Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Biochemistry 4: Cellular Biochemistry Ulrike Gaul Thomas Becker Julia von Blume Ralph Böttcher Veit Hornung Carsten Grashoff Markus Moser Boris Pfander Teaching assistance: Sara Batelli Topics of the lecture course Biochemistry 1 – Introduction and biochemistry of cellular processes Biochemistry 2 – Metabolic and catabolic processes in the cell Biochemistry 3 - Macromolecules (Protein, DNA, Interactions) Biochemistry 4 – Cellular Biochemistry Introduction: internal organization of cells Visualization of cells: light and electron microscopy Membrane biology Protein sorting into compartments and organelles Cell-cell communication Cytoskeleton and intracellular transport Cell cycle Cell death Cell adhesion, junctions, polarity and migration Technical matters Course hours Monday Tuesday 9:00-10:30 9:00-10:30 Lynen Auditorium, Building A Slides Description of course and Powerpoint presentations available on the Gene Center website: http://www.genzentrum.lmu.de/lehrplan/Course/19 Login: lectures Password: lect2004 Reading material / Textbooks Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts, Walter Essential Cell Biology 3rd edition, 2010, Garland Science Alberts, Johnson, Lewis, Raff, Roberts, Walter Molecular Biology of the Cell 5th edition, 2008, Garland Science Pollard and Earnshaw Cell Biology - Das Original mit Übersetzungshilfen 2nd edition, 2008, Spektrum Akademischer Verlag Cells Using microscopy, biologists discovered 170 years ago that all living things are made of cells Simplest forms are solitary cells Higher organisms (plants, fungi, animals) consist of many different cell types, often organized into organs, such as brain, muscle, bone gut, liver, kidney, which are designed to perform highly specialized functions Differences between cells sizes and shapes nerve cell in cerebellum paramecium section through a young plant stem small bacterium human white blood cell (neutrophil) eating a red blood cell environmental requirements oxygen yes/no air – sunlight – water – minerals complex mixture of foods made by other organisms. Commonalities between cells/organisms discoveries of molecular biology and biochemistry: DNA RNA protein DNA acts as store of genetic information Proteins serve as structural support, chemical catalysts, molecular motors All cells require energy to maintain themselves Commonalities between cells/organisms Commonalities at molecular level: gene families shared by all organisms Three major domains/divisions of life bacteria – archaea – eukaryotes derived of a common ancestor cell (based on rRNA sequence relationships) Bacteria and archaea are prokaryotes Shapes and sizes of some bacteria Prokaryotic cells live in enormous variety of ecological niches, very varied in their biochemical abilities… 99% unexplored Eukaryotes In contrast to prokaryotes, eukaryotes have a nucleus (eu karyon) which contains the cell’s DNA. Shapes of some single cell eukaryotes (protists) Protist on the move Eukaryotic cell organization – overview Eukaryotic cells possess a variety of organelles, subcellular structures, that perform specialized functions. Eukaryotic cell organization – nucleus Chromosome condensation during cell division Eukaryotic cell organization – mitochondria Mitochondria generate usable energy from food to power the cell TEM reveals two membranes, outer and inner, inner is folded. Mitochondria perform endoxidation: sugars are oxidized using oxygen, ATP and CO2 are produced Eukaryotic cell organization – mitochondria Mitochondria have their own DNA most likely evolved from engulfed bacteria Eukaryotic cell organization – chloroplasts Chloroplasts capture energy from sunlight Large green organelles, only found in plants and algae, not in animals or fungi. Two surrounding membranes, internal stacks of membranes that contain chlorophyll. Photosynthesis: chlorophyll traps energy from sunlight and use energy to make sugars, oxygen is released as a byproduct. Eukaryotic cell organization – chloroplasts Like mitochondria, chloroplasts have their own DNA most likely evolved from photosynthetic bacteria that were engulfed by an early eukaryotic cell Eukaryotic cell organization additional compartments within eukaryotic cells: membrane-enclosed organelles involved in import and export of materials Eukaryotic cell organization – endoplasmic reticulum (ER) ER – irregular maze of interconnected spaces enclosed by a membrane the site where most cell membrane components, as well as materials destined for export are made Eukaryotic cell organization – Golgi apparatus Golgi apparatus – stack of flattened discs, receives and chemically modifies the molecules made in the ER and then directs them to the exterior of the cell or to various locations inside the cell Eukaryotic cell organization Lysosomes – small acidified (pH 5) organelles for intracellular digestion release nutrients from food particles and break down unwanted molecules for recycling or excretion Peroxisomes – peroxide-containing organelles for oxidation Endocytosis and exocytosis continuous exchange of vesicles between the different cell compartments and the plasma membrane Eukaryotic cell organization – cytosol Apart from all the membranous compartments, there is the cytosol the largest compartment of most cells full of