorder (i.e., degrees of freedom (DF)) Entropy change (DS)= change in DF= change in number of possible states o + DS: increase in DF and is favorable o - DS: decrease in DF and is unfavorable  Hydrophobic effect offsets entropy loss o Main driving force for protein folding Increased H2O disorder= increase DS Proteins are dynamic molecules Undergo con.nuous structural fluctua.ons between different conforma.ons/states with different energy levels DG determines rela.ve popula.on distribu.on of each state o E.g., conforma.ons of lower free energy= more stable and more populated Transi(on state energy: rate of transi.on between states determined by height of energy barrier between states Enzymes alter rate of reac.on via lowering ac.va.on energy Structure Dictates Func.on The shiL in conforma.on popula.on distribu.on of single molecules that governs cellular phenotype (i.e., behavior) Intrinsically Disordered Proteins (IDP) Proteins that do not have a stable ter.ary conforma.on No large minimum free energy change (DG) upon folding o No large DG= no ter.ary structure Amino acid composi(on determines whether stable folded protein or IDP IDP/IDR Most proteins are combina.on of folded domains and intrinsically disordered regions (IDR) that lack stable structure o IDRs are hot spots more muta.ons IDP/IDR are highly sensi.ve to external factors= leads to different structures, dynamics and func.ons o Thermodynamically easier to change structure and func.on IDP is sensi.ve to environmental changes (e.g., pH, PTM, ionic strength); can act as a sensor Ordered proteins are frequently involved in catalysis and transport (areas where precision is required) IDPs are commonly involved in regula.on and control of signaling processes Advantages of Mul.-step Signal Transduc.on Pathways Energy efficiency Mul.ple points to modify responsiveness of pathways Amplifica.on of signal Signal Transduc.on: Conceptual Framework Friday 10/18/2024 Hourglass Conundrum: cells only use a few signaling pathways Timing and loca(on are key for achieving selec.ve signal transduc.on Achieving Selec.ve Ac.va.on Controlling signaling component loca.on to facilitate or inhibit probability of interac.ons o Scaffolding proteins form signaling nodes that help to relocate signaling § Binding to scaffold elicits conforma.onal change in signaling protein o Compartmentaliza.on: can also be used to relocate Phosphoryla.on of KSR scaffold blocks Raf binding= decreasing pathway output Biomolecular Condensates Highly dynamic and reversible membrane-less organelles are formed through liquid- liquid phase separa.on Network Dynamics Temporal control via connec.on and interac.on amongst signaling components o Cross-talk: allows integra.on of input s.muli o Combinatorial signaling o Feedback loops  o Feed-forward loops Thermodynamic of Ligand-Target Interac.ons Magnitude of DG is directly related to affinity and inversely related to KD For ligand to bind to target, the net DH and DS must be favorable (i.e., lower DG) o Hydrophobic effect= driving force for L-T binding How Signal Transduc.on Pathways Change the Phenotype of a Differen.ated Cell Alter expression level of effector and regulatory proteins o Transcrip.on and degrada.on Ac.vate molecular switches to alter protein ac.vity, loca.on, and/or protein-protein interac.ons o Ras family o Calcium- calmodulin system PTM o Reversible covalent addi.on or removal of a modifying group to the amino acid side chains o Occurs in IDR  o Affects protein expression o Regulates protein-protein interac.ons Post Transla(onal Modifica(on Residue modified Acetyla.on Methyla.on Phosphoryla.on Ser (S); Thr (T); Tyr (Y) Prokaryotes: His (H); Asp (D) Ubiqui.na.on Sumoyla.on S-nitrosyla.on