Exam 1 Review Notes PDF

Fundamentals of Cellular and Membrane Structure Monday 8/26/24 Dr. Fliesler Lecture Summary Cells are...

Fundamentals of Cellular and Membrane Structure Monday 8/26/24 Dr. Fliesler Lecture Summary Cells are compartmentalized o Anatomically: via membranes · compart: o Func?onally: conven?onal vs current defini?ons - ↑ Uxn efficiency. Membrane compartmentaliza?on: - avoid futile cycles o Increases reac?on efficiency = o Avoids “fu?le cycles” Prokaryotes do NOT have internal organelles Polare (hydrophilic) charged head , RNP (RNA + protein)= organelles that lack membranes Chydrophobic, non-charged tail Membranes are amphipathic a o Polar (hydrophilic), charged head group lipids · o Non-polar (hydrophobic), non-charged tail FA chains GPLs - Cytosol is the largest compartment in the cell sterols - - cytosol = Cell membranes are made up of: (4) sphingolipids & - largest all. 1. Lipids § Glycerophospholipids (GPLs) protein · compartment C-1: saturated FA carbs · C-2: unsaturated FA glycoproteins - C-3: esterified glycerol backbone glycolipids- § Sterols RNA · 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 ↑ protein : lipid ratio = bio activity. 3 Increase FA chain length= increase melt temp. ↑ FA chain = 4 Tm. Increase dbs= decrease effec?ve chain length ↑ dbs o Membrane thickness would be reduced = ↓effective chain & § Unsaturated chains (dbs) take up less space than saturated chains (no dbs) Increase FA unsatura?on= increase dbs= decrease melt temp. a unsort ↑dbs ↓ im = = Increase carbon number= increase melt temp ↑ Carbon # ↑ Tm = Physical proper'es of phospholipids tend to be conferred by their cons'tuent FAs T p: / Of PL tend # Physical props. ↑ protein : lipid ratro = ↑ bio. activity to be conferred by their FA ↑ Carbon# =↑ FA chain = ↑ Im ↑ unsat =* dbs = chain = I Tm reffective  · hydrophopic effect : ↳ mini · free energy NP-NP interactions ↳ max. Polar-polar & 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 · PL cylinders = = o Phospholipids + water= form bilayers BILAYERS o Phospholipids are cylindrical and form bilayers, liposomes/vesicles NOT micelles · wedge = o FAs are wedge-shaped and form micelles NP pass quicker through membrane than polar molecules micus a 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 · peripheral (extrinsic) o Peripheral (extrinsic) loosely associated - § Loosely associated with bilayer mild +X to Deremoved -. § Require mild tx to remove them from membrane Integral (intrinsic) O · integral (intrinsic o O § Strongly associated with bilayer -strongly associated narshty to be removed - § 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 · bilayer distribution of proteins , Detergents: lipids & carbs is ASYMMETRICAL o SDS: unravel protein and denatures them SDS harsh 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 choline o Tend to have more saturated FAs ↑ extra naturate Amino-PLs (PE, PS) prefer inner (cytoplasmic) leaflet of bilayer v o Tend to have more PUFAs · Choline-PLs - extraceuclar - have more saturated FAs · amimo-PLs - intracacular have more PUFAs - ↑ aminos are introverts & PUFA up Physical state of membrane lipids depends on composi?on and temperature & o More unsatura?on = more fluid - - ana lipid o Higher temperature = more fluid funidity (temp) = ↑ unsat. = 4fluidity summer K ↑ activity ↑ temp. = 4 fluidity Humidity  TM = < fluidity J Cholesterol as < Tm ↓ fluidity a fluidity buffer ↓ = 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 a rotational diffusion rapid = T transverse = Lateral and rota'onal diffusion of lipids and flexing of PL acyl chains are rapid d extremely Transverse (“flip-flop”) diffusion of lipids is extremely slow in pure lipid environment Slow 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) cold dmumt = Membrane fluidity is influenced by TEMP. and COMPOSITION & glycolipids o Decrease temp= decrease fluidity > - ↓ temp. ↓ = fluidity not still f glycosphingolipids outer leaflet of bilayer Kunsat = fluidity ↑ =. unsat = ↑ Guid 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 lipid rafts Sphingolipids · = o Serve as plauorms for signal transduc?on components ↓ Cholesterol Membrane thickness varies with faXy acyl chain composi?on of phospholipids Membrane proteins can be classified according to their: o Associa?on with membrane (e g., peripheral vs. integral. 