Published on May 8, 2008
Organization of the Cell: Organization of the Cell Lecture Topics: Lecture Topics Cells: the basic unit of life Methods for studying cells Cell organization Organelles Cytoskeleton Extracellular matrix The Cell Theory: The Cell Theory Cells are the smallest parts of any organisms that can still be clearly identified as either being that organism, or originating in that organism - Matthias Schleiden (botanist) and Theodor Schwann (zoologist) All cells are derived from cells - Rudolf Virchow All cells can be traced back to a common ancient ancestor - August Weissman (an extension of Virchow’s concept) Pasteur Refuted Spontaneous Generation: Pasteur Refuted Spontaneous Generation Popular concept: Life was thought in some cases to arise spontaneously. e.g., Mice from piles of hay; maggots from rotting meat Pasteur tested this: Knew that broth would grow fungus, bacteria Knew that he could kill microorganisms by boiling Tested to see if the growth of microbes was due to the broth spontaneously growing them, or if they came from the environment Pasteur’s Experiment: Pasteur’s Experiment Used ‘retorts’: flasks with long arms he could seal off Boiled (sterile) broth in sealed retort If seal maintained, or if the retort outlet (arm) very long, no growth If the retort arm was broken, opening retort to the environment, then growth Therefore, microorganisms grew from previous organisms in environment Cell Function and Structure Is Conserved Across All Living Systems: Cell Function and Structure Is Conserved Across All Living Systems All cells compartmentalize their contents away from the environment with a plasma membrane All cells contain a DNA-based information system All cells maintain an internal homeostasis All cells have metabolism: use energy from environment to build structure All cells reproduce: grow and increase in numbers All cells respond to stimuli All cells move All cells evolve and adapt to environment Cells Are Small: Cells Are Small Most cells ~1mm (1/106 m) to 1 mm in diameter Some are bigger The Small Size of Cells Is Beneficial: The Small Size of Cells Is Beneficial The smaller the cell, the greater the surface:volume ratio Improved transmembrane transport for given cell volume Cell Structure Reflects Function: Cell Structure Reflects Function Cellular structures have evolved because of a need for a particular function, for example: Neurons have axons to transfer information to other cells Sperm have whiplike tails to enable them to swim Eggs have large quantities of nutrients to aid early embryonic development Early History of Microscopy: Early History of Microscopy Hooke first observed cells in cork (book Micrographica, 1665) and named them after small study rooms used by monks. He did not realize the tiny ‘rooms’ were living structures His microscope very rudimentary - poor quality image Antony van Leewenhoek: Dutch eyeglass maker, remarkable skill at grinding lenses Made unusual small microscopes – very good quality (1670) First reported observing living unicellular organisms he called “animalcules” His skill not passed on Microscopes greatly improved in late 19th century and early 20th century Improvements in Microscopes: Improvements in Microscopes Improved glass making and grinding allowed investigators to clearly identify living cellular structures Combination of improved chemical knowledge with microscopy: revealed cellular details Improved fixation techniques (killing and immobilizing), staining, and embedding techniques gave rise to histology and cytology (study of cellular structure) Magnification and Resolution : Magnification and Resolution Magnification is important Ratio of observed size to actual size Allows observer to see detail Maximum magnification about 1000X Resolution is very important Resolution is smallest distance two objects can be separated and observed to be distinct Critical to producing usable, interpretable image Resolution is dependent on wavelength of light Shorter wavelength, better resolution Thus, blue light provides better resolution than red light Best possible resolution with modern microscope is 0.2 mm (2 X 10-7 m) using green (560 nm) light Stereo (Dissecting) Microscopes: Stereo (Dissecting) Microscopes Allow user to