Hydroxyla.on o Type and number of PTMs and temporal sequence are cri.cal determinants for signaling Protein Phosphoryla.on Protein kinases (PK) hydrolyze ATP -> ADP Creates docking site for protein-protein interac.on Alters protein structure PK recognizes Ser, Thr, Tyr, or His w/in specific sequence on substrate IDR of substrate allow greater surface interac.on between substrate and enzyme Phosphoprotein Phosphatase (PPP) PK= regula.ng signal amplitude, PPP= regula.ng signal dura.on o PPP rate constant > PK rate constant PPP is subject to regula.on by PTM Two types of PPPs o Protein serine/threonine phosphatases (PSP) o Protein tyrosine phosphatases (PTP) and dual specificity phosphatases (DSP) Ubiqui.na.on: mul.-step addi.on of ubiqui.n Ubiqui.n is ac.vated by E1 Ub-ac.va.ng enzyme Response to ubiqui.n depends on amount of ubiqui.n and how they are linked Sumoyla.on SUMO- small ubiqui.n-like modifier SENPs (sentrin/SUMO-specific proteases) hydrolyze the. Bond between SUMO and the SUMOylated target Acetyla.on Weakens histone-DNA and histone-histone interac.ons Increase protein stability by preven.ng ubiquityla.on and thus degrada.on Alters nuclear receptor signaling Methyla.on In DNA, alters chroma.n structure and recruitment of nonhistone proteins Hydroxyla.on Prevalent in secreted proteins and involved cellular oxygen sensing machinery S-nitroslya.on NO covalently aoached to thiol group of cysteine residues o Low level of NO= normal neuronal func.on and ac.vity o High level of NO= pathophysiology Signal Transduc.on: Receptor I Monday 10/21/2024 Know the following: The subfamilies of LGIC differ in subunit composi.on and be able to explain the effects of these differences The LGIC desensi.ze at different rates and to different extent o Desensi.za.on is influenced by the agonist used and PTM Piezo receptors are trimeric ion channels that open in response to mechanical s.muli The par.culate guanylyl cyclase generates the 2nd messenger cGMP from GTP Each class of receptors exists as different subtypes and isoforms with different proper.es (e.g., affinity, localiza.on, expression level) Receptors may have divergent effects due to being coupled to mul.ple signal transduc.on pathways (e.g., canonical vs non-canonical) The different receptors and signaling pathways “cross-talk” (i.e., exert posi.ve or nega.ve effects on other signal transduc.on pathways) Final cellular response may be regulated by mul.ple receptors and signaling pathways i.e., ul.mate cellular response is an integra.on of all the signaling-induced changes Nuclear Receptors (NR) Alter gene regula.on to change protein levels o Either increase or decrease transcrip.on of mul.ple genes that regulate various body func.on Provide a “low-cost efficient” way to alter many body func.ons Either directly bind to DNA or indirectly bind to TFs o Homodimeriza.on allows for binding to specific steroid-response element (SRE) § This changes the transcrip.onal ac.vity but NOT for agonist binding o Receptors are kept in cytosol via the heat-shock protein (HSP) Dimerized NRs= nuclea.on center Non-genomic Ac.ons of Steroid Receptors Steroid can elicit rapid cellular effects that do NOT require binding to DNA, RNA synthesis, or protein synthesis Ac.va.on of a PK inhibits that cell’s transcrip.onal response to a glucocor.coid Ligand-Gated Ion Channels (LGIC) Agonists bind at orthosta.c site (where endogenous ligand binds) altering: o Probability of channel opening o Dura.on of channel opening Amplitude of current flow of a single LGIC is fixed (e.g., either open or closed) o Dose-dependent effects depend on probability of ion channels being open Prolonged exposure to agonist= current decay (decrease in conductance and