This transmission electron micrograph to the right shows the microtubules in longitudinal ultrathin section. Note, the tubulin molecules cannot be visualized in this preparation. Early electron microscopists found that in order to preserve microtubules, they had to fix the cells in glutaraldehyde at room temperature. Why do you think the
temperature conditions were important? What might happen if they fixed the cells for 30 min in the cold? |
The extensive distribution of microtubules can really be appreciated in the light microscope after immunolabeling for tubulin with fluorescein-labeled antibodies. This micrograph shows cells in culture labeled for tubulin. The labeling is so fine, the small microtubules can be delineated. |
Microtubule Formation
The first stage of formation is called "nucleation". The process requires tubulin, Mg++ and GTP and also proceeds at 37 C. This stage is relatively slow until the microtubule is initially formed. Then the second phase, called "elongation" proceeds much more rapidly. During "nucleation", an alpha and a beta tubulin molecule
join to form a heterodimer. Then these attach to other dimers to form oligomers
which elongate to form protofilaments. Each dimer
carries two GTP molecules. However the GTP that appears to function binds to the
beta tubulin molecules. When a tubulin molecule adds to the microtubule, the GTP is hydrolyzed to GDP. Eventually the oligomers will join to
form the ringed microtubule. The hydrolysis of GTP of
course is facilitated at a temperature of 37 C and stopped at temperatures of 4
C. This figure shows that, as the oligomers assemble, they form a series of
rings, 25 nm in diameter. In cross section, each ring consists of 13 beads. The
rows of beads in longitudinal section are called protofilaments. |
Tests have shown that microtubules will form normally with nonhydrolyzable GTP analog molecules attached. However,
they will not be able to depolymerize (see below). Thus, the normal role of GTP
hydrolysis may be to promote the constant growth of microtubules as they are
needed by a cell. |
Microtubules may vary in their rate of assembly and disassembly. Tubulin half life is nearly a full day, however, the half life of a given microtubule may be only 10 minutes. Thus, they are in a continued state of flux. This is believed to respond to the needs of the cell and is called "dynamic instability". Furthermore, there are regulatory processes that appear to control this in a cell. Microtubule growth would be promoted in a dividing or moving cell. However, microtubule growth would be more controlled in a stable, polarized cell. As described in your text, the cell can provide a GTP cap on
the growing end of a microtubule to regulate further growth. This happens when
the tubulin molecules are added faster than the GTP can be hydrolyzed. Thus, the
microtubule becomes stable and does not depolymerize. It may also be encouraged
to continue growing. Once the GTP is hydrolyzed, it begins to shrink, however.
Another way of capping a microtubule is to put a structure at its end, such as a
cell membrane. |
Furthermore, it is believed that some of these MAPs may bind to special sites on the alpha tubulin that form after it is in the microtubule. These are sites where a specific molecule is acetylated or the tyrosine residue is removed from the carboxy terminal. These sites are important marker sites for stabilized microtubules, because they disappear when microtubules are depolymerized. This figure shows a 3-D view of a neuron with its processes containing
microtubules. At higher magnifications, the vesicles are seen attached to MAPs
and moving along the microtubule conveyer belt. The MAPs include kinesins and dynein which "walk" along the microtubules in
opposite directions.The kinesins move the vesicle along
towards the plus end and dynein walks towards the minus end. In neurons, as the
microtubules grow from the cell body through the processes, the plus end is more
peripheral. These proteins have head regions that bind to microtubules and also
bind ATP. The head domains are thus ATPase motors. The tail domain binds to the
organelle to be moved. It is not known how the energy from ATP breakdown is
converted into vectorial transport. |
One can label beads with kinesin or dyneins and watch the direction of
movement in a cell at the light microscopic level. What would happen if the
beads were simply labeled with "cytoplasmic extract"? This cartoon shows the
motility process in vitro. The tubule is moving along a
negatively charged glass surface and the vesicle moves along the tubule.
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