Working With Cells: Visualization, Isolation & Manipulation - Topic 2 - F2024 PDF

Live lectures may be recorded with Echo360!  Introduction into cell biology Working with cells: Visualization, isolation and manipulation Membrane structure Cell communication Cytoskeleton Membrane tr...

Live lectures may be recorded with Echo360!  Introduction into cell biology Working with cells: Visualization, isolation and manipulation Membrane structure Cell communication Cytoskeleton Membrane transport of small & signaling pathways Cell cycle molecules & the electrical Apoptosis properties of membranes Intracellular Compartments and Protein sorting Review Intracellular Vesicular Traffic Visualization (chapter 9): Light microscope Different techniques of light microscopy Fluorescence microscopy & fluorescent labels Electron microscope Transmission EM Scanning EM STEHM Isolation of cells from tissues (chapter 8) Cell cultures Transgenic organisms At the end of topic 2, you will - know the most common light microscopy techniques and be able to identify techniques used - understand the principle of fluorescent microcopy and the different labeling approaches - know the differences of light and electron microscopy and differences within electron microscopy - know how to isolate specific cells/organelles/proteins - know about the use of cell cultures and transgenic organisms Early microscopes Garry Larson  The Light Microscope What kind of light microscopes do you know? Do you know different techniques used in light microscopy? What can you see with the light microscope? Fig. 9-2B Molecular Biology of the Cell, 7th edition Most objects (cells, organelles) are not visible with the human eye Fig. 9-1 Molecular Biology of the Cell, 7th edition Figure 9-2 Molecular Biology of the Cell 6th edition 13th century: spectacles were being used Janssen (1590) builds the first compound microscope Giovanni Faber coined the name microscope 1625 Zeiss and Abbe (19th century) make commercial microscopes What is the conventional resolution limit of a light microscope? Figure 9-2A Molecular Biology of the Cell 7th edition Figure 9-3 Molecular Biology of the Cell 7th edition Figure 9-4 6Molecular Biology of the Cell 7th edition Light waves travel slightly different routes -> optical diffraction  When do we reach the limit of resolution? 1. single point à ______________________________ 2. two points close together à___________________ Figure 9-29 Molecular Biology of the Cell 7th edition  When do we reach the limit of resolution? 0.61 l Figure 9-5 partly Molecular Biology of the Cell 7th edition resolution = n sin q numerical aperture resolution depends on 1. 2. Is there a difference between resolution and detection??? 1. air (n=1), q=20, white light (l=530nm) 2. air (n=1), q=60, white light (l=530nm) 3. air (n=1), q=60, white light (l=390nm) 4. oil (n=1.51), q=60, white light (l=390nm) HOMEWORK!!!! n sin q = numerical aperture 1. Bright field microscope 2. Dark field microscope 3. Phase contrast microscope 4. Differential-interference contrast microscope Figure 9-7A Molecular Biology of the Cell, 7th edition fibroblast in cell culture Problem: Solution: Fixation: immobilize, kill and preserve cells àembedding in waxes or resins to support fragile structure à1-10 µm thick sections Staining ‘fast green’ stains Figure 9-9B Molecular Biology of the Cell 7th edition cellulosic cell walls ‘safranin’ stains lignified cell walls Plant root cross section Figure 9-7D, 9-6B Molecular Biology of the Cell 7th edition opaque disc http://tiny.cc/72ghq  N. Radiolaria Hughes holoplanktonic zooplankton protozoa Borrelia Figure 9-7B, 9-6C Molecular Biology of the Cell 7th edition Fibroblast in cell culture Nobel Prize 1953 Frits Zernike  similar to phase contrast microscopy system employs polarized light à 3D image Fibroblast in cell culture Figure 9-7C Molecular Biology of the Cell 7th edition https://encrypted- tbn1.gstatic.com/images?q=tbn:ANd9GcRhWp8Ya6cHZ9N- dsM2cuqGq9N5bpB3Dpm9UtSdffUZa1mfcmEzO6q1-DRP What affects resolution? What can you change to increase resolution? How do we detect small objects that are below the resolution limit of conventional light microscopes? 1. 2.  