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C2006/F2402 -- Lecture 2 C2006/F2402 '14 Outline Of Lecture #2 -- Updated 01/22/14 02:32 PM (c) 2014 Deborah Mowshowitz, Department of Biological Sciences, Columbia University, New York NY Want to see some pictures with the scanning EM? See Becker, fig. 16-6, 16A-1 (in box 16A), 16-25, 17-12 (7th ed), etc. For pictures of Immunofluorescent Cytoskeleton, See texts,or PP slides* (for lecture 2) on Courseworks or Google Images. Problems to do have been added in magenta italic bold. All the assigned problems are posted online. As soon as the complete problem book is ready, we will tell you. We apologize for the delay. Handouts: 2A* -- Pictures of IF, MF, MT on Courseworks. (= figs. 4-24, 15-24 & 15-25 from Becker 6th ed plus one picture from another text.) See figs. 4-24 & 15-23 (15-24) in 8th (7th) edition. 2-B -- Table with Major Features of Cytoskeleton 2-C -- Immunofluorescence These slides and handouts contains (c) material, so they are only accessible to registered students. * (Students: Use links on the left menu of Courseworks to access Powerpoints or CW Handouts. CW handouts may not be posted until tomorrow. I. Immunofluorescence, Antibodies, & the Cytoskeleton A. What is the cytoskeleton? (see Becker figs. 4-23 & 4-24 or Sadava fig. 5.14 (5.17) 1. Three components made of 3 different protein (More details on Handout 2B) Component abbreviation shape Microtubules MT hollow Microfilaments MF Intermediate filaments IF Made of: Diameter tubulin 25 nm solid rod actin 5-9 nm cable Different proteins; all similar (in same family); Found in cells of multicellular organisms only 8-12 nm 2. Functions a. Support/strength -- weight bearing, shape determining. b. Movement (MF & MT) -- can change shape of cell (as in muscle or amoeba) and/or help move large structures (such as chromosomes) or organelles within cells. c. Localization of other factors -- act as peg board or framework for attachment of organelles, enzymes, etc. (Cytoplasm is not simply a "bag of enzymes.") 3. How discovered? How do you locate proteins that have no enzymatic activity? By immunofluorescence (see below) using antibodies to tubulin and/or actin. B. How Immunofluorescence is Used to Visualize Cell Parts. See Handout 2C & Becker table 15-2. Allows you to visualize location(s) of particular proteins. 1. The "immuno" part: How antibodies are used as reagents to identify proteins (& other substances) file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect2.14.html[1/22/2014 4:35:30 PM] C2006/F2402 -- Lecture 2 a. What are Antibodies & Antigens? Antigens are foreign or unusual materials that trigger an immune response Antibodies are made by vertebrates in response to antigens Antibodies are always proteins Antigens can be proteins (as in all cases discussed here) or other substances. Usual abbreviations are Ab = antibody; Ag = antigen b. Specificity. Each antibody (against a protein) binds to a specific small part of one protein (called an epitope or antigenic determinant.) Therefore each antibody binds to only one protein or a very small number of similar proteins. See Becker fig. 15-9 (15-10) for an example with a metal labeled antibody; table 15-1 for examples with fluorescently labeled antibodies. c. Antibody Structure. (1). Each antibody has a variable part -- complementary in fit to epitope (in this case, epitope = small part of a protein.) (2). Each antibody has a constant part -- constant in all antibodies of that class from that species. d. Why use Antibodies for detection? Many methods identify (or characterize) proteins by their function; antibodies identify proteins by their structure (irrespective of function). Therefore antibodies are often useful for detection of proteins that have no enzymatic activity (such as components of cytoskeleton). e. Detection of Ab-Ag binding. How can you tell if an antibody has bound to its antigen? Clumping and complex formation is one way. (See top of handout 2C.) 2. Role of Fluorescence See Becker Appendix, A-8 to A-10 (A-11); for pictures see Becker table 15-1 or Sadava figs. 5.3, & 5.14 (5.17). How do you find (visualize) the bound antibody? a. Fluorescent materials emit light of one wave length when irradiated at a different wave length. b. Fluorescent material can be located easily by irradiating sample (at one wave length) and seeing what part of sample "lights up" (emits light at a different wave length). c. Small fluorescent groups can act as "tags" or probes -- can be attached to larger molecules such as antibodies, usually to one end, often without altering function of macromolecule. Some tags are added chemically after the protein is made by a cell. Some tags (like GFP -- a small, fluorescent protein) are incorporated into the protein file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect2.14.html[1/22/2014 4:35:30 PM] C2006/F2402 -- Lecture 2 when it is made in a cell. The resulting protein contains a fluorescent domain that acts as a built in tag. Synthesis of a protein with a built in fluorescent tag requires genetic engineering of the corresponding DNA -- more details on this later. If you want to know more about GFP now (& see pictures) go to the Nobel Site. 3. Methods: How Fluorescent Antibodies are used (See handout 2C) a. General Principle: Add fluorescent antibody, wash off unattached antibodies (not bound to target protein), irradiate, and look for light emission (aka 'signal') = site of fluorescent antibody = location of target protein. b. Direct Immunofluorescence -- Antibody with tag (fluorescent) sticks to target. c. Indirect Immunofluorescence.