8.2 Microtubules and MAPS

Question 1

What is the difference between microtubules and actin?

Answer 1

MICROTUBULES: tubulin dimer (alpha and beta heterodimer) subunits make up microtubules
ACTIN: actin monomers make up actin filaments

Microtubules: Bigger than actin 55kDa
Actin: 40kDa

Microtubule: Binds GTP
Actin: Binds ATP

MICROTUBULES: Use motor proteins Kinesin (+ end) and dyneins (- end)
ACTIN: uses motor proteins myosin

MICROTUBULES: can assemble and disassemble at plus end
ACTIN: assembles more readily at plus end but not readily at minus end

Question 2

What is the difference between the GTP in alpha tubulin and beta tubulin?

Answer 2

The GTP molecule that is attached to the α-tubulin subunit is not hydrolyzed and is buried between the two proteins. However, the GTP that is bound to the βsubunit can be hydrolyzed and this hydrolysis plays a role in the assembly of microtubules

Question 3

What happens when tubulin assembles?

Answer 3

When tubulin assembles, the dimers form rows known as protofilaments and all of the dimers point in one direction. So, one end of the microtubule has exposed αtubulin subunits and the other end exposes β-tubulin subunits.

Question 4

How many protofilaments are needed to complete a microtubule?

Answer 4

It takes thirteen protofilaments to form a complete microtubule which is then a structure about 24nm in diameter with a channel down the middle that is about 10nm in diameter

Question 5

How do we know bacteria/prokaryotes has protein similar to tubulin?

Answer 5

In bacteria, there is a protein called FtsZ, which stands for “filament temperature sensitive mutant Z”, which was the strain of bacteria where FtsZ was first identified. FtsZ plays an important role in the division of some bacteria and, most interestingly, FtsZ also plays a role in the replication of mitochondria and chloroplasts and this is another good piece of evidence that mitochondria and chloroplasts are descendents of free living prokaryotes.

Question 6

What are MAPs? Which two will we focus on for this lecture?

Answer 6

Microtubule associate proteins (MAPs) paffect stability, nucleation and turnover of microtubules.

MAPs are proteins that bind along the length of microtubules and stabilize or bundle microtubules. They also modify the underlying ability of tubulin to assemble and disassemble AKA microtubules associated proteins
Focus: MAP2 and Tau

Question 7

Assembly and disassembly occur preferentially on which side of the microtubule?

Answer 7

Microtubule assembly and disassembly occurs preferentially at the plus end. The beta subunit faces the plus end, exposing it’s GTP binding site.

Question 8

Both alpha and beta subunits bind GTP. Which side is exposed and hydrolyzed?

Answer 8

Only beta is hydrolyzed and exposed. This makes sense b/c it points to the plus side of the microtubule

Question 9

What happens when GTP binds to beta subunit on a microtubule?

Answer 9

It facilitates the addition of the subunit onto the end of a growing microtubule. After a short period in the microtubule, the GTP that is bound to the β-subunit gets hydrolyzed to GDP

Question 10

What happens when GTP is hydrolyzed to GDP on the beta subunit in microtubule?

Answer 10

As long as the microtubule is growing rapidly, the GDP-bound subunits will be buried in the microtubule by addition of new GTP-bound tubulin dimers and the newly added GTP-bound subunits form what is known as GTP cap. It’s a cap of GTP bound tubulin dimers
subunits have weakened interactions with other subunits and the protofilaments begin to splay out from each other

Question 11

What happens after protofilaments begin to splay out from each other?

Answer 11

the microtubule can’t regrow and it will depolymerize completely unless some other factor is around to stabilize the protofilament. Splaying happens b/c of loss of GTP cap

Question 12

In actin filament, polymerization is rapid at the plus end and depolymerization is frequent at the minus end. How is this different from microtubules?

Answer 12

both polymerization and depolymerization can be occurring at the plus end of a microtubule

Question 13

What does GTP-tubulin cap do? What happens w/o cap?

Answer 13

It protects growing microtubules. Loss of the GTP cap causes conformational change and microtubule disassembly. Protofilaments begin to splay. Once the protofilaments splay out from one another, the microtubule can’t regrow and it will depolymerize completely unless some other factor is around to stabilize the protofilament.

Question 14

What is dynamic instability?

Answer 14

Dynamic instability is the behavior displayed by microtubules where you’ll see some growing and others shrinking at the same time even though the population as a whole might have a constant amount of polymer and free dimer.

