Microtubules and tubulin

K.J. Böhm, Leibniz Institute for Age Research - Fritz Lipmann Institute, Jena, Germany

Microtubules are obligate proteinaceous elements found in nearly all eukaryotic cells. In co-operation with other components of the cytoskeleton, namely with actin microfilaments and intermediate filaments, microtubules are involved in several basic cellular processes as segregation of genetic material, intracellular transport, maintenance of cell shape, positioning of cell organelles, extracellular transport by means of cilia, and movement of cells by means of flagella and cilia. Irreversible elimination of microtubules causes cell death.

Microtubules can be characterized as long hollow cylinders with outer diameters between 20 nm and 30 nm and an inner diameter of about 14 nm. The main constituent of microtubules is the tubulin, which is a globular protein. In the native state, it exists as a heterodimer of an alpha and a beta form. Both forms have a molecular mass of approximately 50 kDa and a diameter of about 4 - 5 nm.

 

 

Microtubules of mammalian cells disintegrate at temperatures below 10 °C. On the other hand, they reconstitute from tubulin in vitro at physiological temperature in the presence of GTP (at equimolar concentrations to tubulin) and magnesium ions.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Microtubule formation can be recorded by turbidity measurements at 360 nm using a spectrophotometer equipped with a temperature-controlled cuvette holder.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Assembly of tubulin in the presence of MAPs. Disassembly was induced after 30 min by shifting the temperature from 37° C to 2° C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

On the basis of the reversibility of cold-induced microtubule disassembly, tubulin can be easily purified by so-called temperature-dependent disassembly/reassembly cycles. This procedure was introduced by Shelanski et al. (1973) to obtain microtubule protein from brain tissue, which is known to be rich in tubulin. It includes the following preparation steps:

 

                -    cooling tissue homogenates/microtubule suspensions

                -    separation of non-microtubule material by high-speed centrifugation

                -    re-warming after co-factor addition (GTP, Mg2+)

                -    sedimentation of reassembled microtubules.

 

After three cycles, a pure preparation of tubulin is obtained, which is only accompanied by a set of microtubule-binding proteins, shortly called MAPs. For mammalian brain, two main groups of high molecular weight MAPs around 300 kDa (MAP1 and MAP2) and the group of tau proteins (between 55 and 70 kDa) are known.

       

 

MAPs are believed to stimulate the formation of microtubules and to stabilize them. The kind of MAPs bound to microtubules is tissue- or cell-specific and seems to be correlated with microtubule function.

The high molecular weight MAPs form about 100-nm long filamentous side projections on the microtubule surface.

Electron images: a - Microtubule formed from MAP-free tubulin; b- Microtubules formed from tubulin in the presence of MAPs (arrows)

 

 

 

Especially when whole-mount samples are viewed, the microtubules reveal longitudinal striations which represent the tubulin protofilaments.

These filamentous subunits can be described as chain-like association products of alpha and beta tubulin.

 

 

 

 

 

Because of the dimeric character of tubulin and the strong alternation of alpha and beta, one end of the protofilament is terminated by an alpha subunit and at the opposite one by a beta subunit. This provides the protofilament a certain kind of polarity. Within the microtubule, the protofilaments are associated laterally with same polarity. Therefore, the microtubule also appears as a polar structure with a plus and a minus end. Under conditions of steady state, at the plus end more dimers are added than lost and at the minus end more dimers are released than new ones re-associate. Polarity is a very important feature for microtubule functioning. It is the basic property for direction-dependent cellular events, e.g., vesicle transport.

            

 

 

 

 

 

The number of protofilaments (microtubule cross sections) observed in vivo ranges from 11 to 18, whereas most of the cytoplasmic microtubules of animal or plant tissues have 13 protofilaments. Eichenlaub-Ritter and Tucker demonstrated that in some ciliates the protofilament number changed during mitosis, suggesting that the protofilament number is a dynamic property which is obviously correlated with microtubule function.

 

 

 

Cross-sections of microtubules formed in vitro.

 

 

For more information see review: Unger E., Böhm K.J., Vater W.: Structural diversity and dynamics of microtubules and polymorphic tubulin assemblies. Electron Microsc. Rev. 3, 355-395
 


 

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