Microtubule Structure & Function

Question 1

Define microtubules.

Answer 1

Microtubules are long, hollow, unbranched tubes composed of subunits of the protein tubulin.

Question 2

Define microfilaments.

Answer 2

Microfilaments are solid, thinner structures, often organized into a branching network and composed of the protein actin.

Question 3

Define intermediate filaments.

Answer 3

Intermediate filaments are tough, ropelike fibers composed of a variety of related proteins.

Question 4

What type of bonds holds together the cytoskeleton?

Answer 4

Cytoskeletal filaments (which are polymers of protein subunits) are held together by weak noncovalent bonds.

Question 5

Define protofilament.

Answer 5

A protofilament is the structure that composes the wall of a microtubule, made from globular proteins arranged in longitudinal rows. They are aligned parallel to the long axis of the tubule.

Question 6

Describe the structure of a- and B-tubulins in a protofilament.

Answer 6

The protofilament is asymmetric with an a-tubulin at one end (the minus end) and a B-tubulin at the other (the plus end). These two subunits fit together tightly.

Question 7

How is the microtubule-binding activity of MAPs controlled?

Answer 7

This process is controlled by the addition and removal of phosphate groups from various amino acid residues. MAPs generally increase the stability of microtubules and promote their assembly.

Question 8

How do motor proteins and microtubules interact within an axon?

Answer 8

Microtubules mediate the movement of vesicles along an axon. They also serve as tracks for a variety of motor proteins that generate the forces required to move objects within a cell.

Question 9

How is the energy necessary to fuel a motor protein’s activity provided?

Answer 9

As the protein moves along, it undergoes a series of conformational changes that constitute a mechanical cycle. This cycle is coupled with a chemical cycle together to fuel the activity of the motor protein.

Question 10

What are the steps in the chemical cycle of a motor protein?

Answer 10

1. The binding of an ATP molecule to the motor.
2. The hydrolysis of the ATP.
3. The release of the products (ADP and P) from the motor.
4. The binding of a new molecule of ATP.

Question 11

What does it mean to say that kinesin is a plus end-directed microtubular motor?

Answer 11

It means that as the motor protein moves along microtubules, it moves towards its plus end. Because the minus ends face the cell body, kinesin transports vesicles and other cargo toward the synaptic terminals.

Question 12

Which direction in the cell does dynein usually go?

Answer 12

Cytoplasmic dynein moves processively along a microtubule toward the polymer’s minus end—opposite of kinesins.

Question 13

What are the two functions of cytoplasmic dynein?

Answer 13

1. As a force-generating agent in positioning the spindle and moving chromosomes during mitosis.
2. As a minus end-directed microtubular motor with a role in positioning the centrosome and Golgi complex and moving organelles, vesicles, and particles through the cytoplasm.

Question 14

What is dynactin, and what function does it fulfill?

Answer 14

Cytoplasmic dynein does not interact directly with the membrane-bounded cargo but requires an intervening adaptor; this is usually the multisubunit protein dynactin. Dynactin may also regulate dynein activity and help bind the motor protein to the microtubule, which increases processivity.

Question 15

What happens if an individual organelle binds kinesin and dynein at the same time?

Answer 15

Because kinesin and dynein move in opposite directions, this gives the organelle the ability to move in opposite directions.

Question 16

Describe the structure of the centrosome.

Answer 16

The centrosome is a complex structure that contains two barrel-shaped centrioles surrounded by amorphous, electron-dense pericentriolar material (PCM).

Question 17

What role do MTOCs play in the cell?

Answer 17

MTOCs (microtubule-organizing centers) control the number of microtubules, their polarity, the number of protofilaments that make up their walls, and the time and location of their assembly.

Question 18

How are dramatic changes in the spatial organization of microtubules accomplished?

Answer 18

Two separate mechanisms work to accomplish this change:
1. The rearrangement of existing microtubules
2. The disassembly of existing microtubules and reassembly of new ones in different regions of the cell.

Question 19

Explain how GTP is used for microtubule assembly.

Answer 19

Assembly of tubulin dimers requires that a GTP molecule be bound to the B-tubulin subunit. B-tubulin is not only a structural protein but an enzyme: a GTPase. The GTP is hydrolyzed to GDP shortly after the dimer is incorporated into a microtubule, and the resulting GDP remains bound to the assembled polymer.

Question 20

What two observations does the phenomenon of dynamic instability explain?

Answer 20

Dynamic instability explains
1. that growing and shrinking microtubules can coexist in the same region of a cell
2. that a given microtubule can switch back and forth, unpredictably between growing and shortening phases.
Dynamic instability is an inherent property of the microtubule, and more specifically, of the plus end of the microtubule.

Question 21

What does dynamic instability allow the microtubule to accomplish?

Answer 21

It provides a mechanism by which the plus ends of microtubules can rapidly explore the cytoplasm for sites of attachment, allowing the cell to build its complex cytoskeletal networks. It also allows cells to respond rapidly to changing conditions that require remodeling of the microtubular cytoskeleton.

Question 22

What are the four functions of the cytoskeleton?

Answer 22

1. Structure and support
2. Intracellular transport
3. Contractility and motility
4. Spatial organization

Question 23

How do a- and B-tubulins differ with respect to how they interact with GTP?

Answer 23

Both a- and B-tubulins bind to GTP, but only B-tubulins act as a GTPase, meaning they hydrolyze the GTP to GDP.

