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Cell biology 2014 (revised 20/1-14) Lecture 1: “Recommended reading” Chapter 8 Chapter 9 501-505 571-572 Alberts et al 5th edition 579-589 592-593 604-610 1 The tree of life Microbiology Microbiology & Cell biology (prokaryotes) Nucleus Eubacteria Eukaryotes Archaea Cytosol 2 Biology Molecular biology Cell biology Organism biology Met Ser Arg Pro Nanometers Micrometers Millimetres Meters 3 The starting point of cell biology: microscopy I am seeing atoms Let's call them cells (1665) Robert Hooke (1635 – 1703) Cellulae, little room Sliced cork 4 Conceptual breakthroughs in cell biology Mikroskopische Untersuchungen über die Übereinstimmung in der Struktur und dem Wachsthum der Tiere und der Pflanzen (1839) - All organisms consist of one or more cells - The cell is the basic unit of structure Die Cellularpathologie (1858) - All cells arise from preexisting cells On the Origin of Species by Means of Natural Selection (1859) - All cells have a common ancestor Zellsubstanz, Kern und Zelltheilung (1882) - Chromosome (thread) segregation during mitosis (i.e. precise partitioning/transport of defined cell structures) 5 All eukaryotic cells are in principle very similar - Organelles - Cytoskeleton - Nucleus - Chromosomes Key questions in cell biology • Structure and functions of cellular components • How do cells communicate? • Which signals trigger cell cycle entry? • How is cell duplication coordinated? • How is one cell split into two? 6 Multicellular eukaryotes – not just cells The extra cellular matrix (ECM) works as a scaffold in metazoans supporting cells in various ways 7 Animal tissues mainly consisting of (different) cells - Epithelia Protective covering of surfaces, both outside and inside the body - Muscle Force generating cells (contraction) Animal tissues consisting of cells and ECM - Connective • Hard tissues of bone and teeth • Transparent matrix of the cornea • Ropelike organization of tendons 8 How to study individual animal cells Primary cell cultures Secondary culture Explants Proliferation (growth factors) Complete tissue section Only cells Tumor patient Immortalization (e.g. by oncogenes) Cell line, with indefinite 9 proliferative potential I. How to study the function of a protein in cells Depletion/mutation of endogenous protein Normal (Control) Overexpression of protein (ectopic expression) 10 II. How to study the function of a protein in cells Central dogma of molecular biology - Loss-of-function mutations - Gain-of-function mutations - Overexpressed (trans)gene DNA Transcription - RNA interference mRNA Translation Protein -Inhibitory (pharmaceutical) drugs new field ”chemical genetics” RNA interference – depletion of a specific protein ds short RNA (synthetic or expressed as shRNA) 12 RISC mRNA Duplex formation mRNA detroyed Normal cell RNAi treated cell DNA: mRNA: mRNA degraded! Protein: Already existing proteins decay over time Systems for overexpression of a protein Transient transfection Stable transfection (plasmid DNA is not replicated) (Chromosomal integration) Plasmid + Quick (4 – 6 hours) High expression level - Heterogeneous cells Small amount of transfectants drug resistance + Homogenous cell line Unlimited amount of transfectants - 4 – 6 week to establish a cell line Impossible if gene product causes a cell cycle block 13 The development of microscopy ~1900 Zacharias Janssen (1580 -1638) The first microscope Today 14 The three principle tasks of microscopy - Produce a magnified image (magnification) - Separate the details in the image (resolution) Resolution: the smallest distance between two objects at which the two objects can be seen as separate units Maximal resolution = l/2 - Render the details visible (contrast) 15 Bright field microscopy Ocular Objective Stage Condenser Lamp 16 Specialized bright field microscopy Enhances the contrast between intracellular structures Bright field Phase contrast Differential interference contrast (DIC) 17 Creation of contrast in bright field microscopy Unstained cell Stained cell Classical stains 18 Preservation of biological structures by fixation Process in which cellular structures are preserved and fixed in position by chemical agents Glutaraldehyde Formaldehyde Extensive protein cross-linking Alcohols Protein denaturation Fixation may introduce structural artifacts 19 Shortcoming of bright field microscopy Okay this was interesting..... ...but where is the protein of interest? 20 Raising antibodies against specific proteins Polyclonal antibodies Purify antibodies from the blood of the animal Epitope Protein X Monoclonal antibody 1. Take out antibody producing B cells Protein X 21 + 2. Molec models. 25.2-antibodies Fuse with myeloma cell to generate a hybridoma Detection of specific proteins with antibodies Primary antibody Specific to epitope on protein X Protein X Secondary antibody Specific to the primary antibody, conjugated with e.g. a fluorochrome Protein X Protein X The primary antibody (e.g. rabbit) is recognized by many secondary antibodies (e.g. goat anti-rabbit) Signal amplification 22 Principle behind a fluorochrome Fluorochrome Excitation - Emission - - - - Fluorochrome # 1 - - Fluorochrome # 2 A fluorochrome absorb light of a particular wavelength and re-emit light of a longer wavelength 23 How it works in reality Emission filter Filter cube Excitation filter Beam splitter - - - 24 Electron microscopy (EM) Maximal resolution = l/2 400 700 nm Maximal resolution 200 nm Resolving smaller structures demands something with a much shorter wavelength e- + 100 000 V el= 0.004 nm Resolution 0.002 nm (0.1 nm in reality) 25 Transmission Electron Microscopy (TEM) Electron gun e- eVacuum! Very thin section of a cell stained with heavy metal Detector Supporting grid 26 Scanning Electron Microscopy (SEM) Visualizing surface features The specimen is coated with metals to deflect electrons Sequential scanning Electron gun e- e- e- e- Cell with metal coating 27 Different forms of microscopy Bright field microscopy cell organelles large molecules Electron microscopy Fluorescence microscopy Location of molecules Different techniques – different ”windows” 28 The fluorescent protein revolution YFP DsRed - 29 - GFP - Aeqourea victoria Protein X GFP Protein X GFP Transient or stable expression Detection in either live or fixed cells Video 02.3-brownian_motion.mov Video 10.6-FRAP Visualization of signaling in live cells (NFAT): Video 12.2-nuclear_import.mov -