2.19.2010

1. Microtubule polymerization slide 9, 10, 11a. Nucleation – the limiting step.b. Non covalent binding of tubulin monomersc. Dimers add to plus end of microtubule.d. Polymerization is favored at the plus end, minus is depolymerization favorede. Plateau phase

i. Equilibrium when dimmers add and come off equally.f. GTP bound dimer

i. Both alpha and beta have GTP bound to them.g. Microtubules can be stabilized.

i. Plus ends carry the GTP and they form a ‘cap’ when GTP is bound, which stabilizes the microtubule ofr a short time.

1. Cap allows growth in plus directionii. The cap will continue to move down the length of the tubule until it

reaches minus end and hydrolysis of GTP takes place.iii. Treadmilling

1. Theory of dynamic instabilitya. Capping of microtubules with GTP bound dimmers, which

stabilizes, but a catastrophic event can take place at the plus end, which can provide a source for tubulin for other tubules elsewhere.

iv. If the GTP bound dimer concentration is high, elongation is favored, if low, then depolymerization is favored

v. Loss of cap results in GTP bound tubulin and rapid catastrophe occurs.1. Growing end unwinds2. Shrinkage can be stopped by increased concentration of tubulin

dimers bound to GTP.3. Provides a source of GDP dimers to be regenerated into GTP

bound dimmers.4. Shrinkage allows exchange of GDP and GTP dimers for other

microtubules.h. GEFS squeeze out GDP and then GTP is favoredi. GAPs – GTPase Activate Proteins for making GTP GDPj. Tubulin can be an intracellular signal

2. Where are microtubules?a. Centrosomes

i. A microtubule organizing center1. Nerve cells have stabilized microtubules that

is responsible for the orientation of them along the axon

2. Ciliated epithelial cells have basal bodies (similar to centrosomes) that attach to minus end of tubules and extend toward the apical end

3. Marginal bundle of microtubule – plus and minus are randomly assorted

b. Centrosome is made of a pair of centriolesi. Centrioles are microtubules

1. Arranged in triplets, linked together.2. 9 triplets make centriole

ii. they contain ‘gamma-tubulin ring complex’ that make up the circular features around the peri-centriole-complex, a ton of proteins around the centrosomes to hold them. They stabilize microtubule growing filaments by binding the minus end. As long as there is enough GTP bound dimers to add on.

iii. the grip proteins bind up the minus end of a microtubule, and that allows the tubule to grow rapidly at the plus end as long as there are GTP bound dimers. The grip proteins allows the orientation to take place to drive mitosis

iv. Microtouble organizing centers – MTOCs1. Nerve cells are polar w/long axons and short dendrites, so we

have stabilized microtubules that have centrosomes that are responsible for the polarity. The dendrites don’t have a polarity or MTOC so the microtubules line up in opposite if not random ways.

2. Apical and basal ends, plus end at end, apical, and MTOCS attach to minus end and stabilize minus end of MT

v. Basal bodies help orient polarity of epithelial cells.vi. MT help organize the polarity of the cell

c. Motor proteinsi. Kinesin – move toward the plus end

1. Drive movement of organelles and proteins along tubules.ii. Dynein – toward the minus end

1. Drive movement of organelles and proteins along tubules.2. Globular heads that link up to MT that requires ATP hydrolysis

and a walking motion occurs. They have an ATP binding site, which specifically bind onto B tubulin subunit.

iii. Light chain interacts with cargo, and heads have atp binding sites, which bind to beta tubulin subunits during movement.

1. Have different types for different cargo2. Growth factors are taken in by receptor mediated endocytosis and

can be moved by motor proteins into the celliv. Slow and fast transport (don’t worry too much)

3. Microtoubules – slide 17a. Important for where organelles are in the cell.b. Tubules associate with proteins of endoplasmic reticulum and help shape/form

in cytosplasmc. Some proteins can cap the tubules and determine the structure of the cell.

i. Shape/movement are important aspects as well.d. Polarity affects function of cell

i. MT can be capped and stabilized

ii. Unstable MT can remain dynamic

4. Slide 18a. 2 types of microubules that form outer doublet that surrounds central pair of

microtubules. A and B form outer doublet.b. Same tubules in centerc. Outer doublets are held together by stabilizing proteins, such as nexin, and the

inner dynein arm, which allows outer tubule doublet to slide along one another and yet be held together to create a whip-like movement, such as cilia or cilia-like structures.

d. Slide 19i. Linked by dynein motor proteins that bind ATP, and with ATP the motor

proteins shift the microtubules in separate directions.ii. Or linking proteins that use shift of tubules, to allow the tubules to bend.

When ATP is hydrolyzed, one shift ups and one shifts down for a bending, which creates movement.

5. Actin Filaments – Slide 20a. Found just underneath plasma membraneb. Made of actin monomers instead of tubulin monomers, such as microtubules.

i. Nucleating proteinsii. Sequestering monomer proteins

iii. Bundling proteins that bind actin filaments.iv. Cross-linking proteins

c. Two actin filaments form a helical shape.d. Polymerization of actin is bound by ATP. Actin can be bound by ATP or ADP

i. ATP: stable and addition is favoredii. ADP bound actin undergoes conformational change and associates with

other actin monomers less tightly and falls off actin filament. iii. Works like tubulin, but ATP instead of GTP is involved

e. Types of Actin Filaments and associated proteins:i. Nucleating proteins

1. Allow places for new actin filaments to growii. Bundling proteins

1. Bind individual actin filaments and align them so that filopodia can function

a. Filopodia are fingerlike projects at the edge of the cell. Protrude out of the membrane. Provide an ability to feel out in space and find things to take up for movement or adhesion with neighboring cells.

2. Bundling in filopodia is important for elongation.iii. Motor proteins

1. Allow for movementa. Myosin I helps actin slide along itself

iv. Capping1. No longer polymeize

v. Cross-linking

1. Form a matrix and increase stability under plasma membrane instead of paralleling

vi. Severing proteins1. Sever actin

f. Microtubules-specific drugsi. Phalloidin – tagged with fluorescent dyes to visualize actin filaments.

1. Used in cell culturesii. Cytochalasin – caps filament plus

iii. Taxol, colchicine – stabilize microtubules (and prevents polymerization)g. Myosin proteins are used to stabilize actin in cells.

i. In the head, there is an actin binding domain and tail region hangs off end and regulated by other binding proteins.

ii. Slide 25 – muscular cells.iii. Sarcomere will shorten as actin filaments are broguh together.iv. Cell pinching is driven by actin-myosin association.

h. Myosin-I is important for allowing actin-filament dynamics to take place at peripheral edge of membrane. Can move to distal end of cell membrane