see 3D structure – useful for surgery, dissection Very well-made, adjustable magnifying system Good for studying overall structure OK for studying only the largest cellular structures (not organelles) Transmitted Light Microscopes: Transmitted Light Microscopes Brightfield (oldest modern design; typical student microscope) Depends on staining for best contrast Phase contrast Enhances contrast by enhancing destructive and constructive interference in the image Designed in 1940s by Otto Zernicke Allows study of living cells without stain Differential interference Enhances contrast by enhancing interference, but has fewer artifacts in image than phase contrast Sees gradients in thickness and refractive characteristics Has very clear 3D image, thin focal plane, very good detail Designed by Nomarski (different versions by other scientists) in early-mid 1960s Allows study of living cells without stain Fluorescence Microscopes: Fluorescence Microscopes Fluorescence microscope Much like transmitted light microscope, but sends light down objective to excite fluorescent molecules Excitation light causes fluorescent molecules to glow Reveals location of specific molecules: investigator usually labels the molecules Cells may be fixed (dead) or alive; depends on application of technique Confocal fluorescence microscope Computer-driven, special fluorescent light microscope – some use laser to illuminate specimen Provides exceptionally clear fluorescent images Electron Microscopes: Electron Microscopes Use beams of electrons boiled off hot filament to form images Have tall column down which electron beam moves – air pumped out to allow electrons to move unimpeded Electron beam accelerated down column by high voltage between filament and cap with a hole … beam continues down evacuated column by inertia Very high magnification: up to 500,000X For most biological applications, up to ~100,000X Very good resolution: ~ 1 nm (10-9 m) Comparison of Light and Electron Microscopes: Comparison of Light and Electron Microscopes Transmission Electron Microscopy(TEM): Transmission Electron Microscopy (TEM) Sample is thinly sectioned (sliced), plastic-embedded cells/tissues stained with metal atoms Electron beam absorbed, deflected by metal in sample Image is a shadow: electron beam “shadow” cast on phosphorescent ceramic plate or film view with ‘binoculars’ Film developed as normal black and white negative, and printed to produce the positive print Scanning Electron Microscopy (SEM): Scanning Electron Microscopy (SEM) Looks at surface of specimen Surface coated with metal Primary beam hits and scans over surface Detector ‘reads’ secondary electrons, scans surface Voltage signal is read as function of time and played out on special high-resolution ‘slow scan’ TV set (= cathode ray tube) Biochemical Tools to Study Cells: Biochemical Tools to Study Cells In order to study cellular chemistry, must gain access to cellular compartments: Break cells open Disrupt in a grinder/glass homogenizer Resulting mixture of organelles and cytoplasm is the homogenate Centrifugation uses high gravitational force – G-force – to separate different cellular compartments Centrifugation: Centrifugation Used to separate cellular components Homogenate is poured into strong centrifuge tube which is revolved rapidly Heavier organelles form a pellet; fluid above the pellet is the supernatant Supernatant is centrifuged at greater G-force, a new supernatant is recovered and subjected to greater G-force, and so forth Separates out cellular components based on size and density Centrifugation: Materials to be separated (such as a homogenate of cell components) are loaded into centrifuge tubes Tubes are loaded into rotor (‘hinged bucket’) and spun by powerful motor. Heavier material is driven by centrifugal force to the outer periphery of the circle described by rotating centrifuge tubes. Centrifugation Differential Centrifugation: Differential Centrifugation Homogenization disrupts cells, releases organelles Centrifugal force separates larger, heavier components from smaller, lighter components Stepwise process using increasing centrifugal force in each successive step Density Gradient Centrifugation: Density Gradient Centrifugation Pellet placed on top of a