CELL 1360 Membranes Spring 2022 Ken Lerea, Ph.D. I. Introduction to Cell Biology Cell Biology Identifies cellular structures and their architecture/organization allowing for spatial and temporal regulation of cellular processes. • Adaptability – responding to external environment • Variability – cell to cell variability Credit BIOPHOTO ASSOCIATES /SCIENCE PHOTOLIBRARY Hurbain I., Romao M., Bergam P., Heiligenstein X., Raposo G. (2017) Analyzing Lysosome-Related Organelles by Electron Microscopy. In: Öllinger K., Appelqvist H. (eds) Lysosomes. Methods in Molecular Biology, vol 1594. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6934-0_4 Ciechanover A. Nature Reviews. Molecular Cell Biology 6:79 (2005) Visualization of intracellular compartments: membranous and nonmembranous organelles viewed as independent structures Interrelationships among compartments Techniques / strategies • Conventional methods • Subcellular fractionation • Disruption of cells CANVAS Posting - Guide to the disruption of biological samples - 2012 Random Primers, Issue No. 12, Jan. 2012, Page 1-25 (updated June 4, 2012) Techniques / strategies • Conventional methods • Subcellular fractionation • Disruption of cells • Mechanical shear forces Dounce Homogenizer Potter-Elvehjem with PTFE Pestle Techniques / strategies • Conventional methods • Subcellular fractionation • Disruption of cells • Mechanical shear forces • Non-mechanical methods • Detergents • Hypoosmotic shock • Freeze-thawing • Enzymatic Sonication: high frequency Force through small opening: high pressure http://stevegallik.org/cellbiologyolm_fractionation.html ‘mild detergent’: poking holes in the PM Shear cells between close-fitting plunger and glass vessel Detergents used for sample preparation and their properties. Detergent Type Characteristics Use Level SDS (sodium dodecyl sulfate) Anionic Strong detergent used to disrupt membranes and denature proteins Commonly used between 110% Sodium Deoxycholate Anionic Derived from bile salts. Effective at solubilizing proteins and disrupting protein-protein interactions. Common use level is 0.5%. CTAB (cetyltrimethylammonium bromide) Cationic Popular cationic detergent used for the isolation of DNA from plants. Polysaccharides associated with plants are insoluble in CTAB and high concentrations of NaCl. This can be used to effectively separate DNA from plant carbohydrates. For DNA isolation buffers, typical use level is 2%. NP-40 (nonyl phenoxypolyethoxyl ethanol) Non-ionic Generally mild surfactant which can dissolve cytoplasmic membranes but not nuclear membranes. Useful for isolating nuclei. Use at 0.1 to 1%. Non-ionic This mild surfactant is useful for disrupting cytoplasmic membranes of cultures cells, but lacks the strength to emulsify nuclear membranes. Consequently it can be used to harvest cytoplasmic proteins and analytes. Nonidet P-40 (octylphenoxy polyethoxyethanol)² Use at 0.1 to 1%.(P) Triton X-100 Polysorbate 20 (Tween 20, Polyoxyethylene (20) sorbitan monolaurate) Non-ionic This is a mild surfactant/surfactant that has polyethylene oxide as a hydrophilic group and a tetramethylbutyl phenyl group as the hydrophobic portion. For lysis solutions, up to 5%. In wash solutions, 0.10.5%. Non-ionic This surfactant is a heavily modified sorbitol in which polyoxyethylenes serve as the hydrophilic group and a 12 carbon lauric acid as the hydrophobic end. It is a very biomolecule friendly surfactant, being used in foods, pharmaceuticals, and in wash solutions for assays. Typically used at very low concentrations of 0.1%. https://opsdiagnostics.com/applications/samplehomogenization/homogenizationguidepart4.html Techniques / strategies • Conventional methods • Subcellular fractionation • Disruption of cells • Mechanical shear forces • Non-mechanical methods • Detergents • Hypoosmotic shock • Freeze-thawing • Enzymatic • Separation techniques • Differential centrifugation • Density-gradient centrifugation Differential centrifugation Nuclei Mitochondria, lysosomes The rate of sedimentation: density, size Ribosomes, exosomes Differential centrifugation Cell Homogenate cf 1,000 x g S P cf 13,000 x g P S cf 50,000 x g S P cf 100,000 x g S P Density-gradient centrifugation • • Media – sucrose, Ficoll (polysucrose & dextrans), Percoll Migration to the region where density equals surrounding medium From: Section 5.2, Purification of Cells and Their Parts Cover of Molecular Cell Biology Molecular Cell Biology. 4th edition. Lodish H, Berk A, Zipursky SL, et al. New York: W. H. Freeman; 2000. Techniques / strategies • Recent methods • Immunoaffinity purification • Ligands - antibodies https://www.sinobiological.com/res ource/protein-review/proteinpurification-by-ac Techniques / strategies • Recent methods • Immunoaffinity purification • Ligands - antibodies Bioanalysis. 