C2006/F2402 '04 Outline Of Lecture #2 -- Updated
01/22/04 03:14 PM
© 2004 Deborah Mowshowitz, Department of Biological Sciences, Columbia University, New York NY
Problems to do have been added in magenta italic bold.
-- Immunofluorescence & Freeze Etch ;
1B. You'll also need to refer to 1B -- Cytoskeleton
A. Overview of Components -- MT, MF, IF . See handout 1B & Becker Table 22-1 for summary of properties; see Becker fig 22-1 for pictures and handout 2B for diagrams. In Purves, see 4.21.
B. General Functions of Cytoskeleton
a. Support/strength -- weight bearing, shape determining.
b. Movement (MF & MT)-- most movement involving MF & MT occurs by sliding of one fiber or organelle relative to another. (Changes in length due to polymerization and depolymerization do occur.)
c. Localization of other factors -- act as peg board or framework for attachment of organelles, enzymes, etc.
C. How Components are Visualized See Becker Table 22-2.
1. Antibodies are used as reagents to identify proteins (& other substances)
a. What are Antibodies & Antigens? Antibodies are made by vertebrates in response to foreign materials (antigens). Antibodies are always proteins; antigens can be proteins (as in all cases discussed here) or other substances.
b. Specificity. Each antibody (against a protein) binds to one protein or a very small number of similar proteins. (See Becker fig. 22-12 for an example.)
c. Antibody Structure.
(1). Each antibody has a variable part -- complementary in fit to part of target protein.
(2). Each antibody has a constant part -- constant in all antibodies of that class from that species.
d. Use of 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.
2. Role of Fluorescence See Becker Guide to Microscopy p. 8-11 (appendix, p. 824-827) for details; for pictures see Becker fig. 22-1 or Purves 4.3, 4.21 & 4.22.
a. Fluorescent materials emit light of one wave length when irradiated at a different wave length.
b. Fluoresc. material can be located easily by irradiating sample and seeing what part of sample "lights up" = emits light.
c. Small fluorescent groups or GFP (green fluorescent protein) can act as "tags" or probes -- can be attached to large molecules such as antibodies, often without altering function of macromolecule.
(1). GFP -- genetic engineering used to change gene for protein of interest, so altered protein (including GFP) is made internally by the cell. Cell makes its own fluorescently tagged version of the protein.
(2). Small group -- fluorescent material added to cell (or to isolated protein) from outside.
3. Methods: How Fluorescent Antibodies are used (See handout 2A)
a. General Principle: Add fluoresc. antibody, wash off unattached antibodies (not bound to antigen), irradiate, and look for light emission = site of fluorescent antibody (= site 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 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.
(a). Different primary antibodies used for each target protein. (Not labeled.)
(b). Same labeled secondary antibody binds to all (unlabeled) primary antibodies.
D. How Cytoskeleton Discovered
1. Large, permanent structures made of tubulin and actin well known in specialized cells. 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 added to living cells.
2. Antibodies to tubulin bind to ("light up") MT in cytoplasm of almost all eukaryotic cells.
3. Antibodies to actin bind to MF in cytoplasm of almost all euk. cells.
4. 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.
b. Permanent -- form stable specialized structures such as cilia, flagella and muscle fibers. See Purves 4.24 or Becker fig. 23-7 & 23-8 for nice pictures
5. IF found later in multicellular organisms.
Look at problem 1-1.
E. Structure, Function & Properties of each component of Cytoskeleton -- IF, MF and MT.
1. Properties & Structure: See handout 1B (for properties) & Becker fig. 4-24 and/or Purves fig. 4-21 for diagrams of structure. Nice pictures and additional details (which go beyond the scope of this course, but might be of interest) are in Becker Chap. 22.
2. How each monomer forms a polymer (See handout 2B)
a. Actin/MF. Globular monomer (G actin) forms chain of beads. Two chains twist around each other ---> polymer. Looks symmetric (in standard picture) but isn't -- has "+" and "-" ends (like a chain of pop beads does). Nice pictures of how MF provide structural support to microvilli are in Purves 4.23 and Becker figs. 22-18 & 22-19 (22-19 & 22-20).
b. Tubulin/MT. Globular monomers are 2 types, alpha and beta. Alpha + beta ---> dimer ---> chain of dimers (protofilament) ---> rings of chains (usually 13 chains/tubule)
(1). Chains and tubules grow primarily by addition of dimers to "+" end.
(2). MT usually anchored at (and grow away from) structure at "-" end called a microtubule organizing center (MTOC) or centrosome. (For examples, see Becker Fig. 22-10)
c. IF. Monomers (extended chains, not globular) ---> dimers; 2 dimers --> tetramer = protofilament (fibrous basic subunit). Staggered protofilaments --> flat cable, 8 across ---> twist into final structure. (fig. 22-25 of Becker = handout 2B.)
Look at problems 1-7 & 1-8 (A & B).