gradient of solute, usually sucrose Lowest density of sucrose at top of tube – highest at bottom Steps or continuous gradient of higher sucrose down the tube Fractions of the homogenate migrate to form band at the position which matches density Other Methods: Other Methods Column chromatography: methods for separation and purification of molecules, usually using beads By size – passing through a molecular mesh By charge – ionic molecules bind to charged surfaces of beads Electrophoresis: movement of molecules in electrical field Determine size of molecules Can examine ‘native’ (unperturbed) molecules Can ‘break’ molecules and examine denatured molecules Proteins, nucleic acids, and carbohydrates can be studied Techniques to identify molecules ‘Western’ blotting – proteins identified with antibodies Nucleic acids identified with ‘Southern’ and ‘Northern’ blotting Techniques to assay function Identify unique activity – e.g. - breakdown of hydrogen peroxide by catalase (catalase was the first fully characterized enzyme) Cells and Non-Cells: Cells and Non-Cells Life has made enormous changes to the Earth Earth is covered with many different forms of Life and non-living materials that are of biological origin, but are not alive It is very important to understand the difference between actively living, and biological, but non-living, things Living things are made of cells, ALWAYS! Viruses: NOT alive, NOT cells: Viruses: NOT alive, NOT cells Viruses are not alive They are dependent on cells for their existence They are a compartment (protein capsid) that contains a bit of nucleic acid as an information system Cells are Compartmentalized : Cells are Compartmentalized All cells are bounded by a plasma membrane Protects cellular reactions from the environment All cells have cytoplasm Fluid compartment of the ‘body’ of the cell called cytosol Internal organelles found within the cytosol Cells have internal compartments Keep different reactions apart Keep compatible reactions together Eukaryotic cells have membrane-bounded organelles Membranes are very important to the cell Cellular membranes Transport materials in/out of cell Locate/hold reactions (in/on organelles) Distinguishing Features of the Two Major Cell Types: Distinguishing Features of the Two Major Cell Types Bacteria (Prokaryotes): Bacteria (Prokaryotes) Cell wall Plasma membrane Cytoplasm Nucleoid DNA, genome Mesosome Internal membrane Energy metabolism Ribosomes 70S Flagellum Single protein, flagellin An Animal Cell: An Animal Cell A pancreatic cell that makes digestive enzymes A Plant Cell: A Plant Cell A photosynthetic plant cell Eukaryotic Endomembrane System : Eukaryotic Endomembrane System Nucleus: Birthplace of the endomembranes DNA held in chromosomes: cellular library of information Nucleolus: birthplace of ribosomes Endoplasmic reticulum (rER and sER): cell’s factories Rough endoplasmic reticulum (rER) makes secreted proteins and integral membrane proteins (IMPs) and modifies proteins Smooth ER (sER) makes lipid molecules, such as steroids, and adds lipids to proteins Golgi apparatus Modifies proteins made in the rER Lysosomes The “destroyer”: proteolytic, lipolytic enzymes, acid pH Vacuoles and microbodies Storage & concentration of cellular waste, nutrients, and enzymes The nucleus: The nucleus Nucleus Chromatin Condensed heterochromatin Noncondensed Nucleolus Birthplace of ribosomes Double-layered membrane of nuclear envelope Nuclear pores Endomembranes and Secretion: Endomembranes and Secretion The nucleus produces the Rough Endoplasmic Reticulum (RER), a system of flattened membranes The RER makes proteins that are secreted or inserted into membranes The RER passes its protein products via transport vesicles to the Golgi Apparatus, which are also flattened membrane systems, which processes them . . . And sends them to various locations, including the plasma membrane Note that proteins produced inside the RER stay inside the Golgi and transport vesicles, but are expressed on the surface of the plasma membrane Ribosomes: Ribosomes Made of 3 RNA strands and 75 different proteins Free: suspended in cytosol Bound: associated with rough endoplasmic reticulum Assemble proteins by dehydration synthesis Smooth Endoplasmic Reticulum: Smooth