Question 24

The plus-end of a microtubule corresponds to what type of protofilament?

Answer 24

B-tubulin

Question 25

The minus-end of a microtubule corresponds to what type of protofilament?

Answer 25

a-tubulin

Question 26

In most animal cells, what nucleates the microtubules?

Answer 26

The centrosome, which is the microtubule organizing center (MTOC).

Question 27

Describe the structure of centrosomes.

Answer 27

Centrosomes contain a pair of centrioles surrounded by the denser pericentriollar material (PCM). These centrioles are built from microtubules (called 9+0 microtubule triplets) and are set at right angles to each other.

Question 28

Where is the only place that new microtubules can be formed?

Answer 28

The centrioles, which are surrounded by pericentriolar material.

Question 29

Describe gamma-tubulin ring complexes.

Answer 29

These complexes are like the foundation of a house in that you can build new microtubules on these foundations, but only on these foundations. These ring complexes have directional growth, polarity, and the plus-end sticks out.

Question 30

How is gamma-tubulin different from a- and B-tubulin?

Answer 30

It is essentially the same except for the fact that it is only found in the foundation that is gamma-tubulin ring complexes.

Question 31

What happens to the ER and Golgi when the microtubular skeleton is disassembled by a drug such as nocodazole?

Answer 31

Microtubules provide a scaffold for organizing the ER and Golgi, and the Golgi is contained in space by a cage of microtubules. When the microtubular cytoskeleton is disassembled, the ER and Golgi have no framework and fragment.

Question 32

In a steady state, microtubules can exist in one of two states. Describe these two states.

Answer 32

1. Rescue. The microtubule is growing.
2. Catastrophe. The microtubule is shrinking.

Question 33

What happens to a microtubule when the rate of subunit assembly is quicker than the rate at which the GTPase acts?

Answer 33

There will be a GTP cap at the end of the microtubule. This stabilizes the microtubule and ensures its growth.

Question 34

What happens to a microtubule when the rate of hydrolysis exceeds the rate of addition?

Answer 34

The GTP cap will disappear. This means that all of the GTP is bound to B-tubulins and there is no GTP cap bound at the end of the microtubule to protect it.

Question 35

When there is only a GDP molecule bound to the end of a microtubule, what happens?

Answer 35

The protofilaments become structurally unstable. It causes each of the protofilaments to take on a profound curvature, causing the microtubule to peel away and shrink.

Question 36

What are the two scientific techniques for demonstrating the dynamic instability of microtubules?

Answer 36

1. Labeled tubulin with fluorescence for one minute.
2. Fluorescently labeled end-binding protein (a tubulin-associated protein) EB1

Question 37

Give two examples of MAPs that stabilize the microtubule.

Answer 37

MAP2 and the tau protein.

Question 38

Describe the process by which the dendrites of a neuron are formed.

Answer 38

When a neuron is developing, it must send out long projections that will become dendrites. Normally microtubules are in a state of dynamic instability regulated by the GTP cap. To create the dendrites, you will need the extensions of microtubules to extend past their normal state. This requires MAPs that stabilize the microtubule and confine it to a single region (where the dendrites will emerge), generating polarized growth.

Question 39

Describe the chains that make up kinesin and detail their function.

Answer 39

Kinesin is composed of two heavy chains and two light chains. The heavy chains bind to microtubules, hydrolyze ATP to walk, and act as ATPases. The light chains bind to the transport vesicle and cargo.

Question 40

Which direction does kinesin travel in?

Answer 40

Kinesin powers anterograde transport in the direction of the synapse (in a neuron) using hydrolysis to walk towards the plus-end.

Question 41

Describe the chains that make up dynein and detail their function.

Answer 41

Dynein is composed of two proteins: heavy chains and some light chains. The heavy chains do the walking, and the light chains are responsible for binding cargo with a high degree of specificity.

Question 42

Which direction does dynein travel in?

Answer 42

Dynein powers retrograde transport to the cell body using ATP hydrolysis to walk towards the minus-end.

Question 43

How does a lack of kinesin affect the mitochondria of a cell?

Answer 43

Without kinesin, the mitochondria cluster near the center of the cell. This happens because the mitochondria are dependent on the intracellular transport processes of microtubules.

Question 44

How do you build a flagellum or cilium from microtubules? Describe the resulting structure.

Answer 44

Motile flagella/cilia core is a 9+2 arrangement of microtubules. There are 9 doublets on the outside and a pair of microtubules on the inside. Proteins link the doublets of the axoneme.

Question 45

What role do nexin proteins and dynein play in flagellum/cilium movement?

Answer 45

Dynein works as it normally does to move two things relative to each other, but nexin converts sliding to bending, causing only one of the doublets to move. This bending back and forth (which is powered by the ATPase motor) causes the movement of the flagellum or cilium.

Question 46

How would you experimentally disassemble a flagellum or cilium?

Answer 46

You would incubate the de-membraned cell with EDTA (chelates magnesium). This causes the arms of the dynein microtubules to fall apart from the soluble ATPase portions of these molecules. That allows you to isolate either the motor proteins or the non-motor portion of these structures independently of each other.

Question 47

What are the two microtubule binding sites of dynein?

Answer 47

Dynein creates a tether that binds to A-subfiber and its head walk along the adjacent B-subfiber. The goal of the dynein protein is not to move cargo down the microtubule but to move two microtubules relative to each other.