Bi1_2009_Lecture2_full - Common functional and structural...

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Unformatted text preview: Common functional and structural properties of all cells • Membrane (boundary between the cell and environment) • Cytosol (fluid in which biological reactions take place) • DNA (hereditary material)/RNA (message made from DNA) • Ribosomes (translation of genetic information into proteins, the molecules that perform the metabolic functions of the cell) • ATP (universal energy currency) How we study cells The light microscope Can magnify cells up to 1000x and resolve details of 0.2 m. Transmission Electron Microscopy Can magnify up to 1x106 times and resolve details of 2-6 nm. Sizes in biology Higher resolution now possible Clicker question Do all cells have organelles? A) Yes B) No Bacteria (procaryotes) have no organelles Procaryote = “before nucleus” membrane Eucaryotic cells contain membrane-enclosed organelles distributed within the cytoplasm Eucaryote = “truly nucleus” Evolution of membranous organelles? Lipid bilayers enclose the cell and organelles within the cell Little Alberts, Figures 11-3 and 11-4c Clicker question Why do lipids form bilayers? A) The hydrophobic tails stick together B) The hydrophobic head groups stick together C) The hydrophilic tails stick together D) The hydrophilic head groups stick together E) Previous Bi1 students told them to The Cytoskeleton The cytoplasm of eukaryotic cells contains three classes of dynamic protein filaments, which together form the cytoskeleton. Intermediate filaments -- enable cells to withstand mechanical stress. Microtubules -- create a series of tracks on which vesicles and organelles can move. Actin filaments -- essential for movements (e.g., macrophage engulfing of particles). Clicker question The nucleus contains: A) Ribosomes B) Mitochondria C) Chromosomes D) Farandolae Nucleus Enclosed within a double membrane that forms the nuclear envelope. Contains DNA, the genetic information of the organism. Individual chromosomes are not visible in the nucleus because the DNA is dispersed at this point in the cell cycle. Little Alberts, Figures 1-15 and 1-16 Chromosomes become visible just before cell division. DNA packaging Chromosomes are visible only when cells prepare to divide Clicker question Fill in the blanks: Human somatic cells* have __ chromosomes. Human germ cells* have __ chromosomes. A) 46; 23 B) 23; 46 C) 46; 46 D) 23; 23 * Somatic cell: Any cell in a plant or animal except for the reproductive (germ) cells. * Germ cell: An ovum or a sperm cell or one of their developmental precursors. Genes are carried on chromosomes Humans have 22 pairs of chromosomes plus the X and Y. Females are XX; Males are XY. Chromosomes have been “painted” with fluorescent dyes. Chromosomes have been arranged to form pairs. Little Alberts, Figure 5-12 Packaging DNA into chromosomes Mitochondria Double membrane -- inner and outer membrane Contain their own DNA -- will be important later when we discuss anti-HIV drugs Generate chemical energy by oxidizing sugar molecules to produce ATP Cellular respiration: Consume oxygen and release CO2 Smooth outer membrane, convoluted inner membrane. Inner membrane contains most of the proteins involved in cellular respiration. Little Alberts, Figure 1-18 Endoplasmic reticulum (ER) Site of synthesis of membrane-bound and secreted proteins. Carbohydrates are added to newly synthesized proteins while in ER. Rough ER is coated with ribosomes, molecular machines that perform protein synthesis. ER is contiguous with membrane of the nuclear envelope. Alberts et al., Figure 1-22 and 15-23 Misfolded proteins are retained in the ER. Properly folded proteins travel to the Golgi. Addition of sugars (glycosylation) in the ER Oligosaccharides can be transferred to asparagine (Asn) side chains if the Asn is present in the tripeptide sequence Asn - X -Ser/Thr. The envelope protein of HIV, gp120, contains many Asnlinked carbohydrates. Alberts et al., Figure 15-22, p. 517 Golgi Apparatus Consists of stacks of flattened membrane-enclosed sacs. Receives and modifies molecules made in endoplasmic reticulum (e.g., the Golgi processes carbohydrates added in the ER). Cargo travels between endoplasmic reticulum and Golgi in vesicles that pinch off from the membrane of one compartment, then fuse with the membrane of another. Alberts et al., Figure 1-23 Clicker question In the book A Wind in the Door by Madeline L’Engle (a sequel to A Wrinkle in Time), Meg Murry visits which intracellular organelle inside one of Charles Wallace’s cells? 