Cytoskeleton, microtubules

Question 1

Describe microtubules in general.

Answer 1

• tubulin subunits
• hollow - provides rigidity and strength
• polar
• dynamic - population of tubules and subunits constantly exchanging
• highly conserved among organisms, meaning it is super important
• railways for motor proteins - kinesin, dynein which walk by hydrolyzing ATP and directed by polarity

Question 2

Describe actin filaments in general.

Answer 2

very similar to microtubules

• made from actin subunits
• non-hollow
• polar
• dynamic
• highly conserved
• railways for motor proteins

Question 3

Describe intermediate filaments in general.

Answer 3

• not very conserved
• various types of cell-specific intermediate protein subunits
• non-hollow
• non-polar
• non-dynamic - not many free subunits available in the cell
• diverse
• not railways for motor proteins
• less essential to the cell than microtubules and actin. Only become very important if one of the other two types become defective.

Question 4

How are intermediate filaments used as markers of disease?

Answer 4

Because they are cell-type and location specific.

Question 5

What is an experimental method that is useful for studying functions of microtubule-associated proteins?

Answer 5

RNA interference

function-blocking antibodies

Question 6

Is GTP hydrolysis required for microtubule assembly?

Answer 6

Hydrolysis is not required, but GTP attached to tubulin is required.

Question 7

How does free tubulin exist?

Answer 7

As GTP-tubulin

Question 8

What is the polarity of microtubules in neurons?

Answer 8

The plus end of microtubules is oriented away from the cell body (towards end of axon)

Question 9

How can microtubules and actin be visualized together?

Answer 9

Cells can be made to express fluorescent hybrids of the subunits that fluoresce at different wavelengths. Images can then be merged to view the structures simultaneously.

Question 10

How is it that cytoskeletal elements sometimes overlap in function?

Answer 10

because both actin and myosin play roles in coordinating the cell and dividing labor.

Question 11

Describe the subunits of microtubules.

Answer 11

• subunits exist in as a dimer of alpha and beta tubulin
• cells contain both microtubules and free tubulin dimers, which are constantly in exchange
• each ring of the microtubule consists of 13 subunits around

Question 12

Describe the polarity of microtubules and how polarity is established.

Answer 12

• polarity has nothing to do with chemical charge!
• plus and minus end because subunits are heterodimers
• plus end is favored for assembly and disassembly (more dynamic!)
• polarity exists all along the length of the microtubule
• alpha subunit revealed at minus end, and beta subunit is revealed at the plus end

Question 13

Describe how microtubules are non-equilibrium polymers.

Answer 13

There is first a lag phase of microtubule polymerization, followed by an exponential growth phase, and finally a steady state where subunits are coming on and off

This is a non-equilibrium polymer because tubulin must be above a critical concentration before polymerization can take place, much like salt cannot crystallize in water until it reaches a critical concentration.

Question 14

What conditions are needed for a microtubule to form?

Answer 14

1. GTP
2. 37 degrees
3. magnesium
4. tubulin above critical concentration

Question 15

How was microtubule polarity discovered?

Answer 15

imaging of flagella revealed that the two microtubule ends have different critical concentrations for assembly, plus end having a lower critical concentration and the minus end having a higher critical concentration.

Question 16

What is an example of an organelle which is distributed by motor proteins on microtubules?

Answer 16

Golgi. The motor proteins involved are highly processive.

Question 17

Describe microtubule treadmilling.

Answer 17

Treadmilling is an illusion of microtubule movement which is observed in vitro. It is the tubulin subunit concentration is at a point in which polymerization at the plus end equals depolymerization at the minus end, so the microtubule appears to move through solution. This does not seem to happen in vivo.

Question 18

What does the dynamic instability mechanism of microtubules describe?

Answer 18

It says that individual microtubules in a population are doing very different things at the same time (e.g. one might be assembling while another is disassembling). Dynamic instability is governed by the GTP cap, catastrophe, and rescue.

Question 19

What is the homology between alpha and beta tubulin? How do their functions differ?

Answer 19

There is 40% homology between them. Both bind GTP, but only the beta tubulin GTP is hydrolyzable.

Question 20

Describe the growth and breakdown of microtubules

Answer 20

• GTP tubulin adds to plus end, generating GTP cap. As long as there is a GTP cap, microtubule will continue to assemble
• GTP hydrolysis occurs only after GTP-tubulin is incorporated into the microtubule. Allows energy to be stored in the microtubule lattice.
• If GTP hydrolysis catches up to assembly, GTP cap is lost and microtubule disassembles through catastrophe
• If GTP cap is re-established before microtubule is completely gone, we get rescue

Question 21

What length microtubule has a better chance of rescue after catastrophe?

Answer 21

Longer microtubules take longer to be totally hydrolyzed, so they have a better chance of rescue.

Question 22

Why is GTP hydrolysis of GTP-beta tubulin in microtubules necessary?

Answer 22

GTP hydrolysis stores energy in the microtubule lattice so that microtubule disassembly can do work, such as assisting in separation of chromosome during mitosis.

Question 23

Describe selective stabilization of certain microtubules.

Answer 23

There are two methods of selective stabilization:

1. search and capture of the plus end: microtubules grab the edge of the cell and are stabilized. Also polarizes the cell.
2. proteins bind microtubule lattice to prevent disassembly: Tau, MAPs 1-8, doublecortin, STOP, plectins

Question 24

Describe the role of Tau protein in alzheimer’s disease.

