We now take a look at a dynamic network of structural and associated proteins that play important roles in maintaining cell integrity and in generating cell movements. 

Cell shapes and sub-cell shapes are directly influenced by the cytoskeleton.  Amoeba pseudopodia, epithelial microvilli in the small intestine, and biconcave erythrocytes are just a few examples.

Movement by such proteins are evident in striated muscle contraction, mitosis, amoeba, flagella, and cilia.

Cytoskeletal Elements (CSE) include:
        microtubules (24 nm diameter)
        intermediate filaments (10 nm diameter)
        microfilaments (7 nm diameter)

Common Themes of Cytoskeletal Elements

1) soluble, globular protein subunits make up insoluble, linear fibrous polymers by covalent assembly
2) rapid exchange between a subunit pool of quaternary globular proteins and a polymer (self-assembly process) "dynamic instability" that is not in equilibrium.
3) association of subunits involved in non-covalent interactions
4) microtubules (MT) and microfilaments (MF) the form filaments that are helical with periodically placed interaction sites.  This provides multiple equivalent binding sites.
5) MT and MF form filaments that are polar and all CSEs have readily distinguishable ends.  (The cell knows which way it needs to be moved in relation to (+) and (-) ends.
6) Demonstrate hierarchy of interactions (protein-protein interactions)
7) are evolutionary conserved - however they often exist as multiple gene families
8) can be LABILE (sensitive to disassembly) and permanent (or semi-permanent)
9) functioning of CSE involves ASSOCIATION WITH OTHER PROTEINS
        -microtubule-associated proteins (MAPs)
        -actin-associated proteins (actin binding proteins)
        -intermediate filament associated proteins (IFAPs)

Types of processes mediated by associated proteins include:
1) affect assembly, disassembly, and stability
                                    - bind to monomers, prevent assembly
                                    - initiate assembly
                                    - promote assembly
                                    - cause disassembly
                                    - subunit modification
                                    - length determination
2) mediate interactions
                                    - cross-link filaments by forming bundles or networks
                                    - link filaments to the cellular structure (including membranes)
                                    - bind along side to strenghen filaments
3) generate movements by mechanoenzymes called "motor proteins"
                                    - convert chemical potential energy of ATP to mechanical energy

Microtubules (MT) are made up of various subunits are different levels...

Polymers of tubulin are made of alpha and beta tubulin.  Each are 55,000 daltons.

At the quaternary structure are alpha-beta tubulin dimers (110,000 daltons, 8 nm long).

Non-covalent interactions between dimers create PROTOFILAMENTS

Protofilaments associate side to side to form MICROTUBULES

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These microtubules have dynamic properties.  They are reversible by re/depolymerization.

High temperatures = association of MTs
Low temperatures = dissociation of MTs

In a test tube, a MT's length over time will go through lag, log, and stationary phases.  The lag phase is nucleation of a sheet to form a ring.  Log is the exponential growth that quickly arises after the initial nucleation centers have been created.  The presence of native or the addition of MT pieces will speed up the process by overcoming the lag.

A unique "treadmilling" property is found in a MT where the (+) end is the addition end with a "net assembly."  The (-) end has a "net disassembly."

Antimicrotubule drugs have been used in the fight against cancer by interfering with mitotic spindle fibers.
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colchicine, colcemid, nocodazole                                              inhibits addition of tubulin to MT, increases polyploidy
vinblastine, vinoristine (from Madagascar periwinkle)                aggregate the tubulin dimers (used against testicular cancer)
taxol (from the bark of ewe trees)                                            stabilizes MTs by binding to them; the cell is unable to get rid of
                                                                                               MTs and mitosis goes haywire (1st drug with major success
                                                                                                                                                against ovarian cancer)

In the cell context, MT don't exist with both + and - ends free.
The microtubule organizing center (MTOC) lengthens and shortens the + end only.  In cells, the minus end of a MT is usually embedded in the MTOC and is not a site of tubulin assembly.
The MTOC is the major location of a 3rd tubulin.  This isotype is known as gamma tubulin.  Gamma tubulin serves to nucleate MTs.

