Microtubule Cytoskeleton

Front 1

α-β Tubulin Dimer Structure

Back 1

Alpha and beta Tubulin bind GTP. The GTP bound to alpha tubulin is“locked in” between the dimer and is not exchanged or hydrolyzed. The GTP boundto beta tubulin is exchangeable and can be hydrolyzed. NOTE: actin binds ATP,tubulin bind GTP.

Front 2

MicrotubuleAssembly

Back 2

Tubulin dimers are the individualpieces that are assembled to make microtubules. Alpha and beta tubulin existonly as dimers in the cell. Microtubules have a plus and minusend (they are polarized). Polymerization and depolymerization canoccur at either end in vitro. In cells, polymerization occurs only at the plusend (they are polarized).13 protofilaments make up a microtubule.

Front 3

Microtubule Assembly

Back 3

Like actin, microtubules…

•are polarized

•extend from the plus end in cellsBut unlike actin, microtubules …

•are more stiff

•exhibit dynamic instability andless treadmilling•contain subunits that bind GTP/GDP

Dynamic instability is that theplus ends can both depolymerize or polymerize, depending on conditions in thecell. This makes it harder to control microtubule length.

Front 4

Protofilament

Back 4

Microtubules are made by assemblingtubulin dimers into a filamentcalled a protofilament. 13protofilaments associate laterally to make a microtubule with anempty centralcore. Note, the protofilament is not assembledfirst, rather alpha and beta heterodimers are added to each protofilament sothey grow together.pository0 *H

Front 5

Microtubule Assembly in vitro

Back 5

Microtubule assembly in vitro. Note the timescale…it takes ten minutes before nucleation occurs spontaneously! Inside of acell, the conditions are not favorable for spontaneous nucleation ofmicrotubules. We will see that cell will keep careful control of where and whenmicrotubules nucleate.

Front 6

Gamma Tubulin

Back 6

Gamma tubulin is the protein thatdrives microtubule nucleation, but is not required for polymerization.

Front 7

Dynamicinstability: tubulin as GTPase

Back 7

Microtubulesneed to be dynamic in living cells so that they can change. Otherwise, once youmade a microtubule, it would be very difficult to disassemble. Once again,individual microtubule components are held together by weak non-covalentinteractions. When Beta tubulin is bound to GTP (remember that alpha tubulinalways bound to GTP, but cannot exchange it and never hydrolyzes it), it favorsassembly into a microtubule. GDP-bound tubulin will assemble into a microtubuletoo, but the concentration has to be 100 times higher. Inside a cell, theconcentration of tubulin is intermediate such that GTP-bound tubulin favorassembly and GDP-bound tubulin favors disassembly.

Front 8

Dynamic Instability

Back 8

Microtubulesthat are capped with GTP-tubulin at the end are stabilized, even thoughGDP-tubulin may be found the entire length of the rest of the microtubule.Microtubules with GTP caps continue to have tubulin dimers added. If the cap islost, because the tubulins hydrolyze their GTP and GTP tubulin is not added,the microtubule is destabilized and begins to depolymerize from the plus end(this is called catastrophe). Return of the cap and re-initiation ofpolymerization is called rescue.ExtensionName":""}

Front 9

Organizing Centers

Back 9

Note thatmicrotubules all emanate from a single point in most cells, the microtubuleorganizing center. Microtubules are captured there by their minus ends and withtheir plus ends point away from the organizing center. Growth in the cell alloccurs at the plus end, as does most depolymerization.Microtubule length is controlled by polymerization or depolymerization atthe plus end, a process termed dynamics instability, that is controlled byother proteins. Some treadmilling ofmicrotubules also occurs.ţP*:

Front 10

Gamma (2)

Back 10

The third type of tubulin protein,gamma, is found in the MTOC. It initiates mictrotubulenucleation and tethers microtubules to the MTOC.Long microtubules are flexible and resemble spaghetti (cooked).

Front 11

MicrotubuleOrganizing Centers (MTOCs),or centrosomes

Back 11

In the MTOC, also called thecentrosome, are the centrioles. Two proteinaceousstructures arranged at normal angles toone another. These structures are duplicated during cell division, giving riseto two pairs of centrioles and two MTOCs that make the poles of the spindleapparatus. Thecentrioles are the organizing center and are surrounded by pericentriolar material. The gammatubulin occursin gammatubulin ring complexes that are arranged around the centrioles(light spots found around the centriolesin the pictures above) in the pericentriolar material and are the nucleation site ofmicrotubules.DNA (nucleic acid) and centrioles (protein)are the only two things in the cell where the copy number is preciselycontrolled. DNA and centrioles must be copied once, and only once, during thecell cycle. Making additional copies or insufficient copies is highly toxic.The mechanisms that regulate centriole duplication is still being worked out.

Front 12

Higherorder MT structures

Back 12

Higher order structures are morelimited formicrotubulescompared to actin.

Front 13

Microtubule-binding proteins modulatedynamics

Back 13

Just like actin filaments, thereare a number of proteins that stabilizeor destabilize microtubules. The proteins that interact with microtubules arecollectively called microtubule-associated proteins (MAPs). Catastrophe factorbind microtubules and is thought to pry them apart, lowering the energyrequired for disassembly.

Front 14

Different Micro-tubules

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You should be familiar with the gamma-tubulin ring complex(gamma-TuRC above), Kinesin-13, Katanin, MAPS, and XMAP215. Katanin is easy to remember (if you speakJapanese) as it the japaense word for sword (cuts microtubules).Thestathmin knockout mouse is a “fearless” mouse.This protein is abundant in the area of the brain responsible for our fearresponse and researchers are trying to see if natural polymorphisms in the genemake people more or less prone to experiencing fear.