2010 April ; 2(4): 769–790. doi:10.4155/bio.10.31 Techniques / strategies • Recent methods • Immunoaffinity purification • Ligands - antibodies • Ligands – other chemical ligands • Biotin-avidin • Annexin – phosphatidylserine non-covalent interaction (Kd = 10-15M) Avidin – 66-69 kDa Biotin – vitamin B7 https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learningcenter/protein-biology-resource-library/pierce-protein-methods/avidin-biotin-interaction.html Techniques / strategies • Recent methods • Immunoaffinity purification • Ligands - antibodies • Ligands – other chemical ligands • Biotin-avidin • Annexin V– phosphatidylserine • Flow cytometry • characterization based on their light scatter and fluorescence properties 2. Review of Plasma Membrane structure Question: Can you visualize the PM at the level of the light microscope? Table 2: Size of cellular structures & resolving power of microscopes A. Microscope Light microscope electron microscope B. Cellular structures •Cells •Nucleus •Microvilli •Mitochondria •Lysosomes •Peroxisomes •Golgi vesicles •Cytoskeletal elements •Plasma membrane Resolution 0.2 μm 2 nm (20Å) Size 10 – 100 μm 3 – 10 μm 1.4μm 1μm (length)-0.5μm (width) 0.5μm 0.5μm 50 nm 8 – 25 nm 5 – 10 nm Porter & Bonneville (1968) Fine Strucute of cells and tissues, 3rd edition, p7 Porter & Bonneville (1968) Fine Structure of cells and tissues, 3rd edition, p7 Components of plasma membranes • Lipids (phospholipids, glycolipids, cholesterol) • Sugars (N-linked vs O-linked glycosylation) • Proteins (integral, peripheral, lipid anchored) (Which is amphipathic?) Phospholipids – Glycerol based Do plasma membrane A and plasma membrane B exist in live cells? Tools within our freezer/ techniques within our lab: •Annexin v •Methyl--cyclodextrin •Triton X-100 •SDS •FRET •Fluorescence microscopy •Centrifugation •Flow cytometry Flow cytometry: fluorescence-activated cell sorter (FACS) Number of cells Membraneimpermeable fluorescent probe negative positive Fluorescence intensity Modified from: http://www.invitrogen.com/etc/medialib/en/images/ics_organized/References/the-handbook/Probes-Lipids-Membranes/Sphingolipids-Steroids-Lipopolysaccharides.Par.68068.Image.-1.0.1.gif Annexin v phosphatidylserine Phospholipids – sphingosine- based ceramide/ sphingomyelin - choline Where do these lipid modifications occur? Where do proteins modified by each reside? Thioester bond long-chain saturated fatty acid What type of modification? 16 carbons What type of modification? 14 carbons • Alpha amino group of an N-terminal glycine residue • Targets to endomembranes and plasma membranes What type of modification? vs GPI-linked proteins Dynamic regulation – how to control localization - Explain Citation: Triola G (2011) The Protein Lipidation and Its Analysis. J Glycom Lipidom S2:001. doi:10.4172/2153-0637.S2-001 http://www.omicsonline.org/the-protein-lipidation-and-its-analysis-2153-0637.S2-001.php?aid=2542 Components of plasma membranes • Lipids (phospholipids, glycolipids, cholesterol) • Sugars (N-linked vs O-linked glycosylation) • Proteins (integral, peripheral, lipid anchored) (Which is amphipathic?) Ruthenium red (polycationic dye) Components of plasma membranes • Lipids (phospholipids, glycolipids, cholesterol) • Sugars (N-linked vs O-linked glycosylation) • Proteins (integral, peripheral, lipid anchored) (Which is amphipathic?) Place the following proteins X, Y, and Z correctly? Leaflet O Leaflet I = Nllinked sugar X Y GPI Z SH Membrane Proteins Integral Membrane Proteins Single-pass membrane proteins Multipass transmembrane proteins Lipid Anchored membrane proteins • Lipid chain-anchored membrane protein • GPI-anchored membrane protein Peripheral membrane protein Integral membrane proteins are asymmetrically distributed: •Sugars (N-linked) face •Intrachain disulfide bonds •Sulfhydryl groups Plasma membranes are fluid structures Heterocaryon T= 0 min Filter: Double T= 40 min Fluorescein T= 40 min Rhodamine Singer & Nicolson 3. Membrane microdomains ‘Fluid—Mosaic Membrane Model’ Lipid rafts Nocolson GL. DISCOVERIES 2013, Oct-Dec; 1(1): e3. DOI: 10.15190/d.2013.3 2013 1972 Levental I, Grzybek M, Simons K Biochemistry 2010, 49, 6305-6316 State a hypothesis suggested by the following: Non- Historical Perspective: •Cholesterol, sphingolipids, GPI linked proteins remain in DRMs (1980’s) • Caveat – Detergent specificity • Caveat – GPI-linked protein subset Historical Perspective: •Cholesterol, sphingolipids, GPI linked proteins remain in DRMs (1980’s) • Caveat – Detergent specificity • Caveat – GPI-linked protein subset •From the beginning, cholesterol was suggested to be the essential component of rafts. (1970’s & 1980’s) • methyl cyclodextrin, saponin, cholesterol oxidase sphingomyelin sphingomyelin NH NH O H O Ro´g and Pasenkiewicz-Gierula Biophysical Journal 91(10) 3756–3767 (2006) NH O H Ro´g and Pasenkiewicz-Gierula Biophysical Journal 91(10) 3756–3767 (2006) How would you determine if protein X or complex Y associates with lipid raft/caveolae structures? phospholipid sphingolipid GPI-linked protein Cholesterol Flotillin Caveolins Irina S. Babina1, Simona Donatello1, Ivan R. Nabi2 and Ann M. Hopkins1. Lipid Rafts as Master Regulators of Breast Cancer Cell Function 0.5% Triton/ sucrose gradients https://www.mun.ca/biology/scarr/Gr10-23.html 0.5% Triton/ sucrose gradients Detergent-resistant membrane markers. Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 Oly- ostreolysin (oyster mushroom)- cholesterol interacting Cav- caveolin Flot- flotillin TrfR- transferrin receptor Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 Oly- ostreolysin (oyster mushroom) Cav- caveolin Flot- flotillin TrfR- transferrin receptor Dsc2- desmocollin 0.5% Triton/ sucrose gradients Detergent-resistant membrane markers. Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 Oly- ostreolysin (oyster mushroom)- cholesterol interacting Cav- caveolin Flot- flotillin TrfR- transferrin receptor 0.5% Triton/ sucrose gradients 12 11 10 9 Detergent-resistant membrane markers. Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 Oly- ostreolysin (oyster mushroom)- cholesterol interacting Cav- caveolin Flot- flotillin TrfR- transferrin receptor Question – Do desmosomal proteins associate with lipid-raft structures? Desmosomes: A light microscopic and ultrastructural analysis of desmosomes in odontogenic cysts https://www.jomfp.in/article.asp?issn=0973029X;year=2014;volume=18;issue=3;spage=336;epage=340;aulast=Raju;type=3 Question – Do desmosomal proteins associate with lipid-raft structures? Approach – isolation of buoyant detergent-resistant membranes https://plasticsurgerykey.com/noninfectious-vesiculobullous-and-vesiculopustular-diseases/ Dsc2 associates with DRMs of MDc-2 cells. Desmocollin-YFP desmocollin desmoglein desmoplakin cytokeratin Is this an issue of overexpression? Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 Oly- ostreolysin (oyster mushroom) Cav- caveolin Flot- flotillin TrfR- transferrin receptor CK- cytokeratin MDc-2 cells – clone of MDCK cells, epithelial Madin- Darby canine kidney cells Dsc2 associates with DRMs of MDc-2 cells. Desmocollin-YFP desmocollin desmoglein desmoplakin cytokeratin Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 Oly- ostreolysin (oyster mushroom) Cav- caveolin Flot- flotillin TrfR- transferrin receptor CK- cytokeratin Question – How would you further prove that desmocollin associates with rafts? Approach – Manipulate cholesterol Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 Question – How would you further prove that desmocollin associates with rafts? Approach – Manipulate cholesterol Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 Question – How would you further prove that desmocollin associates with rafts? Approach – Manipulate cholesterol Cholesterol oxidase Predict the structural change with cholesterol oxidase Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 CO-Cholesterol Oxidase Question – How would you further prove that desmocollin associates with rafts? Approach – Manipulate cholesterol Cholesterol oxidase http://www.personal.psu.edu/faculty/r/x/rxn/enzyme-catalysis.html Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 Explain this data What would you expect if you treat with methyl -cyclodextrin? Dsc2 – desmocollin Oly – ostreolysin Resnik N et al. J. Biol. Chem. 2011;286:1499-1507 What domain determines whether a protein associates with lipid raft structures? http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/shimomura-slides.pdf http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/tsien-slides.pdf http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/shimomura-slides.pdf Osamu Shimomura DISCOVERY OF GREEN FLUORESCENT PROTEIN, GFP http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/chalfie-slides.pdf http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/tsien-slides.pdf Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916 acylation acylation prenylation acylation Madin-Darby canine kidney (MDCK) cells Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916 Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916 Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916 Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916 Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916 Edidin M (2003) Lipids on the frontier: a century of cell-membrane bilayers. Nature Reviews Molecular Cell Biology 4, 414-417
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