-- Antibody #1 (without tag) sticks directly to target; secondary labeled (tagged) antibody sticks to constant part of first antibody. Advantages of indirect: (1). Gives an amplification effect -- more tag or label ('signal') per molecule of target protein. (2). Requires only one labeled antibody to identify many proteins. Same labeled secondary antibody can be used to bind to ("light up") many different proteins. (Preparation of labeled antibody is more difficult and expensive than preparation of ordinary, unlabeled, antibody.) (a). A different primary antibody is used for each target protein. (Not labeled -no tag.) Variable part of primary antibody binds to specific part of target protein. (b). The secondary antibody binds to the constant part of the primary antibody. Therefore a sample of the same (labeled or tagged) batch of secondary antibody can bind to many different (unlabeled) primary antibodies. C. How Cytoskeleton Discovered 1. How did scientists get (labeled) antibodies to components of MT and MF in the first place? Large, permanent structures made of tubulin and actin are well known in certain specialized cells. (Structures are big enough to be easily detectable.) Tubulin (from cilia & flagella) & actin (from thin filaments of muscle) purified; antibodies made to them. Fluorescent probes were attached to the antibodies, and antibodies were injected into living cells. 2. Cytoskeleton of MF & MT Identified -- During interphase, structures made of actin & tubulin found in cytoplasm of almost all eukaryotic cells. a. Antibodies to tubulin bind to ("light up") MT. (1). MTs are present in interphase, not just in spindle fibers during mitosis (2). MTs are present in ordinary cells, not just in specialized cells with cilia & flagella. b. Antibodies to actin bind to MF. MF found in ordinary cells, not just in specialized cells such as muscle. 3. Implies two possible states of tubulin and actin: a. Temporary (dynamic) -- no fixed state; monomers and polymers are in dynamic equilibrium as in cytoskeleton, spindle fibers, cleavage furrow. file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect2.14.html[1/22/2014 4:35:30 PM] C2006/F2402 -- Lecture 2 b. Permanent -- form stable specialized structures such as cilia, flagella and muscle fibers. See Sadava fig. 5.17 (5.20) or Becker fig. 16-7 & 16-8 for nice pictures of cilia. 4. IF found later in multicellular organisms. (Not present in unicellular eukaryotes.) Look at problem 1-1. D. Overview of Cytoskeletal Components -- See handout 2B or Becker table 15-1. 1. Major structural features of each component. See table on 2B for size, subunits, shorthand, and shape. For a summary, see table at start of lecture. For more details, see below. 2. Polarity & Movement. MT & MF are polar & are involved in movement; each is associated with a corresponding 'motor molecule.' IF are not polar and are not involved in movement. (Note: polar here does not refer to solubility, or separation of charges, but to having a distinct 'head' and 'tail' end so the molecule has a direction.) II. Microfilaments & Microtubules -- a closer look See Handout 2B A. Overview of Structure -- For pictures & a summary of properties, see handout or Becker Table 15-1. For diagrams, see handout 2A (& Becker 4-24). In Sadava, see fig. 5. 14 (5.17). Many nice pictures and additional details (which go beyond the scope of this course, but might be of interest) are in Becker Chap. 15 & 16. B. How each monomer forms a polymer 1. Actin/MF. a. Monomer. One type of globular monomer = G actin. b. How polymer (filament) forms: Globular monomer (G actin) forms chain of beads. Two chains twist around each other → polymer. c. Polymer has polarity. Polymer looks symmetric (in standard picture) but isn't -- it has "+" and "-" ends (like a chain of pop beads does). Nice pictures of how MF provide structural support to microvilli are in Sadava fig. 5.16 (5.19) and Becker fig. 15-17 & 15-18 (15-18 & 15-19). d. Growth: Polymer grows (and shortens) preferentially at "+" end. (FYI: Growth requires ATP.) e. How drugs affect polymerization. See table on handout. (1). Many drugs 'sequester' the monomers or polymers. These drugs bind to either the polymer or the monomer, and stabilize or trap the form they bind to. (a) Drugs can bind to and stabilize the polymer. These