small and large molecules – water based gel catabolic and metabolic processes; protein biosynthesis (ribosomes) Eukaryotic cell organization – cytoskeleton Cytoskeleton: actin, microtubules, intermediate filaments generate morphology movement of the cell movements within the cell such as distributing chromosomes in dividing cells Where did eukaryotes come from? Animal – plant – bacterial cells Summary Cells are the fundamental units of life. All present-day cells are believed to have evolved from an ancestral cell that existed more than 3 billion years ago. All cells grow, convert energy from one form to another, sense and respond to their environment, and reproduce themselves. All cells are enclosed by a plasma membrane that separates the inside of the cell from the environment. All cells contain DNA as a store of genetic information and use it to guide the synthesis of RNA molecules and of proteins. The simplest of present-day living organisms are prokaryotes: although they contain DNA, they lack a nucleus and other organelles and probably resemble most closely the ancestral cell. Different species of prokaryotes are diverse in their chemical capabilities and inhabit an amazingly wide range of habitats. Two fundamental evolutionary subdivisions are found: bacteria and archaea. Eukaryotic cells possess a nucleus and other organelles not found in prokaryotes. They probably evolved in a series of stages. An important step was the acquisition of mitochondria, which are thought to have originated from bacteria engulfed by an ancestral eukaryotic cell. The nucleus is the most prominent organelle in most plant and animal cells. It contains the genetic information of the organism, stored in DNA molecules. The cytoplasm includes all of the cell’s content outside the nucleus. It harbors a variety of membrane-enclosed organelles with specialized biochemical functions. Mitochondria carry out oxidation of food molecules. In plant cells, chloroplasts perform photosynthesis. The endoplasmic reticulum and Golgi apparatus permit cells to synthesize complex molecules for export from the cell and for insertion in cell membranes. Other vesicles (endosomes, lysosomes, peroxisomes) are required for import and digestion of large molecules. The cytosol contains a concentrated mixture of large and small molecules that carry out many essential biochemical processes. The cytoskeleton extends throughout the cytoplasm. This system of protein filaments is responsible for cell shape and movement and for the transport of organelles and molecules within the cell. Free-living single-cell eukaryotes include some of the most complex eukaryotic cells known, and they are able to swim, mate, hunt, and feed. Animals, plants and fungi consist of diverse eukaryotic cell types all derived from a single fertilized egg cell; the number of cells in a large multicellular organism runs into the billions. Cells in a multicellular organism, though they all contain the same DNA, can be very different. They turn on different sets of genes according to their developmental history and cues they receive from their environment. Biochemistry 4: Cellular Biochemistry Ulrike Gaul Thomas Becker Julia von Blume Ralph Böttcher Veit Hornung Carsten Grashoff Markus Moser Boris Pfander Teaching assistance: Sara Batelli Visualization of cells by light and electron microscopy Today’s topic Cell biology started with light microscopy … and is still very important in the form of fluorescence microscopy (in various forms), but its resolution is limited by the wavelength of visible light. By using a beam of electrons instead, electron microscopy can image the macromolecular complexes within cells at almost atomic resolution. Today, we will discuss both types of microscopes and specimen preparation, beginning with light microscopy. Scale – from macroscopic structures to atoms resolving power of eye, light and electron microscope Light microscopy Light waves travel by slightly different routes, so that they interfere with one another and cause optical diffraction effects. At high magnification, an edge appears as a series of lines, a point of light as a concentric pattern Light microscopy – spatial resolution Resolution limit is determined by features of the objective lens (numerical aperture, NA) and the wavelength of the light () numerical aperture (NA) Working distance α barely resolved resolved α α α = 7º α = 20º α = 60º NA = n sin(α) n: medium refractive index n(air) < n(water) < n(oil) α: ½ of the objective angular aperture radius airy disk (=lateral resolution) rAiry 0.61 NA Rayleigh criterion: Two adjacent object points are defined as being resolved when the central diffraction spot (airy disk) of one point coincides with the first diffraction minimum of the other point in the image plane Example: for NA = 1.3; λ = 546 nm lateral resolution = 0.61λ/NA = 260 nm Types of light microscopy Bright field Dark field - only scattered light enters objective Phase contrast Differential interference contrast (DIC, Nomarski) - exploits interference effects Sample preparation Intact tissues are usually fixed, stained and sectioned before microscopy Fluorescence microscopy can be used to visualize specific molecules within cells certain dyes and proteins (!) can be excited by absorption of light; they revert to the ground state