3. Role in movement (See Purves 4.25 or Becker fig. 23-3 & 23-6)
a. IF not involved in movement (don't lengthen or shorten); Do assemble/disassemble during cell cycle.
b. MF, MT involved in movement. Primarily using a "motor molecule" (see handout 1B) that slides down fiber using a rachet mechanism (& splitting of ATP).
(1). Major motor molecules for tubulin/MT:
Dynein carries material "in" toward the nucleus/cell body -- Dynein draws things in (toward the - end of MT).
Kinesin carries material "out" toward the edges of the cell -- to the cell "Korners" (toward the + end of MT).
(2). Major motor molecule for actin/MF = myosin
c. Two major types of movement using motor molecules
(1). Can have 2 fibers sliding past each other (motor is part of one fiber or in between the two)-- overall effect is to shorten/lengthen structure. Examples:
(a). Anaphase -- MT slide "out" to give longer spindle fibers and separate chromosomes.
Note: Individual MT may shorten or lengthen by loss/addition of tubulin subunits, but overall movement of chromosomes seems to depend primarily on sliding of fibers past each other, not change in length of individual fibers. For details, see Becker.
(b). Telophase -- MF slide "in" forming cleavage furrow that divides cell in half.
(2). 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:
Neurons are nerve cells with long extensions (axons) that make connections (synapses) with other cells at the end of the axon. Materials are carried up and down the axon along MT between nucleus/cell body and synapse at end of axon. (See Becker fig. 23-1 & 23-2 or Purves 4.25) Direction of movement depends on whether motor molecule is dynein or kinesin.
Look at problems 1-9 to 1-11. See handout 1-B for effects of drugs mentioned.
F. IF's represent a protein/gene family (See Becker fig. 22-24) = handout 2B.
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. See Purves Ch. 24 esp. pp. 442-446 for more on evolution of gene families.
a. Why do copies stay so similar? Sections of the protein (& corresponding sections of the gene) that were essential to IF formation were preserved in all duplicates -- mutations that caused loss or serious alteration of these sections were lost (= conservative selection against mutations that ruin function = selection against organisms that function worse than average) .
b. Why do copies diverge? Alterations that allowed useful variations in function were preserved (= innovative selection for mutations that improve function = selection for organisms that function better than average).
4. Lamins are a type of IF found in the nucleus; do not confuse them with laminins (proteins found in the extracellular matrix). Lamins are the same in all cell types.
5. Cytoplasmic IF's (not lamins) are tissue specific -- origins of cancers can be traced from the type of IF's they contain. (Becker, box 22A, p. 774). Different tissues transcribe ("express") different IF genes.
II. Membrane Structure -- Any part of this not finished will be covered in lecture #3.
A. Lipid part/bilayer
1. Amphipathic nature of lipids (See Purves. 5.2)
2. Lateral diffusion (fast -- secs) vs. flip-flop (slow -- hrs) -- need enzymes (flippases) to speed flip flop. See Becker 7-10 & 7-11. Animation of lateral diffusion.
3. Two sides of bilayer (leaflet) can have different lipids.
a. All the lipid is made and inserted on one side.
b. An individual type of lipid can be "flipped" so it equilibrates on both sides; remain on original side, or be translocated (using energy) preferentially to the "other" side. Flipping or translocation requires enzymes.
B. Fluid mosaic model -- where are the proteins? Is it a "unit membrane?"
For "unit membrane" See Becker fig. 7-4 ; for fluid mosaic model see Becker fig. 7-5 or Purves 5.1.
1. Use of freeze fracture/freeze etch procedure
a. E vs P faces of bilayer = surfaces you see if you crack bilayer open = inside of bilayer
(1). E face = inside of the monolayer that is closer to extracellular space
(2). P face = inside of the monolayer that is closer to protoplasm
b. What do you see on inside? (see handout 2A, bottom & Becker fig. 7-16 or Purves 5.3).
(1) Inside is not smooth -- shows proteins go through bilayer (implies "mosaic" model not unit membrane)
(2). More bumps (proteins) on P face than E face -- shows more proteins anchored on cytoplasmic side.
2. Types of membrane Proteins -- peripheral vs. integral
Type of Membrane Protein
Protein Removed From Membrane By
Location/Attachment of Protein
salt, pH changes
On one 1 side of bilayer; non covalently attached to lipid
disrupting lipid bilayer
Goes through bilayer* or Covalently attached to lipid on one side (Lipid-anchored)**
* A small number of integral proteins do not go all the way through the membrane; they will be largely ignored in this course. For examples see Becker fig. 7-19 (first protein on left) or Purves 5.1 (last protein on right).
**Note that lipid-anchored proteins can be considered a type of integral protein or a separate category. See Becker fig. 7-19.
3. Transmembrane proteins (See Purves 5.5 and/or Becker fig. 7-19 & 7-21)
a. Single pass vs multipass
b. Domains -- intracellular, extracellular, transmembrane
c. Location of carbohydrates -- all in extracellular domain
d. Anchored or not? Some proteins are anchored to cytoskeleton; some float in lipid bilayer
Next Time: More details of membrane structure; then on to transport -- how are small molecules moved across membranes?