responsiveness)  LGIC exist in mul.ple states: o Closed: w/o agonist= closed= no ion conductance o Open: agonist binding= open= ion conductance o Desensi(zed: ligand bound= closed= no ion conductance § Buys.me for channel to return to res.ng state Rate and extend of desensi.za.on depend on: LGIC and subunit composi.on Specific agonist and PTMs can influence recovery Subunit composi.on affects loca.on and signaling LGIC subunit composi.on and stoichiometry affects channel ga.ng proper.es Orthosteric site: Where endogenous agonist binds Allosteric site: Any other site from the orthosteric site Posi(ve allosteric modulators (PAM): poten.ate effects of agonists Nega(ve allosteric modulators (NAM): inhibit effects of agonists Many ion channels are polymodal Gated by different types of s.muli (e.g., electrical, chemical, mechanical forces) Transient receptor poten.al (TRP): non-selec.ve ca.on channels sensi.ve to temp and chemical compounds K+ channels are sensi.ve to voltage and mechanosensi.vity Piezo channels are mechanosensi(ve channel and allow (+) ions to enter cell o Mechanosensi.vity: temperature, stress temperature Enzyme Linked Receptors: they all have the same general sturture- Single transmembrane protein Ligand recogni.on site on ECM Enzyma.c acidity in the cytosolic domain Types of ELRs Par.culate guanylyl cyclase o Catalyzes GTP to 2nd messenger cGMP Receptor tyrosine kinase (RTK) o Mediates ac.ons of growth factors o Ac.vated RTK= scaffold for docking and ac.va.on of signaling pathways o Helps decode input signals o Mechanism: § At basal state= TK is inhibited § Ligand (e.g., GF) binding= dimeriza.on (e.g., conforma.on change)= disinhibi.on= increase TK ac.vity= cross phosphoryla.on § Phosphorylated RTK= docking site for cytosolic proteins containing SH2 or PTB Ini.a.ng ac.va.on of: PL-gamma, Ras, MAPK, PI-3K/Akt pathway § RTK undergoes trafficking= affec.ng signaling and responsiveness o MAPK (i.e., ERK1/2 subfamily) § Dimeriza.on -> phosphoryla.on -> scaffold -> ac.va.on of a GEF -> ac.vates Ras protein ac.vates Rab -> ac.vate MEK -> ac.vate MAPK: Can either phosphorylate proteins Or enter nucleus to ac.vate transcrip.on § Cascade allows for: Specificity and control Amplifica.on Adaptability § Phosphoryla.on ac.vity of substrate can be done: Directly: phosphoryla.on by ERK Indirectly: through MAPK ac.va.on § Gene expression of substate can be done: Induc.on of phosphatases § Dura.on and amplitude of MAPK ac.va.on are cri(cal determinant of cellular effects and are regulated by network dynamics § ERK can influence EGF- induced prolifera.on via posi.ve feedback loop= phosphoryla.on= stable cFos= increase gene expression of AP-1 NGF-induced differen.a.on via nega.ve feedback loop= phosphoryla.on does NOT stabilize cFos= decrease gene expression of AP-1 o PI3K/Akt § Catalyzes phosphoryla.on of PIP2 (PIP2 -> PIP3) § PI3K has an SH2 binds to ac.vated receptor -> phosphorylates PIP2 -> PIP3 -> phosphoryla.on of Akt -> stabiliza.on -> increase ac.vity Phosphatases turns off pathway § Ac.va.on of Akt= signal amplifica.on  Jak-Stat receptors (JAK: janus kinase, STAT: signal transducer and ac.vators) o Cri.cal for stem cell maintenance, hematopoiesis and regula.ng immune system o JAK itself is not an ac.ve receptor but it is bound to a family of receptors o There are several different JAK receptors and then there are different STAT subtypes w/in each JAK § Each STAT subtype has their own set of TFs o Turn Signaling OFF: § Ac.va.on of protein tyrosine phosphatases (PTP) § Induc.on of Suppressors Of Cytokine Signaling (SOCS): Blocks STAT recruitment Targets degrada.on receptor Inhibits JAK kinase ac.vity Targets JAK degrada.on -- Wednesday 10/23/24 Receptor