How do MTs lengthen ans shorten at the same end (known as dynamic instability)?
    GTP, yes GTP, is required for MT assembly, and GTP hydrolysis accompanies this but with a slight dealy.   GDP bound tubulin has a higher rate of disassembly while GTP bound tubulin has a higher rate of assembly.

A growing MT has a GTP cap (single ring on + end) on GTP tubulin.  If hydrolysis catches up with assembly, a GDP tubulin cap is present, and MT rapidly shortens.

Let's look at the actin-subunits of actin microfilaments.

    Globular (G) actin make up filamentous actin (F-actin microfilaments) polymers (45,000 daltons).

Intermediate filaments (IF) have fibrous, hydrophobic cores with globular ends that give variation.  They can be found as dimers and tetramers.

Motor proteins help maintain cell shape via MT and actin based motors.

Cell/Organelle Shape
        1) Erythrocyte membrane skeleton - the behavior of the network of actin is understood and used to predict how blood cell shapes come about in hypotonic and hypertonic solutions.  These anucleated cells maintain their biconcave discs despite the harsh conditions of being squeezed through capillaries.  They do this by storing energy in its erythrocyte membrane skeleton (like a spring, as Melanie mentioned in class).  Proteins involved in the plasma membrane of an erythrocyte include:
    Band 3 - a carbohydrate-containing, membrane-spanning protein present as a dimer with 2 identical subunits which span the membrane at least a dozen times each.  It allows for passive exchange of anions across the membrane.  Its name comes from its position in an electrophorectic gel.
    Glycophoryin A - a carbohydrate-containing, membrane-spanning protein that spans the membrane once and contains 16 oligosaccharide chains.
    Ankyrin, Spectrin, Actin, and Tropomyosins are other noteworthy proteins.
        2) Dystrophin - binds to actin and dystrophin associated protein (in the membrane) to the extracellular matrix.  It has been involved with an inherited, x-link recessive disorder (part of X chromosome is missing in patients with multiple problems), but why does it have a relatively large number of occurences in people with no history of this disorder?  It is the largest human gene so it will statistically have a larger chance of mutation than other genes.  The problem stems from a weakness where dystrophin is not connected to the cell.
        3) Microvilli - an end cap maintains its shape
        4) Nuclear lamin - a network that makes up the nuclear lamina and supports the nuclear envelope.  How does it keep its shape?
It is the target of a cyclin dependent kinase.  Phosphorylation will cause its disassembly.  Human mitosis requires break down of the nuclear envelope by phosphorylation.  Surprise, dephosphorylation and lamins help to reassemble it.
 

As you can see, CSEs affect cell shape and sub cell shape.  The plasma membrane is not just dictated by hydrophobic interactions.

Cilia and flagella must be anchored.  The plasma membrane actually surrounds the flagella.  Except for bacterium flagellum, the rest have a "9+2" arrangement.  This includes 9 outer doublets that circle a central doublet of 2 singlets when looking at the cross section of core of the cilium or flagellum, called the axoneme.  This makes a total of 20. The circling doublet is made of a complete A tubule and an incomplete B tubule.  A radial spoke extends from the A tubule.  The nexin bridge connects the two types of tubules.  The A tubule also has the motor protein, axonemal (ciliary) dynein.  It helps to slide the B tubule to the negative direction (relative to the A tubule for which it is attached).
Flagella movement involves MT and axonemal (ciliary) dynein.
For the nerve cell, the MTOC (-) is located in the cell body and the MTs extend outward.
Anterograde movement is created by kinesin.  It has 2 heads (reversibly ATP bound) with a hinged stalk.  The tail attaches to the cargo (vesicle).  The kinesin receptor on a cell tells it to go anterograde.
Dynein is larger and also has 2 ATPase heads (heavy chain) and a stalk.  In addition, it has light chains.  Dyneins act as the retrograde motor protein.