Download C2006/F2402 `14 Outline Of Lecture #2 -

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)
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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
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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.
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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
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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
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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
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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.
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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?
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