1) 2) 3) 4) 5) Nucleus Farandolae Golgi Apparatus Mitochondria Endoplasmic reticulum Other membrane-enclosed organelles Lysosomes are degradative compartments containing proteases (enzymes that cleave other proteins). Peroxisomes provide a contained environment for reactions involving H2O2. Vesicles transport materials from one membraneenclosed organelle to another. Lysosomes are membranous sacs of hydrolytic enzymes that are active at acidic pH Lysosomes include enzymes that digest proteins, nucleic acids, carbohydrates, and phospholipids. Acidic pH is maintained by proton pump. Alberts et al., Figure 15-34 Endosomes are low pH compartments involved in trafficking of endocytosed cargo Alberts et al., Essential Cell Biology, 2nd Edition, Fig 15-32, p. 526 Endocytosis Exocytosis Cells import external material via fluid-phase (not receptormediated) endocytosis. Synthesized materials can be released via exocytosis. Low density lipoprotein (LDL -- aka “bad cholesterol”) is internalized via receptor-mediated endocytosis. LDL dissociates from its receptor inside endosomes and then is transferred to lysosomes, where it is degraded to release cholesterol. See =180 Transport vesicles carry soluble proteins and membrane between compartments The enclosed space within each compartment is topologically equivalent to the outside of the cell. Some viruses (e.g., influenza, but not HIV) are taken into endosomes via endocytosis, then low pH activates their fusion machinery. Alberts et al., Figure 15-17, p. 512 Fusion of a transport vesicle to a membranous organelle is similar to fusion of an enveloped virus to a host cell membrane Alberts et al., Figure 15-21 Transmission Electron Microscopy A 2-D projection Can magnify up to 1x106 times. This is also a 2-D projection image Computed tomography (CT) -a medical imaging method Tomography: 3D reconstruction of an object from a series of projections Set of 2D projections recorded while tilting the object. Calculate the backprojection for each projection. The sum of all the backprojections represents the density of the original object. Baumeister et al., 1999, Electron tomography of molecules and cells. Trends Cell Biol. 9: 81-85. Electron tomogram Model of tomogram ~0.5 m 2D images of mitochondria from textbooks suggest an incorrect 3D model Tomogram of mitochondria Segmented model derived from tomogram of mitochondria 3D reconstruction of Golgi 6-8 nm resolution Colors indicate different Golgi cisternae ER: blue-gray Ribosomes: purple spheres ER-Golgi intermediate compartment: yellow Golgi cisternae: C1: green C2: purple C3: rose C4: olive C5: pink C6: bronze C7: red Ladinsky et al. (1999) J. Cell Biol. 144: 1135-1149 High resolution 3D electron tomography of pancreatic beta cell line Boulder Laboratory for 3D Structure of Cells, University of Colorado ER: yellow membrane-bound ribosomes: blue free ribosomes: orange microtubules: bright green mitochondria: dark green dense core vesicles: blue clathrin-negative vesicles: white clathrin-positive compartments and vesicles: red clathrin-negative compartments and vesicles: purple 500 nm Marsh et al. (2001) PNAS 98: 2399-2406 High resolution 3D electron tomography of pancreatic beta cell line Boulder Laboratory for 3D Structure of Cells, University of Colorado ER: yellow membrane-bound ribosomes: blue free ribosomes: orange microtubules: bright green mitochondria: dark green dense core vesicles: blue clathrin-negative vesicles: white clathrin-positive compartments and vesicles: red clathrin-negative compartments and vesicles: purple 500 nm ~0.5 m Marsh et al. (2001) PNAS 98: 2399-2406 From macroscopic to microscopic Light or fluorescence microscopy (can be done on live cells) Electron microscopy (cannot be done on live cells) or X-ray crystallography 0.1 nm = 1 Ångstrom Clicker question A fluorescent molecule that absorbs a photon of light at one wavelength 1) always emits a photon at a shorter wavelength 2) always emits a photon at a longer wavelength 3) can emit a photon at a shorter or a longer wavelength depending on the type of fluorophore Fluorophores • component of a molecule that causes it to be fluorescent • Fluorescein isothiocynate (FITC) FITC Absorb: 495 nm (blue) Emit: 521 nm (yellow green) – -N=C=S is reactive group allowing coupling to proteins • Rhodamine Rhodamine Absorb: 515 nm (yellow green) Emit: 546 nm (red) Alexa Fluor dyes -- more photostable, more colors Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/The-Alexa-Fluor-Dye-Series.html Confocal microscopy Insect embryo Conventional fluorescent microscope Confocal fluorescent microscope Conventional fluorescence microscopy results in a blurry image because there are fluorescent structures above and below the plane of focus. A laser scanning confocal microscope uses a pinhole aperature in the detector that allows only fluorescence emitted from the plane of focus to be included in the image. A laser beam is scanned across the specimen to give a sharp 2-D image in each plane of focus. A series of xy planes can be taken at different depths to yield a 3-D image. Confocal microscopy can be used to derive 3D reconstructions of cells Rice et al., 2009, J. Mol. Biol. 386; 717-732 5m Live cell imaging Intracellular vesicles can move 1-2 m/sec Rice et al., 2009, J. Mol. Biol. 386; 717-732 Live imaging of HIV spread Scientists-film-HIV-spreading-for-first-time.html Clicker question Viruses can be seen with: A) B) C) D) E) The naked eye A light microscope A fluorescent microscope An electron microscope A telescope Size comparison • • • • Typical eucaryotic cell: 10s of microns ( m) Typical bacteria: one or a few microns Protozoa (e.g., Cryptosporidium or Giardia): 3-6 m Viruses: less than 1 m ( 100 nm) • Water filters: 0.1 to 1 m pore size. Will NOT filter out most viruses. ...
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