Endoplasmic Reticulum The smooth endoplasmic reticulum (SER) is tubular, unlike the RER. It receives membrane from the RER. Is tubular, (unlike the RER, which consists of flattened membranes) Mitochondria are shown here also Non-Endomembrane Organelles: Non-Endomembrane Organelles Mitochondrion Energy organelle Aerobic respiration Derived from an ancient bacterium engulfed by a heterotrophic prokaryotic ancestor of the eukaryotes (’urkaryote’) Ancient bacterium became an endosymbiotic organism Chloroplast Light-energy collection organelle Makes oxygen Photosynthetic bacteria and chloroplasts changed the world Production of oxygen changed ancient atmosphere Derived from bacterium, like mitochondrion Mitochondria : Mitochondria Sausage-shaped Outer and inner membranes Inner membrane infolded to form cristae Chloroplast: Chloroplast Similar in structure to mitochondrion Photosynthetic membranes in thylakoids Mitochondrial and Chloroplast Functions Are Complementary: Mitochondrial and Chloroplast Functions Are Complementary The Cytoskeleton: The Cytoskeleton A system of filaments within the cytoplasm; the “internal materials” of the cell Provide structure for the cell Bind the cell together Provide “highways” within the cell Tracks for movement of organelles Microfilaments (7 nm dia) Actin 43 kDa 2 strands, twisted like string Microtubules (little tubes) (25 nm dia) Tubulin 50 kDa 13 protofilaments, arranged in a cylinder Intermediate filaments (10-12 nm dia) Many different types 40-~100 kDa e.g., keratin (hair, fingernails, skin surface) Cytoskeleton: Actin Filaments: Cytoskeleton: Actin Filaments F-actin (filamentous actin microfilament) Two strings of G-actin (globular actin) attached end-to-end 7 nm wide Made of actin: 43,000 molecular weight Filament hydrolyses ATP as it grows F-actin very common - forms shape of cells; allows amoebae to move, to take up food; facilitates muscle contraction, cell division, and cell anchorage Muscles are Actin- and Myosin-Based Machines: Muscles are Actin- and Myosin-Based Machines Muscle sarcomere Myosin is an actin ‘motor’ Myosin (200,000 MW) makes thick filaments in muscle. Myosin hydrolyzes ATP, pulls on the F-actin filaments to contract muscle Myosin and actin slide past each other as contraction occurs Microtubules: Microtubules Microtubule (right) Made of tubulin: 50,000 molecular weight Alpha and beta isoforms of tubulin join together to make dimers, then dimers join to make the tubule GTP hydrolyzed to GDP and phosphate as the tubule grows In picture to right, tubulin dimers move through; dimers add at (+) end, fall off at (-) end: called treadmilling Microtubules Are Important for Eukaryotic Cell Division: Microtubules Are Important for Eukaryotic Cell Division Microtubule Motors: Microtubule Motors Dyneins: 1, 2, or 3 “heads” that cleave ATP Run along microtubules Large, complex: up to 25 polypeptides, 2,000,000 MW Run toward base of cilia (“minus end-directed”) Make cilia wiggle; can move very fast Pull vesicles with nutrients Kinesins: 1 or 2 heads that cleave ATP Also run along microtubules Small, only 2 types of polypeptides, total of 4: 150 kDa Tend to be slow Drag vesicles over microtubules Move in opposite direction to dyneins (“plus end directed”) Anatomy of Eukaryotic Cilia and Flagella: Anatomy of Eukaryotic Cilia and Flagella 0.25 m diameter Made of 9 outer doublet microtubules: special double microtubules 2 central MTs Very complex, ~300 proteins! Dynein arms link doublets together, push tipward and drive the bending of the cilium Kinesin: Kinesin Walks on the microtubule; carries a vesicle Hydrolyses ATP to cause movement Vesicle is adapted to the kinesin molecule by intermediate complex Centrioles: Centrioles Also based on MTs Within the MT- organizing center (MTOC) 9 triplet MTs + 0 MTs in center Near nucleus Important in mitosis Intermediate Filaments: Intermediate Filaments Long fibrous proteins associate side-by-side, then end-to-end No overall polarity, unlike actin and microtubules, which are polarized with specific longitudinal organization Very strong and stable Intermediate Filaments : Intermediate Filaments Extracellular Matrix: Extracellular Matrix A system of proteins and carbohydrates outside of the cell. Includes cell walls.