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 ↑ left we just RBC membrane 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 & proteins lipias domains may be restricted o Membrane specializa5ons (e.g., junc?onal complexes, ?ght junc?ons) also basal side represent dis?nct membrane domains to apicall -> :S3 Usedto State mobility FRAP & FLIP Modern biophysical method (e.g., FRAP, FLIP) provide quan?ta?ve means to study : protein lateral protein lateral mobility Mobility - o FRAP: blast bright light on membrane, measure that spot (?me of recovery) o FLIP: measure distally of bleached area FRAPd FLIP help determine · FRAP and FLIP determine lateral diffusion coefficient (D-trans) lateral diffusion coefficient o Small D-trans= restricted mobility ↳ ↓ D-trans ↑ mobility = Plasma membrane proteins interact with cytoskeleton (cytoplasmic face) and the restriction 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 · caveolar = o Cavelin: scaffolding proteins that binds cholesterol and non-clathrin- signal transduc?on proteins, receptor molecules, etc. mediated endcyto. 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 · rafts & caveolae try not to sombized they have low buoyancy = allows them to Separate from other membrane domains Subcellular frac?ona?on objec?ves: o Maintain organelle/membrane integrity o Isolate organelle/membrane from other cellular components · subcellmar fractionation - maintain integrity - isolate - avoid friction  o Avoid excessive fric?on to minimize membrane fusion Features in media use to disrupt ?ssues/cells: media" (5) o Avoid high [salt] (causes aggrega?on) "perfect · ↓ [salt] - o Avoid Ca2+ (ac?vates proteases) no cast - o Osmolarity (iso-osmo?c condi?ons) iso-osmotic - o pH (7.4-7.5) 7 5 ph - o Protease inhibi?on. - Protease inhibitors Density gradient ultracentrifuga?on - ↑DS = ↑ separation 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 Particle Predia Sink : § Buoyancy of par?cle < buoyancy of medium= par?cle floats Particle PTM = : - o Occurs in ER ↳ occurs in ER ER Glycosyla?on func?on: ↳ you are o Quality control o Facilitate protein delivery given sugar o Mediate cell aXachment in the ER o Influence protein-protein interac?on o Alter protein solubility 2 Types o N-linked: always occurs on Asp residue types of § N-X-S/T glycosylation 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  test. glyco. - uctin - SDS PAGE & - pharm agents point Mutations 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 classes of oligos & 2. Complex oligosaccharides complex - High-mannose oligosaccharides high mannose - Recruitment of COP2 Proteins GTP-bound Sar1 binds to COP2 GTP -> GDP causes Sar1 hydrophobic tails to pop out= coat disassembles ⑭ GTO-cop ⑳ ↳ sand fails pop out= disassembly 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 COP1 resides · ER-proteins are retrieved via ↳ ADEL C-term sequence on. 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 Atp - Microtubules= tubulin 2 Add = association = Gend Polarity of Ac?n Filaments ~ - ADP Associa?on of ATP-ac?n: (+) end => V Deute = dissociation O end = Dissocia?on of ADP-ac?n: (-) end ~ - T o [Free monomers] influences rate of dissocia?on Rate-limi?ng step= availability of ac?n subunits # hydro occurs * hydrolysis occurs AFTER polymeriza?on AFTERPOLY G actin ATPAD - F-actin - ATP. - Ac?n-binding proteins # Regulate polymeriza?on phos. ↑ ↓ hydro Filament length y F-actin - ADP G-actin ADD - Structure Func?on  · F-actin = drags lyso some , carrying Ac?n Molecules cargo, into cel Ac?n= road Myosin= motor vehicle that uses ATP o ATP hydrolysis drives myosin mo?lity (“power stroke”) myosin + ATP - o Myosin + ATP= force = power stroke 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 - dymin endocytosis = Dynein: towards (-) end, retrograde o Endocytosis Kinesin: towards (+) end, anterograde o Exocytosis ↳ nesin exocytosis = · Centrosomes= MTOCs 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