Microtubule Associated Proteins

What are the 4 classes of MAPs

1. Capping-proteins/Nucleators → stabilizers; there are minus-end capping proteins (gamma-TURC) and plus-end capping proteins

2. Depolymerizers → destabilizers; (+) TIPs (promote GTP hydrolysis), catastrophe factors (some kinesins), severing proteins (katanin), GTP-bound tubulin sequestering proteins

3. Cross-linkers → stabilizers; connect microtubules to other microtubules or to membrane of organelles/plasma membrane

4. Molecular motors → movers; utilize free energy from ATP hydrolysis to change conformation and “walk” along surface of microtubule; move vesicle/organelles, and other microtubules; plus-end directed motors (kinesins) and minus-end directed motors (dyneins)

What is the difference between capping-proteins and nucleators?

Capping-proteins stabilize the ends of microtubules by preventing depolymerization of subunits; nucleators promote growth of microtubules by eliminating the lag phase during polymerization; capping-proteins can change the Critical concentration of assembly, nucleators do NOT change Critical concentration of assembly

What is katanin?

A microtubule severing protein; when added to solution, it will cut microtubules, destabilizing them

Do all kinesins walk towards the plus-end of microtubules?

No, most do, but not all

What is the structure of kinesin?

It is a trimer composed of two heavy chains wrapped around each other and a light chain at one end; at the other end, each heavy chain balls up and forms a globular head

Which part of the kinesin binds to the cargo and which part does the walking?

The globular heads walk along the microtubule and the light chain end binds to the cargo

Which part of the microtubule do the kinesins walk along?

They only walk along the beta subunits

How do kinesins “walk” along microtubules?

When ATP is bound to head group, there is a tight association of kinesin with the microtubule; when ADP is bound to the head group, there is a weak association of kinesin with the microtubule; when one head group is ATP-bound, the other head group is ADP-bound and it swings forward, then becomes ATP-bound and attaches to the subsequent beta subunit and the previous head group hydrolyzes its ATP into ADP; each step forward moves about 16 nm; kinesins move at a speed of 0.8-2 microns/second

What is processivity?

A description of how far a molecule can travel before it detaches from its substrate; kinesins are very processive, meaning they can keep their heads on a microtubule for a distance of up to 1 micron

How were kinesins discovered?

They were discovered when researchers were discovering the axons of squids; researchers knew that going from end of axon to cell body was minus-end directed movement (dynein), but did not know what protein was responsible for the cell body to axon plus-end directed movement; they isolated the biologically active axoplasm and attempted to use the same techniques they used to isolate dynein to try and isolate the plus-end directed motor, but these techniques did not work; instead, they did cell fractionation, and put one drop of each fraction onto a glass slide, then placed microtubules onto the glass slides; the microtubules could be seen gliding along the glass slide in the opposite direction of their plus end (kinesin was bound to surface of glass slide)

How many active kinesin molecules do you need to move a microtubule in an in vitro gliding assay?

Only one

How are organelles moved in cells?

By motor MAPS

How are microtubules arranged in cells?

The plus-ends are near the edge of the cell and the minus-ends near center of cell

Which motor MAP moves organelles towards the edge of the cell?

Kinesin

Which motor MAP moves organelles towards the center of the cell?

Dynein

In a melanophore, under conditions of high cAMP, what will be observed? What about conditions of low cAMP?

Under high cAMP, he melanosomes will disperse and the cell will appear a certain color; under low cAMP, the melanosomes will aggregate and the cell will appear clear

Where do microtubules originate from in cells?

Microtubule organizing centers (MTOC), aka a centrosome (in animal cells)

What is the structure of a centrosome?

It is structured like a biomolecular condensate; the center of a centrosome has a pair of centrioles, which are very short triplet microtubules, 9 of which arrange in a circle; the two centrioles are perpendicular to one another; surrounding the pair of centrioles is a structured cloud of ~80 types of proteins, called the pericentriolar material; on the surface of this cloud, there are gamma-TURC ring complexes, and this is where the microtubules emenate from

Do plant cells have centrioles/centrosomes?

No

What is a gamma-TURC complex?

gamma-TURC complexes are nucleating sites for microtubules on centrosomes; gamma-tubulin is found at the minus end of microtubules; the gamma-TURC complex forms a platform that gamma-tubulin attaches to, and alpha and beta-tubulin dimers can then build off of this nucleating site to polymerize into microtubules

How can gamma-tubulin be separated from alpha- and beta-tubulin?

Using differential centrifugation because gamma-tubulin has a different S value than alpha and beta tubulin

Where do microtubules emanate from in cells?

The gamma-TURC complexes on centrosomes; NOT from the centrioles