Answer 24

In a healthy neuron, tau binds the microtubule lattice and stabilizes it from degradation. In a diseased neuron, tau is sequestered away so that microtubules are depolymerized more rapidly.

Question 25

List and describe the microtubule-active drugs.

Answer 25

- nocodazole/colchicine: bnds free GTP-tubulin subunits so microtubules are built more slowly. This shifts microtubule dynamics towards disassembly - taxol/epithilone D: stabilizes the plus end of microtubules and prevents disassembly, stopping microtubule dynamics. This is a common treatment of breast cancer. - vinblastine/vincristine: works similarly to nocodazole.

Question 26

GTP hydrolysis in the microtubule lattice only accomplishes a small bit of work. How are the rest of the cell's demands for work accomplished?

Answer 26

The work done by microtubules is accomplished by motor proteins.

Question 27

Describe the types of molecular motor proteins, and what they are good for.

Answer 27

- Dynein carries retrograde cargo and travels toward the minus end. Processivity is enhanced by dynactin. - kinesis carries anterograde cargo and travels toward the plus end - both consist of dimers, because they move by walking. This allows them to be highly processive. - Both are ATPases that generate forces to create motion in the cell. - both have globular motor domains (dimerized part), cargo domain, and a stalk domain in between that are all part of the same gene product.

Question 28

Which domain is the motor domain in motor proteins?

Answer 28

The motor domain is the ATPase globular domain which actually contacts the microtubule.

Question 29

Why do different cell types have microtubules organized in different ways?

Answer 29

Different microtubule organization allows for different types of cell work to be accomplished.

Question 30

Describe motor protein specificity.

Answer 30

1. direction they move 2. cargo they carry requires organization of microtubules based on their polarity

Question 31

Describe ciliar microtubule arrays.

Answer 31

- microtubule minus end is embedded in the basal body (the cell body). In the basal body, there are no free tubules at the center of the structure, and it consists of 9 +3 triplet centriole. - the plus end is pointed outward along the cilia or flagella. Microtubules are arranged 9+2 around the circumference of the flagella, and are highly crosslinked. Two central singlet microtubules are at the center of the flagella. - These microtubules are very stable. - The flagella beats through flagellar dynein.

Question 32

Give an exmple of a cell type which does not have microtubule organization emanating from a centralized foci (centrosome).

Answer 32

epithelia, neuron

Question 33

Why do neuron dendrites stop growing, but not axons?

Answer 33

This is due to microtubule polarity. in dendrites, microtubules have mixed polarity, allowing organelles and veiscles to circulate rather than move in one direction. Axons need unidirectional vector movement accomplished by kinesin (moves toward plus end, therefore no golgi in axon because that is moved toward minus end).

Question 34

How is microtubule formation begun?

Answer 34

- Microtubules get started by nucleation. This explains the lag period of microtubule assembly, because it is not as energetically favorable as microtubule elongation. - nucleating elements are centrosomes and basal bodies (in cilia/flagella). Nucleation at these structures prevents microtubules from randomly popping up throughout the cell.

Question 35

Describe the structure of the centrosome.

Answer 35

- pair of centrioles - each centriole consists of microtubule triplets - surrounded by pericentriolar matrix - PCM contains rings of gamma tubulin which serve as nucleating sites, where microtubules are very stable

Question 36

Describe gamma tubulin and how it functions.

Answer 36

- in pericentriolar matrix - as different from beta and alpha tubulin as they are from one another - present in the form of gamma turc (gamma tubulin ring complex) - gamma turc establishes protofilament number (13) and polarity orientation of the microtubule

Question 37

How are complex "non-centrosomal" microtubule arrays established?

Answer 37

1. may be multiple centrosomes in the cell not known 2. gamma turc may be distibuted to non-centrosome locations in the cell (can be determined from treatment with nocodazole. Microtubules are destroyed, and where new ones appear tells us where gamma turc is located in the cell) 3. microtubules may break off from centrosome and move around the cell in a new pattern

Question 38

Describe microtubule movement in living cells.

Answer 38

Microtubule movement is entirely relative. If the cargo which is carried along it cannot move because it is attached to a coverslip, the microtubule itself will move along it.

Question 39

Describe the microtubule severing proteins.

Answer 39

microtubules are severed by hexameric ATPases. These work by cutting the microtubule at any position, and pull tubulin through the hexameric pore. This is how microtubules can be released from the centrosome in neurons, for example. - katanin - spastin - katanin-like-2

Question 40

Which type of motor protein transports microtubules down the neuron axon?

Answer 40

cytoplasmic dynein, because dynein moves relatively toward the minus end of microtubules.

Question 41

How is axon branch fomation accomplished in neurons?

Answer 41

Axon branch formation is accomplished through microtubule cut and run, via two distinct methods: 1. katanin mode: widespread, tau makes microtubules more vulnerable to katanin 2. spastin mode: spastin is recruited specifically to the branch location

Question 42

How are microtubules visualized in cells?

Answer 42

1. immunostaining of fixed cells with fluorescent/confocal microscopy: use two different colors of secondary antibodies to look at microtubules and actin together 2. electron microscopy: best for study of microtubule arrangement (transmission) 3. GFP-fusions: good for living cells, can make constructs of tubulin, actin subunits. Can do construct with Plus-end associated proteins to determine when assembly is occuring, because they are only associated with GTP caps during assembly and not catastrophe.