Ser/Ther kinase o Involved in prolifera.on, differen.a.on, apoptosis, development, wound repair and.ssue regenera.on o Mechanism § Contains type 1 and type 2 receptors (cons.tu.vely ac.ve) Theres a variety of type 1 and type 2 Immunophilin sits near receptor and prevents phosphoryla.on in the first step § Ligand binds to receptor -> dimeriza.on of both receptors -> release of immunophilin -> type 2 and phosphorylate type 1= phosphorylated Ser/Thr receptor= act as docking sites for R-Smads -> R-Smads are phosphorylated= translates to nucleus -> alters transcrip.on R-Smad linker region= site of PK phosphoryla.on o Turn Signaling OFF: § Inhibitory- Smads (i-Smads) § Recruit phosphatases= degrada.on § Recruit E3 ligase= Smurf= ubiqui.noyla.on= receptor degrada.on o Other Mechanisms: § Bambi Pseudo receptor Combines to both type 1 and type 2= blocks TGF-beta response § Secrete antagonists Bind to ECM BMPs= preven.ng receptor binding § Non-Smad Ac.vated TGF-B= scaffold Type 2 receptor= phosphorylated tyrosine (pTyr) binds w/ SH2= ac.va.on of MAPK pathway  pTyr also recruits TRAF6= ubiquina.on= ac.va.on of TAK1-> ac.vated p38 and JNK MAPK -> IkB (IKK) -> crosstalk with NFkB o recruitment of TRAF6 can also -> ac.va.on of the Akt pathway o pTyr act as a signaling pathway for many other intracellular signals Receptors w/o Enzyma.c Ac.vity Regulated proteolysis o Notch § Involved in paoern forma.on and cell fate § Maintained of SC and joint integrity § Mechanism: In the absence of ligand (Delta-like or Jagged), Notch is folded up= unable to access ADAM protease Presence of ligand, it pulls Notch allowing binging of ADAM protease= cleavage= Notch intracellular domain (NICD= TF) o NICD alter gene transcrip.on via “de-repression” § S.mula.ng of receptor is “one-and-done” § Binary Cell Fate Determinant: Receiving Cell o [Notch] > [DLL] o More notch= greater ability to receive ligands Sending Cell o [Notch] < [DLL] o More DLL= greater ability to donate ligands § Each receptor generates 1 NICD; no signal amplifica.on § Amount of NICD depend on binding of receptors § NICD has a short t1/2 Its own gene products will come back to turn off the system § 4 isoforms of Notch > 5 types of Notch ligands in 2 class (Jag vs Delta) o Wnt § Glycoprotein § TF: B-catenin § Mechanism: No Wnt s.mula.on= B-catenin is targeted for degrada.on by the (APC-Axin-GSK-3-CCKI) complex- biomolecular condensate o CKI and GSK3 phosphorylate B-catenin -> B-catenin ubiqui.na.on -> B-catenin degrada.on o Muta.on in APC= disrupts condensate forma.on of destruc.ve complex Wnt binds to Frizzled -> dimeriza.on w/ co-receptor LRP -> phosphoryla.on of LRP -> Disheveled and Axin recruitment= disrup.on of destruc.on complex func.on o Wnt must be palmitoylated for binding to Fzd o This prevents B-caternin phosphoryla.on  o Unphosphorylated B-catenin translocates to nucleus to drive gene transcrip.on The secretary Wnt regulators Dkk-1 inhibits canonical but not non-canonical Wnt The secretary Wnt regulator, R-spondins, enhances Wnt signaling Receptors linked to NFkB signaling o Central component in coordina.ng inflammatory responses o NFkB is typically inac.vate in the cytosol because it is bound to IkB § To ac.vate NFkB, IkB must be released o TAK-1 ac.vates IKK-1 which phosphorylates IkB o Toll-like receptor (TLR) § TLRs are subset paoern recogni.on receptor (PRR) § Recognize microbes leading to TAK-1 ac.va.on= immune response o Non-canonical NFkB: § NIK: NFkB-inducing kinase § Basal state: NIK cons.tu.vely degrades § Ac.vated: NIK accumulated GPCR o When GPCR is ac.vated, it is conforma.onally changes, opening the channel, allowing entrance o Inverse agonists: decrease basal state o 4 families of G proteins § Gs: Increases adenylyl cyclase § Gi/o: Decreases adenylyl cyclase Increase PLC Ac.vates K+ channels Decrease VDCa2+ channel § Gq/11: Increases PLC § G12/13 Ac.vated RhoA o Regulators of G-protein Signaling (RGS) § Act as GAPs (hydrolyzes)= increase GTPase ac.vity= turning off signal o Binding of G proteins and GPCRs depends § Density and affinity of GPCRs and G proteins § Localiza.on of GPCR and G proteins § GPCR trafficking o GPCR Desensi.za.on § Phosphoryla.on of GRK allows B-arres.n binding= preven.ng G protein from binding B-arres.n acts as an adaptor and scaffold o Fate of internalized GPCR depends upon if it is recycled or degraded Signal Transduc.on Friday 10/23/2024 GPCRS exert their effects by genera.ng 2nd messengers: Ras superfamily: RhoA o G12/13 ac.vates Rho-GEF= GTP -> GDP o RhoA bound to GDP= inac.ve= maintained in the cytosol § GDI maintains in the cytosol and prevents spontaneous release of GDP § GDF causes GDI to dissociate from RhoA o Ac.vated RhoA effects ROCK (Ser/Thr PK) cAMP o Enzyme that catalyzes ATP -> AMP o Phosphodiesterase (PDE): hydrolyzes 3’ cAMP -> 5’ cAMP and/or 3’ cGMP -> 5’ cGMP § PDE-5 is selec.vely hydrolyzes cAMP § PDE-2 can hydrolyze both cAMP and cGMP § PDE modulates amplitude and dura4on of response to cyclic nucleo.des PDE can act as an enzyma4c barrier or a local drain for cAMP o Mul.ple isoforms of adenylyl cyclase (AC) have mul.ple regula.ons involving Ca2+, beta-gamma § 9 isoforms of AC § AC is also regulated by Ca2+ and beta-gamma o PKA is cAMP ac.vated § cAMP regulates gene expression via PKA phosphoryla.ng CREB o Exchange protein directly ac(vated by cAMP (EPAC): acts as a GEF for Ras family o Popeye-domain-containing (POPDC) proteins: membrane-bound proteins that act as cAMP switch and cAMP buffer § Switch: alter biding interac.on § Buffer: facilitate PDE genera.on of cAMP nanodomains o Selec.vely in downstream func.ons is achieved via compartmentaliza.on o cAMP buffering system § Basal state: cAMP is largely immobile= slowed diffusion= PDE generated nanodomains= increase selec.vity cAMP buffering via biomolecular condensates o PKA undergoes liquid-liquid phase separa.on (LLPS) to form biomolecular condensates Diacylglycerol (DAG) o PLC-Beta ac.vity is s.mulated by Gq/11 and Gi o PLC-B hydrolyzes PIP2 -> two 2nd messengers, DAG and IP3 § PLC -> IP3 -> increase Ca2+ from intracellular storage from ER § PLC -> PIP2 -> DAG -> PKC § There are a variety of PKC subfamilies Ca2+ and Calmodulin o Increase Ca2+ § Influx via:  VGCC TRPC LGC § Release from stores IP3 Ryanodine receptor: ac.vated by Ca2+ to release more Ca2+ o Decrease Ca2+ § SERCA (back into storage) § PMCA § NCX § NCKX o Ca2+ is typically transient, localized and highly controlled o Calmodulin does NOT have intrinsic ac.vity but when bound to Ca2+= ac.vated= alter protein ac.vity o Phosphoryla.on of calmodulin-dependent PK (CaMK)= reduces Ca2+= prolong kinase ac.vity o CaMK also ac.vates CREB o High frequency of CaMKII= staircase effect in ac.vity NO o NO + thiol group oof cysteine residue = S-nitrothiols (SNO) o NO is the physiological regulator of soluble guanylyl cyclase (sGC) § GTP –(sGC)--> GMP § NO does NOT regulate the par.culate GC just the soluble GC cGMP o Ac.vates cGMP-dependent PK (PKG) o S.mula.on of PDE2= decrease cAMP levels Cell and Molecular Immunology Monday 10/26/2024 Immune System (IS): Protects host Recognizes self vs non-self An.gen: ac.vates IS Allergen: body mistakes something as hos.le Both arms have 3 phases (recogni.on, ac.va.on and effector) o Innate (IIS) § Cellular: phagocytes and NK cells § Humoral: complement proteins, acute phase proteins and interferons o Adap.ve (AIS) IS Triggers Foreign Paoern Recogni.on o PAMPs o MAMPs Danger