by emitting a photon of longer wave length First excited Singlet state in most cases, the absorption and emission spectra are relatively broad, but emission is always shifted towards longer wave lengths (Stokes shift) Ground Singlet state Stokes shift Higher vibrational states Triplet state Jablonski diagram: 3 level system Absorption spectra Emission spectra Fluorescence microscopy can be used to visualize specific molecules within cells certain dyes and proteins (!) can be excited by absorption of light; they revert to the ground state by emitting a photon of longer wave length First excited Singlet state in most cases, the absorption and emission spectra are relatively broad, but emission is always shifted towards longer wave lengths (Stokes shift) Ground Singlet state Stokes shift Higher vibrational states Triplet state Jablonski diagram: 3 level system Fluorescence microscopy – Green fluorescent protein (GFP) original source: jellyfish Absorption spectra Emission spectra 400 450 Which dyes/proteins are good for biological experiments? High extinction coefficient (> 30,000 – 40,000 M−1cm−1) High quantum yield of fluorescence For example: Cy5: Φ = 29% Rhodamine 6G: Φ = 95% EGFP : Φ = 60% Little photodegradation (photobleaching) Differential photobleaching in multiply-stained tissue over time Absorption in the far red of the visible spectrum. (limits photodamage to living cells, lower autofluorescence) Immunhistochemistry and multiple fluorescent probe microscopy DNA microtubules centromeres Fluorescence microscopy – wide-field vs. confocal Fluorescence microscopy – wide-field vs. confocal Wide-field microscope Confocal microscope laser laser ˗ Low z-resolution (~ 2-3mm) + High z-resolution (~ 600nm) + High time resolution (up to 5 ms per frame) ˗ Low time resolution (secs to mins per image) Confocal microscopy – following cells/proteins live Sensory neurons cytoplasmic GFP time lapse of epithelial closure myristylated GFP + moesin GFP Fluorescence recovery after photobleaching (FRAP) Use strong focused light beam to extinguish GFP fluorescence in specific region of the cell Analyze how the remaining fluorescent proteins move into the bleached area as a function of time Learn kinetic parameters, such as diffusion coefficients, active transport rates, binding/dissociation rates from other proteins Fluorescence resonance energy transfer (FRET) Used to determine whether two proteins interact inside the cell Proteins attached to different color variants of GFP (fusion proteins) e.g. proteinX-CFP, proteinY-GFP when X and Y interact, excitation of CFP leads to activation of GFP, resulting in green emission fluorophores have to be close (1-10 nm) for FRET to occur. Transmission Electron microscopy (TEM) Light microscope Electron microscope The electron microscope can resolve the fine structures of the cell As with light, resolution limit is a function of the wavelength Electrons accelerated at 100,000 V =0.004 nm Aberrations of electron lenses hard to correct, NA very small In practice: resolution is 1nm = 10 A Electron microscopy specimen preparation Hard fixation (glutaraldehyde, osmium tetroxide) Embedding into plastic resin Thin sections (50-100 nm) using diamond knife Place sections on copper grid Electron microscopy Serial EM sections combined with 3D reconstruction reveals complex cell interdigitations Electron microscopy Specific macromolecules can be localized by immunogold electron microscopy Scanning electron microscopy (SEM) SEM uses electrons that are scattered or emitted from specimen specimen preparation Fix – dry - coat with thin layer of heavy metal Or: rapid freezing, place on cooled stage Great depth of field Scanning electron microscopy (SEM) Summary Many light microscope techniques are available for observing cells. Living cells can be seen with phase contrast, Nomarksi optics, dark, or bright field microscopy. Cells that have been fixed and stained can be studied by a conventional bright field microscopy When labeled by immune histochemistry or when expressing genetically encoded fluorescent proteins, they can be imaged by different forms of fluorescence microscopy, in particular confocal microscopy. The resolution of microscopes is determined by their numerical aperture and the wavelength of the light. Confocal microscopes improve resolution in the z-axis by eliminating out-of-focus information. 3D information can be generated from these thin optical sections by 3D reconstruction. Techniques are now available for detecting, measuring and following almost any desired molecule in a living cell. Fluorescent indicator dyes can be introduced to measure the concentrations of specific ions within cells or specific regions. Virtually any protein of interest can be genetically engineered as a fluorescent fusion protein and then imaged in living cells by fluorescence microscopy. The dynamic behavior and interactions can be followed by a wealth of microscopic techniques, including FRAP, photo bleaching, and FRET. Summary cont. Determining the detailed structure of membranes and organelles in cells requires the higher resolution attainable in a transmission electron microscope. Specific macromolecules can be localized with colloidal gold linked to antibodies. 3D views of cell surfaces and tissues are obtained by scanning electron microscopy.