drugs shift equilibrium (monomer ↔ polymer) to right. Ex: phalloidin for MF. (b). Drugs can bind to the monomer and prevent it from polymerizing. Shift equilibrium (monomer ↔ polymer) to left. Ex: latrunculin A for MF. (2). Drugs can act in more complex ways to shift the equilibrium between monomers & polymers. Ex: cytochalasin B. (This drug binds to and caps the file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect2.14.html[1/22/2014 4:35:30 PM] C2006/F2402 -- Lecture 2 polymer so it can't grow at the plus end. The polymers gradually fall apart at the minus end and can't grow back, thus shifting the equilibrium to the left.) 2. Tubulin/MT. a. Monomer. Two types of globular monomers -- alpha and beta tubulin. b. How tubule forms: Alpha + beta → dimer → chain of dimers (protofilament) → rings of chains forming a tubule (usually 13 chains/tubule). c. Growth: Chains and tubules grow primarily by addition of dimers to "+" end. (FYI: Growth requires GTP.) d. Anchors: (1). Role: MT usually anchored at (and grow away from) structure at "-" end called a microtubule organizing center (MTOC). For examples, see Becker Fig. 15-10 (15-11). (2). Orientation: In most cells, major MTOC and "-" ends are near nucleus; "+" ends extend toward edges of cell. This MTOC is called a centrosome. e. Terminology -- Proteins that bind to microtubules are known as Microtubule-Associated Proteins or MAPS for short. These can affect assembly, structure and/or function of MTs. See Becker chap. 15 if you are interested. Note: Proteins that bind to MF are called Acting Binding Proteins or ABPs for short. (See table on handout.) f. Drugs. Colchicine depolymerizes and taxol stabilizes. The drugs shift the equilibrium (monomer ↔ polymer) to left or right respectively. Either drug blocks mitosis -- both drugs prevent spindle fibers from moving chromosomes. Note: Colchicine is used in treating gout. Its medicinal effects on gout are separate from its effects on mitosis. Look at problems 1-7 & 1-8 (A & B). C. Role in movement -- See Sadava figs. 5.18 & 5.19 ( 5.21 & 5.22) or Becker fig. 16-2 & 16-3. 1. MF, MT involved in movement. Primarily using a "motor molecule" (see table on handout) that 'walks' down a fiber using the energy from splitting of ATP. Motor molecule is attached to something that it pulls along. a. Two major motor molecules for tubulin/MT: (1) Dynein -- Dynein carries material "in" toward the nucleus/cell body -- Dynein draws things in (toward the - end of MT). (2) Kinesin -- Kinesin carries material "out" toward the edges of the cell -- to the cell "Korners" (toward the + end of MT). b. One major motor molecule for actin/MF = myosin file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect2.14.html[1/22/2014 4:35:30 PM] C2006/F2402 -- Lecture 2 2. Two major types of movement using motor molecules a. Can have 2 fibers sliding past each other (motor is attached to a fiber -- it's part of one fiber or in between the two) -- overall effect is to shorten/lengthen structure. See Sadava fig. 5.18 (5.21). Examples: (1). Anaphase -- MT slide "out" to give longer spindle fibers and elongated spindle. Note: Overall movement of chromosomes seems to depend both on sliding of some fibers past each other, and the change in length of other individual fibers. For details, see Becker. (2). Telophase -- MF slide "in" forming cleavage furrow that divides cell in half. Muscles contract when MF slide in. b. Can have vesicle or large structure moving down a fiber (motor attached to vesicle) -fiber acts as "railroad tracks" to direct vesicle toward one end of fiber. Example -- How vesicles move down axons of neurons. See Becker fig. 16-1, 16-2 & 16-5 or Sadava fig. 5.19 (5.22) (1). Structure: Neurons are nerve cells with long extensions (axons) that make connections (synapses) with other cells at the end of the axon. Multiple MT extend through the length of the axon. (2). Vesicle Transport: Vesicles are transported along the MT between nucleus/cell body at near end of axon and synapse at far end of axon. Direction of movement depends on whether motor molecule is dynein or kinesin. (3). Important Notes on relative speeds: (a). Vesicle transport is much slower than nerve signaling. Electrical signals are not transmitted down axons using vesicles -- vesicle transport and signal transport are two separate processes. Electrical signal transport is much faster, by a factor of > 105 , and will be explained later. (b). Vesicle transport is much faster than diffusion through the cytoplasm. For an animation of a vesicle moving down a fiber (& many other fascinating cell features) see the video from Harvard called "The inner life of the cell" at http://multimedia.mcb.harvard.edu/media.html. A shorter version of this video (set to music) is at the same site and at http://www.studiodaily.com/main/searchlist/6850.html. The short version without music is at http://www.moma.org/exhibitions/2008/elasticmind/#/118/. (Go to the section titled 