o DAMPs IIS developed paTern recogni(on receptors (PRR) Innate Non-specific: broad classes of pathogens Recognizes pathogens via PAMPs by PRRs Highly conserved Tolerant: ability to dis.nguish self from non-self No memory Hematopoiesis: blood cell genera.on Connec.on between IIS and AIS NK cells Dendri.c cells Mast Cells Ac.va.on -> cytokines -> IL-4, IL-13, TNF-a -> inflamma.on and increase vascular permeability Ac.va.on -> histamines Ac.va.on -> prostaglandins Macrophages (Mo) Originate from blood monocytes Produce IL-2, IL-6, TNF-a Migrate and circulate to patrol for pathogens o In circula.on, they are called monocytes o Once in.ssue, they become macrophages  Opsoniza.on: coa.ng pathogens allowing them to be recognized o Opsonins: complement proteins Func.ons o Act as an an.gen presen.ng cell o Phagocytose o Generate cytokines -> inflamma.on and repair Granulocytes Pathogen recogni.on mechanisms: o Direct: PAMPs by neutrophil PRRs o Indirect: recogni.on of opsonized microbes by Fc or complement receptors Types: o Neutrophils § Extrusion of nucleic acids to form neutrophil extracellular traps (NETs) o Eosinophils: release oof cytokines- allergy o Basophils: release of cytokines, leukotrienes, and histamines Dendri.c Cells Immature states (iDC) are con.nuously sampling their environment When DC phagocytose the pathogen = ac.va.on = moves into lymph node DC recognize, degrade and present on the cell surface with MHC2 MHC1 Found on every nucleated cell CD8+ T cells From “non-self” an.genic pep.des Chaperon: TAP in ER MHC2 Found in certain immune cells CD4+ T cells From “Self” proteins Chaperone: Invariant chain in ER An.gn presen.ng cell: o MO, DCs, B-cells Organs that a play a role Bone marrow Spleen o White pulp: consists of lymphocytes § DC +Ag -> spleen -> ac.vate T and B cells -> plasma cell -> Abs o Red pulp: destroys RBCs § Splenic artery -> cords of Billroth -> sinusoids -> splenic vein o No spleen § Increased RBCs § Low Abs § Low platelet count § Low immune response Lymph node o Mo releases IL-18 -> ac.vates NK cells -> capture Ag -> shuole Ag to B cell= AMS ac.va.on Liver o Generates complement proteins Cell and Molecular Immunology Wednesday 10/30/2024 Types of Cytokines Autocrine: same cell secretes and receives signal Paracrine: secreted to nearby cells Endocrine: secreted to circulatory system AIS Specific Memory Tolerant Adap.ve - Cellular: T and B cells - Humoral: Ab 5 Types of Ab IgG: highest affinity IgE: causes mast cells to release histamines IgM: highest avidity o First Ab to be generated o Undergoes isotype switching into other Abs IgA: undergoes agglu.na.on IgD Ab diversity and specificity is increased by: VDJ rearrangement o Occurs in variable region o Occurs in DNA o D and J combine first, and then together combine to V region o In light chain, only the V and J combine- there is NO D region Class switching o Occurs in constant region of Ab o Immune response -> B cells may undergo class switching -> gives rise to B cells producing IgG, IgA, or IgE Soma.c hypermuta.on o Occurs in variable region o Occurs in lymph node o The V region of VDJ is mutated o Ac.va.on induce deaminase (AID) converts C -> at switch sites § U is then removed by UNG b/c there’s no U in DNA T Cell Development DN: double nega.ve o Does not have CD4 or CD8 DP: double posi.ve o Has both CD4 and CD8 Stem cell -> thymus –4 DN stages--> gets T-cell receptor (TCR) = DP -> depending on cytokine presence, it will develop CD4 or CD8 -> lymph node B Cell Development Pathogen recogni.on -> opsina.on -> ac.n rearrangement forms cup -> phagocytosis -> phagosome + lysosome -> phago-lysosome contains proteases and high acidity -> pathogen degrada.on -> pep.des loaded to MHC -> taken to membrane -> recognized by appropriate T cells (CD4 ro CD8) -> B cell ac.va.on -> Abs produc.on -> Ab undergoes mechanism (VCJ, class switch, hypermuta.on -> pathogen is degraded Cell Cycle Wednesday 11/6/2024 G1 phase: Cells commit to passage through START (restricBon point) DNA replicaBon/origins are licensed in G1 S phase: DNA replicaBon; from 1 copy of DNA to 2 copies of their genome 2 copies of chromosome are held together via centromere Semi-conservaBve DNA replicaBon: strands are separated and each act a complimentary strand to generated new double-strands of DNA G2 phase: Cell growth and makes sure all DNA and chromosomes are replicated in preparaBon for mitosis Mitosis: 4 phases of cell segregaBon Summary of DNA ReplicaBon 1. Find place to start 2. Separate strands 3. Unwind DNA to progress and form replicaBon fork 4. Release torsional strain ahead replicaBon fork by topoisomerases 5. Use RNA primer (primase) to start synthesis 6. Recruit DNA polymerase (synthesis and proofreading) 7. Coordinate synthesis and unwinding EukaryoBc Origins Origin regions: where replicaBon forks can be found It is not unBl it enters the S phase where the complexes (e.., replicaBon, iniBaBon, and progression complexes) are acBvated Enzymes of DNA ReplicaBon Helicase separate DNA strands o As DNA is separated, the single strand of DNA is coated with RPA Topoisomerase unwind DNA by creaBng breaks in DNA o Releasing tension via unwinding DNA polymerase require and annealed 3’ hydroxyl PRIMER (either made of RNA of DNA) in order to add the next nucleoBde ChromaBn structure must also be duplicated CAF-1 synthesizes H3-H4 dimer to be loosely associated with parent DNA NAP-1 loads H2A-H2B dimer into DNA strand Mitosis: chromosome segregaBon and cell division Interphase: DNA replicaBon, chromaBn is NOT highly condensed or packaged Prophase: chromosomes condense and is organized o Prometaphase: centrioles are sending out MTs while chromosomes are being organized Metaphase: chromosomes are aligned at midline Anaphase: replicated chromosomes are pulled apart o Misalignment is detecBng through sensing tension required for pulling chromosomes apart § Tensions indicates that chromosomes are ready to be pulled by MTs Telophase: chromosomes are completely separated and cytokinesis occurs o AcBn and myosin filaments form the contrac'le ring that causes cells to physically divide o Cytokinesis in animal cell results from the cleavage o Cytokinesis in plant cell: vesicles align at midline to form new cell wall § Golgi secretes molecules to help generaBng new cell wall Kinetochore: Kinetochore: proteins that are centralized in the centrosome o MT spindles will aaach at kinetochore Fluorescent AcBvated Cell SorBng Analysis (FACS) or Flow Cytometry Flow cytometry can be used to look at DNA content within a sample o Label DNA with fluorescent dye 2 Mechanisms for Ordering a Fixed Sequence of Cell Cycle Events Dependent pathway (domino theory): cell cycle events are sequenBal o E.g., this event has to happen first, then the second event Independent pathway (clock theory): cell cycle events are temporal sequence o E.g., ader a certain amount of Bme this happens, then ader 5 mins, this happens, etc.) Budding Yeast During S phase, they form a bud that gets bigger as DNA replicaBon conBnues New chromosomes are segregated so the daughter cell can have info too o Daughter cell is always smaller than mother cell Experiment: increased temperature= cell cycle arrest o CDC28= CDK1 is a regulator of cell cycle progression in buddy yeast Gehng through cell cycle requires a series of signal transducBon pathways CDK is a kinase that phosphorylates other proteins o They’re inacBve unBl bound to a cyclin