Some commonly used fluorescent probes: Figure 9-10A, 9-12 Molecular Biology of the Cell 7h edition Some commonly used fluorescent probes Three main groups: 1. fluorescent dye: DAPI, FITC DAPI: 4',6-diamidino-2-phenylindole FITC: fluorescence isothiocyanate 2. fluorescently labeled antibodies: rhodamine B, Alexa 568, Cy5, Cy3 3. fluorescent proteins: CFP, GFP, YFP, RFP (genetically engineering) Figure 9-12 Molecular Biology of the Cell 7h edition nuclear envelope actin cytoskeleton plasma membrane ER mitochondria peroxisome  How does a fluorescence microscope work? dichroic Figure 9-10C Molecular Biology of the Cell 7h edition Figure 9-24 Molecular Biology of the Cell 7th edition  in yeast mitochondrial compartment trap of Utricularia gibba Figure 9-25 Molecular Biology of the Cell 7th edition How can we detect specific molecules? 1. 2. 3. When would you use which fluorescent probe?  Example: Hoechst dye detects DNA http://upload.wikimedia.org/wikipedia/commons/thumb/6/6c/HeLa_cells_stained_with_Hoechst_33258.jpg/3 00px-HeLa_cells_stained_with_Hoechst_33258.jpg Fig. 9-15 Molecular biology of the cell 7th edition What are antibodies? Who produces antibodies? Homework: If you want to analyse a plant protein, Why use secondary antibodies? which organism would you use to make your primary & secondary antibodies?  Multi-fluorescent-probe microscopy actin filaments (green fluorescent antibody) Fig. 9-13 Molecular biology of the cell 7th edition mitochondria (red fluorescent dye) nucleus (blue fluorescent dye DAPI)  Most common one used: GFP à What is GFP (green fluorescent protein)? e.g. GFP post-translationally modification Fig 9-16 Molecular Biology of the Cell, 7h edition  e.g. GFP What do we detect with these reporter genes? What is the advantage of genetic engineering? Fig 8-62A Molecular Biology of the Cell, 7h edition  A B C D E F What do we detect with cis regulatory sequence - marker gene fusions? Fig 8-62B Molecular Biology of the Cell, 7th edition  coding sequence for protein X peptide location signal coding sequence for reporter protein peptide location signal What do we detect with signal peptide marker gene fusions? What do we detect with protein-marker-fusions? Mammalian cell expressing EB3 fused to BFP à appear to zoom around the cell Fig. 9-18 Molecular Biology of the Cell 7th edition Figure 9-20B, C Molecular Biology of the Cell 7th edition movie 10.6 Molecular Biology of the Cell 6th edition Figure 9-19 Molecular Biology of the Cell 7th edition technique? observe with this Discuss: What do we  Modifications of FRET: 1. to detect Ca2+ Discuss: What do we observe with this technique? Figure 9-22A Molecular Biology of the Cell 7th edition  Modifications of FRET: 2. to detect cAMP Figure 9-22B Molecular Biology of the Cell 7th edition Discuss: How is the emission affected upon binding cAMP?  Fig. 9-32 Molecular Biology of the Cell 7th edition àWhat do we observe with this technique? Fig. 9-36, 37 Molecular Biology of the Cell 7th edition Fig. 9-38 Molecular Biology of the Cell 7h edition formation of clathrin-coated pit and its subsequent endocytosis  When to use what probe....??? What fluorescent probe would you use to detect genomic DNA? How would you detect specific DNA segments (chromosome 1) e.g. for karyotyping? What fluorescent probe would you use to detect a very specific type of protein, e.g. a MAP kinase? When to use what transgenic construct…? When to use which microscope? Figure 9-12 Molecular Biology of the Cell 7th edition  Detect classes of molecules by binding dyes Detect specific DNA segments with labeled DNA probes Detect specific proteins by binding labeled antibodies to protein of interest Fusion with marker genes à transform organism with constructs promoter-marker gene fusion: gene expression signal- marker gene fusion: intracellular localization gene A –marker gene fusion: turn over, localization, protein-protein interaction... Visualization (chapter 9): Light microscope Different techniques of light microscopy Fluorescence microscopy & fluorescent labels Electron microscope Transmission EM Scanning EM STEHM Isolation of cells from tissues (chapter 8) Cell cultures Transgenic organisms Figure 9-26 Molecular Biology of the Cell 5th edition Figure 9-14 Molecular Biology of the Cell 7th edition light microscopy resolution: 200 nm electron microscopy resolution: 1 nm How can EM improve resolution? Figure 9-40 Molecular Biology of the Cell 7th edition What