During dynamic instability growing and shrinking microtubules are present at the same time.
Dynamic instability allows the microtubule network to “probe” the cell cytoplasm for targets like chromosomes as dynamic instability.

Question 15

What is a catastrophe in microtubules and what events follow a catastrophe?

Answer 15

Catastrophe occurs when the GTP cap disappears due to hydrolysis catching up to one end of the microtubule. Loss of GTP cap leads to microtubule disassembly.

Once a catastrophe occurs, the microtubule will shrink rapidly and will depolymerize completely unless it’s rescued by some additional stabilizing factor. If the microtubule does encounter a stabilizing factor, it can repeat the cycle of assembly and disassembly over again as long as free GTP-bound tubulin subunits are available. This cycle of growth and depolymerization is going to occur randomly in a population of microtubules (this behavior is dynamic instability)

Question 16

After catastrophe, what happens if a microtubule with no GTP cap encounters a stabilizing factor?

Answer 16

If the microtubule does encounter a stabilizing factor, it can repeat the cycle of assembly and disassembly over again as long as free GTP-bound tubulin subunits are available. This cycle of growth and depolymerization is going to occur randomly in a population of microtubules. The behavior displayed is called dynamic instability.

Question 17

How do microtubules exhibit treadmilling? Is this a frequent action?

Answer 17

Not frequent

-microtubules can exhibit treadmilling if the assembly at the plus end is balanced by disassembly to the minus end.

For most microtubules in cells, the minus end is attached to some sort of structure and treadmilling doesn’t play a major role in determining the behavior of the microtubule. However, for some very long extensions like those made by nerve cells, microtubules may be nucleated at the cell center but then detach and move down the cell extensions by treadmilling.

Question 18

microtubules can assemble spontaneously, but in cells, most microtubules will need what to grow/assemble?

Answer 18

A nucleation site
In non-dividing cells, there is a SINGLE MAJOR SITE of microtubule nucleation called the centrosome. It is also called them microtubule organizing center, or MTOC

Question 19

How are microtubules nucleated?

Answer 19

By small ring-shaped protein complexes that are scattered throughout the centrosome which form the microtubule nucleation sites. The protein that makes up these rings is (gamma) γ-tubulin. So γ-tubulin generally forms the nucleation of microtubules. Cells are nucleated at the centrosome and the minus end of the microtubule is at the nucleation site.

Question 20

Since most of the microtubules in a cell are nucleated at the centrosome and the minus end of the microtubule is at the nucleation site, this means that the minus end of microtubules is generally found at the centrosome. Where are the plus end of microtubules generally found?

Answer 20

the plus ends of the microtubules will be found out at the edges of cells.

Question 21

What is the difference between centrosomes in plants and animals?

Answer 21

Centrosomes in plants and fungi lack centrioles, but are otherwise analogous to those in animals. In animals some centrisome nucleate microtubules which then detach instead of being anchored in the centrisome

Question 22

What is the difference between centrosomes in non-dividing cells vs. mitotic cells?

Answer 22

Non-dividing cells have one single centrosome

Mitotic cells have 2 since the first steps involved in the division of cells is the duplication of the centrosome

Question 23

What is MAP2?

Answer 23

a relatively long microtubule associated protein that stabilizes microtubules and creates loose microtubule bundles

Question 24

What is Tau?

Answer 24

A shorter MAP and makes tighter microtubule bundles.
Tau is also one of the proteins found in high concentrations in neuronal tangles in the brains of people with Alzheimer’s disease. It’s thought that abnormal regulation or function of Tau may contribute to Alzheimer’s disease.

Question 25

How are MAPs (microtubule associated proteins) regulated?

Answer 25

the function of microtubule associated proteins is going to be regulated through phosphorylation
The phosphorylation of MAPs generally releases them from microtubules and results in microtubules which are more dynamic and less stable. So stable microtubules are generally associated with non-phosphorylated MAPs.

Question 26

Apart from phosphorylation, Microtubule stability is regulated by what?

Answer 26

In moving and non-moving cells - it differs. Moving grow and shrink rapidly while non moving is more stable

Question 27

What are the different ways covered in lecture that cells can destabilize microtubules?

Answer 27

catastrophin protein which causes catastrophe
Katanin protein - cuts microtubules
Stathmin - microtubule sequestering protein

Question 28

What is catastrophin protein?

Answer 28

Destabilizes microtubules
Catastrophin acts on microtubules by pulling the protofilaments apart at the microtubule ends using the energy of ATP. When it does this, this has the same effect as the loss of the GTP cap does. In other words, it causes a catastrophe.