'extreme visualization.') Many of the things shown in the video may be obscure at this point, but by the end of the cell biology section of this term you should be able to identify almost all the items shown. Look at problems 1-9 to 1-11. See handout 2-B for effects of drugs mentioned. III. IF's -- see handout 2-A A. Function -- Primarily for structural support -- provide strength and help maintain shape. Not involved in movement. IF's don't lengthen, shorten or slide. Have no polarity, and other motor molecules don't slide along them. B. How monomers form a polymer file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect2.14.html[1/22/2014 4:35:30 PM] C2006/F2402 -- Lecture 2 1. Monomers: All Monomers are extended, not globular -- see handout. There is more than one type of monomer (see below). 2. Formation of polymer -- how monomers form a cable a. Two monomers → dimer; both monomers point in the same direction. Two monomers may be same (homodimer) or different (heterodimer). b. 2 dimers → tetramer; tetramer = fibrous basic subunit. Two dimers in a tetramer point in opposite directions. c. Tetramers stick to each other by overlapping ends → protofilament. d. Multiple protofilaments form a cable, probably 8 protofilaments across. Exact final structure unclear. One possibility: protofilaments form flat cable that twists into final structure. See fig. 15-23 (15-24) of Becker = handout 2A. Note: The text in Becker equates a tetramer with a protofilament; this does not appear to be the usual terminology. All other texts and the diagram in Becker diagram consider a protofilament to be a long structure formed by many tetramers. 3. Stability -- Some IF cables do disassemble/reassemble during the cell cycle or during cell growth. C. Not all IF's are the same. All IF's have similarities, and form similar structures, as above, but there are multiple IF genes and therefore multiple IF's with significant differences. (See Becker table 15-4 or handout 2A 1. Nuclear IF's -- Lamins. a. Lamins are a type of IF found in the nucleus. b. Do not confuse lamins with laminins. (Laminins = proteins found in the extracellular matrix = outside the cell). c. Lamins are the same in all cell types. (Unlike cytoplasmic IFs). ` d. No other IFs, no MTs & no MFs are found in the nucleus. 2. Cytoplasmic IF's a. All IF's but lamins are cytoplasmic. (NB: All MF & MT are cytoplasmic.) b. Cytoplasmic IF's (not lamins) are tissue specific -- origins of cancers can be traced from the type of IF's they contain. That's because different tissues transcribe ("express") different IF genes. D. IF's represent a protein/gene family. See Sadava Ch. 24 esp section 24.3 for more on evolution of gene families. 1. Proteins tend to occur in "families" -- groups of similar proteins. 2. Examples: all IF's (& their genes) are similar. All globins (Hb alpha chains, Hb beta chains, myoglobin) are similar to each other but very different from all IF's. All antibody chains are similar to each other but not to globins and IF's, and so on. 3. How do families form? All members of a family have a common evolutionary origin -- ancestral gene duplicated and copies diverged → family of related proteins. file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect2.14.html[1/22/2014 4:35:30 PM] C2006/F2402 -- Lecture 2 a. Why do copies stay so similar? Conservative Selection. (1). What is Conservative Selection? Sections of the protein (& corresponding sections of the gene) that were essential to IF formation were preserved in all duplicates. (2). How preserved? Mutations that caused loss or serious alteration of these sections were selected against (= conservative selection against mutations that ruin function) (3). Mechanism of selection: Organisms that function worse than average because of mutations leave less offspring than average, so their mutant genes (or alleles) tend to be passed on less than average. Over many generations, the frequency of the mutations in the population decreases. b. Why do copies diverge? Innovative Selection. (1). What is Innovative Selection? Alterations in the protein that allowed useful variations in function were preserved (2). How preserved? Mutations that improve function are selected for (= innovative selection for mutations that improve function) (3). Mechanism of selection: Organisms that function better than average because of mutations leave more offspring than average, so their mutant genes (or alleles) tend to be passed on more than average. Over many generations, the frequency of the mutations in the population increases. 4. Two ways to 'fine tune' protein structure & get a group of similar, but different, proteins: By gene duplication to give a gene family OR alternative splicing of transcripts from a single gene. Next Time: Membrane structure and then the implications -- how are small molecules moved across membranes? file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect2.14.html[1/22/2014 4:35:30 PM]