are the advantages of an electron microscope? What are the disadvantages? complex specimen preparation: thin sections: 25 – 100 nm àdehydration (rapid freezing, freeze substitution), permeated with resin forming solid block heavy metals can provide contrast Figure 9-41 Molecular Biology of the Cell 7th edition Figure 9-42 Molecular Biology of the Cell 7th edition yeast cell  Detect individual proteins with primary and secondary antibodies à label: colloidal gold particle Figure 9-43 Molecular Biology of the Cell 7th edition  Want to guess??? Figure 9-46 Molecular Biology of the Cell 7th edition  actin filament: helical arrangement of actin monomers clathrin coated vesicles Figure 9-47 Molecular Biology of the Cell 7th edition  Specimen frozen/dried à coated with thin layer of heavy metal entire specimen can be put in microscope (any size limit???) resolution: about 10 nm Figure 9-45 A Molecular Biology of the Cell 7th edition  Scanning transmission Electron holographic microscope: STEHM able to look on surface & inside best resolution microscope ever built like a electron microscope with computer tomography Helium atom à resolution 40 pm estimated/calculated diameter: 31 pm http://tiny.cc/mu5i7 Interested? check out the webpage: http://www.stehm.uvic.ca/facility/i nstruments/#section0-22 Light microscopes are perfect to view cells in action Labeling of single molecules allow to analyse these molecules with the light microscope (detection although molecules are below resolution limit) Electron microscopy allows detailed analysis of intracellular organization (TEM) and surfaces (SEM) new microscope (STEHM) allow to view details on atomic level Visualization (chapter 9): Light microscope Different techniques of light microscopy Fluorescence microscopy & fluorescent labels Electron microscope Transmission EM Scanning EM STEHM Isolation of cells from tissues (chapter 8) Cell cultures Transgenic organisms How do we isolate a specific cell type from a whole tissue? 1. 2. e.g. FACS Alternative: laser capture microdissection Why do you want to isolate specific cell types? from the company ‘Sino Biological’ webpage https://www.sinobiological.com/category/fcm-facs-facs à selects 1 fluorescent à sorts several 1000 cell from 1000 cells cells per second! à homogenous cell population 1907: beginning of cell cultures àneuronal doctrine primary cultures: ______________________ secondary cultures: ____________________ In vivo or in vitro? cells often require solid surface to grow and divide Commonly used human cell line: HeLa Who was Henrietta Lacks? plant tissues produce calli, which regenerate new plants Cassava plant regenerated  Figure 8-2 Molecular Biology of the Cell 7th edition Chick Mouse myoblast fibroblast rat ganglion tobacco nerve cells cells  http://www.youtube.com/watch?v=t1TcDHrkQYg Replicative cell senescence: ____________________________________________________ What is the reason for replicative cell senescence? à à Transformed cell lines: cancer cells  What is one major application Hybridoma cell: for the use of hybrid cells? Figure 8-3 Molecular Biology of the Cell 7th edition  How would you improve purity? Figure 8-5 Molecular Biology of the Cell 7th edition 1000 g 80 000 g 20 000 g 150 000 g (chloroplasts) Figure 8-8, 8-9, 8-10 Molecular Biology of the Cell 7th edition Figure 8-11 Molecular Biology of the Cell 7th edition  Gene-replacement in mice Figure 8-55 parts Molecular Biology of the Cell 7th edition Figure 8-55 Molecular Biology of the Cell 7th edition  Site specific manipulation of the genome: use a system found in bacteria as defense against viruses clustered regularly interspaced short palindromic repeats Figure 7.81 Molecular Biology of the Cell 7th edition  Site specific manipulation of the genome: an use a system found in bacteria as defense against viruses endonuclease light blue: binds to Cas9 dark blue: matches position in genome Figure 8.57A, B Molecular Biology of the Cell 7th edition Figure 8.57C,D Molecular Biology of the Cell 7th edition  What exactly happens at this stage? Figure 8-71 Molecular Biology of the Cell 7th edition Individual cell types can be isolated Cell organelles can be isolated Cells can be cultured in cell cultures Transgenic organisms help to study gene/protein function on the cellular and whole organism level