Question 29

What is the role of Katanin?

Answer 29

Katanin severs microtubules so it performs the same function that gelsolin does in the actin cytoskeleton. In addition to destabilizing microtubules, katanin is also involved in releasing them from the centrosome

Question 30

What is stathmin?

Answer 30

A microtubule sequestering protein
that (acts like thymosin in actin).

Stathmin binds to the tubulin dimer when it is not phosphorylated and sequesters it.

When it becomes phosphorylated, it releases dimers and therefore makes more tubulin available for assembly. This increases rate of microtubule assembly in the cells.

Question 31

What happens when stathmin is phosphorylated?

Answer 31

When stathmin becomes phosphorylated, it releases the dimers and therefore makes more tubulin available for assembly. This has the effect of increasing the rate of microtubule assembly in cells. So stathmin basically performs the function of thymosine β-4 when we talked about the actin cytoskeleton.

Question 32

One of the functions of microtubules is to create paths between different parts of the cell and attach growing microtubules to target sites. How does it accomplish this?

Answer 32

With proteins that anchor the plus ends of microtubules and, in effect, stabilize a path between themselves and the centrosome.
Proteins: TIP (EB1)

Question 33

What is the role of Kar9?

Answer 33

Kar9 is a protein that is localized at the membrane of a budding yeast cell and its function is to anchor one end of the mitotic spindle to the new cell. Kar9 doesn’t do this directly but instead it binds to a protein (TIP) that marks the GTP cap that is at the plus end of the microtubule.

Question 34

What is EB1?

Answer 34

The overall effect would look like a region of EB1 coated microtubules moving from left to right (like treadmilling).

New EB1 subunits bind to the GTP cap as it grows at the end of a microtubule. As the tubulin subunits hydrolyze their GTP to GDP, the EB1 subunits fall off.

Question 35

What are the different motor proteins for microtubules? How do they differ? How are they similar?

Answer 35

kinesin family - move toward the plus end of the microtubule
Dynein family - move toward the minus end of the microtubule
Similar in shape: The globular head domains are the motor end of the protein and the tail domain is variable and it gives each motor a specialized function.

Question 36

What are the functions of kinesin proteins?

Answer 36

all kinesins move along microtubules in the direction of the plus end of the microtubule
if an organelle has kinesin attached to it, it will move along microtubules toward the plus ends of the microtubules

Question 37

What do dynein do in microtubules?

Answer 37

Move along the microtubule in the direction of the minus end.
organelles with dyneins bound to them will always move to the minus ends of the microtubules.
Minus end directed motors (dyneins) are involved in chromosome separation, movement of cilia and flagella and direction of membrane bound organelles toward the cell center (golgi, endocytic vescicles).

Question 38

What is the difference between kinesin and myosin motor function?

Answer 38

Kinesin is more processive
kinesin and dynein motors stay attached to a microtubule for half of the ATPase cycle and since kinesins and dyneins have at least two motor subunits, this means that the protein rarely falls off of a microtubule since one subunit is always attached. The word processive is a term used to describe how long something is likely to continue once it gets started
microtubule motors are highly processive but myosin is not.
Myosin motor proteins, in contrast, spend very little time attached the actin filament. Therefore, they generate all of their force in a very short time. That makes myosin motors good for rapid movements.

Question 39

Organelles that need to be in the center of a cell would attach themselves to microtubules using which motor protein? What about the periphery of the cell?

Answer 39

Center of cell using dyneins
Periphery of cell using kinesin
Kinesins oppose dyneins. Both are involved in directed movements (distribution of mitochondria, endoplasmic reticulum, secretory vesicles, pigment granules.)

Question 40

What are the two motor proteins for microtubules? what do they do?

Answer 40

Kinesin and dyneins and they transport organelles, chromosomes and other cargoes along microtubules.

Question 41

What happens if the addition of GTP subunits slows down at the end of microtubules?

Answer 41

the subunits at the end of the microtubule hydrolyze their GTP and become GDPbound subunits and at that point the subunits have weakened interactions with other subunits an the protofilaments begin to splay out from each other

Question 42

What happens when MAPs are phosphorylated?

Answer 42

Phosphorylation of MAPs

causes them to fall off microtubules resulting in less stable microtubules.

Question 43

What does GTP-tubulin cap do? what happens when it dissociates from microtubules end?

Answer 43

GTP cap protect growing microtubules from disassembling. Loss of cap causes conformational change and microtubule disassembly. Can lead to catastrophe.