Pathways are iniBated by CDK/cyclin complexes o CDK acBvity is dependent on cyclins § Cyclins are degraded ader use Cyclins are what change throughout the cycle CDK acBvity can be enhanced by phosphorylaBon by a CDK-ac S)- Oncogene Ras= onco gene Mitogen acBvates Ras Ras phosphorylates MAPK o AcBvates proteins for gene regulaBon (e.g., Myc) o Myc (oncogene) acBvates G1- CDK, pushing it into S phase ConsBtuBvely acBve Ras= unregulated cell growth Over acBve Myc= acBvaBon of G1-CDK inappropriately Entry into Cell Cycle- Tumor Suppressor Rb= tumor suppressor Normal Rb binds and inacBvates E2F o Rb then gets phosphorylated= releasing E2F § Rb is degraded o E2F helps progress cell cycle (pushing into S phase) MutaBon: E2F is not inacBvated= cell inappropriate enter cycle Inhibitors to target CDK4 and CDK6, that control entry into cell cycle Mad2L2 is an APC-Chd1 inhibitor, prevenBng degradaBon of M-cyclins unBl the end of M phase Shigella (bacteria) IpaB interacts with Mad2L2 and inacBvates it- inhabiBng APC-Cdh1 Shigella slows down cell cycle which buys Bme for infecBon to occur Prokaryotes do not have a nucleus but they do have a cell cycle They do not have CDKs but they have these histone-kinase two-component system o Histone will phosphorylate regulator downstream= turning on TFs= cell cycle progression Cell Cycle Friday 11/8/2024 CDC: growth, DNA synthesis, growth, chromosome segregaBon Cell Cycle Checkpoints (CPs) Ensure cell cycle events are occurring correctly Stop cell cycle progression if something is wrong CPs at the Molecular Level Hydroxyurea (HU) depletes dNTP pools= no DNA synthesis o Hus1 is a CP protein in fission yeast Tx with these drugs (e.g., irradiaBon, nocodazole) = incomplete DNA synthesis= cycle arrest In mutant cells, these cells will try to divide regardless if there is something wrong o However, they are missing DNA and are unable to recover ader a few rounds of replicaBon DNA Damage Cell Cycle CPs Sensors recognize damage o They phosphorylate transducers and then effectors o Effectors actually do something; they have an effect 2 Main DNA Damage DNA breaks o Recognized by Atm § Atm= Ser/Thre PK ReplicaBon inhibitors/bulky lesions o RPA is recognized by Atr o Rad= CP protein PIKK Family Atm and Atr are part of the PIKK family They are kinases that iniBate phosphorylaBon cascades Ataxia Telangiectasia: o Cells from these individuals are sensiBve to DNA double breakage o MutaBons of Atm leads to improper cell cycle arrest § PhosphorylaBon cascade: p53 (DNA repair) leads to cell cycle arrest, recruitment of DNA repair proteins or recruitment of Bax proteins = apoptosis DNA Damage Sensing and Atm Recruitment: Atm Pathways Mre11-Rad50-Nbs11 (MRN) complex binds at break site Atm interacts with MRN complex bound to break site= Atm autophosphorylaBon When Atm is phosphorylated, it phosphorylates other proteins including: o Chk2 leading to cell cycle arrest o H2AX: when phosphorylated, it is called gamma-H2AX, recruiBng repair proteins (e.g., BRCA1) p53 Mechanism Unphosphorylated p53 is bound to Mdm2 o Mdm2 targets protein for degradaBon Checkpoint acBvaBon causes phosphorylaBon of p53, unbinding it from Mdm2 p53 turns on p21 gene o p21 is CDK inhibitor protein (similar to p27) = arresBng cell cycle progression o When damage is repaired, p21 will be degraded and complex will progress Atr Pathwway Summarized Compromising progression of DNA replicaBon or DNA damage results in really long track of single stranded DNA (ssDNA) ssDNA is coated with RPA which is recognized by Atr Atr AcitvaBon ssDNA accumulates at damage site because although polymerase is blocked, the helicase keeps going leaving behind long tracks of ssDNA ssDNA is coated by replica