Microtubules labile

The induction of a change in one protein by interaction with another protein is a phenomenon that is met also in the construction of microtubules, ribosomes, cilia, and myofibrillar assemblies of muscle. It is basic to the assembly of the many labile but equally real cascade systems of protein-protein interactions such as that involved in the clotting of blood (Chapter 12) and signaling at membrane surfaces. 

A prominent component of cytoplasm consists of microtubules which appear under the electron microscope to have a diameter of 24 2 nm and a 13 - to 15-nm hollow core.307-310 However, the true diameter of a hydrated microtubule is about 30 nm and the microtubule may be further surrounded by a 5-20 nm low density layer of associated proteins. Microtubules are present in the most striking form in the flagella and cilia of eukaryotic cells (Fig. 1-8). The stable microtubules of cilia are integral components of the machinery causing their motion (Chapter 19). Labile microtubules, which form and then disappear, are often found in cytoplasm in which motion is taking place, for example, in the pseudopodia of the ameba. The mitotic spindle... 

Each tubulin dimer binds one molecule of GTP strongly in the a subunit and a second molecule of GTP or GDP more loosely in the P subunit. In this respect, tubulin resembles actin, whose subunits are about the same size. However, there is little sequence similarity. Labile microtubules of cytoplasm can be formed or disassembled very rapidly. GTP is essential for the fast growth of these microtubules and is hydrolyzed to GDP in the process.320 However, nonhydro-lyzable analogs of GTP, such as the one containing the linkage P-CH2-P between the terminal and central phosphorus atoms of the GTP, also support polymerization.321 Since microtubules have a distinct polarity, the two ends have different tubulin surfaces exposed, and polymerization and depolymerization can occur at different rates at the two ends. As a consequence, microtubules often grow at one end and disassemble... 

Microtubules with dynein arms or cytoskeletal microtubules to which cytosolic dynein or kinesin may attach dynein forms intertubule bridges during movement tubules are stable in cilia and flagella, but often labile in cytoplasm... 

Protocol 5.2 describes a methanol-acetone fixation technique (Pisano et al. 1993 Cenci et al. 1994) which results in very good preservation of cell morphology. Fixed cells viewed by phase-contrast optics exhibit most of the structural details that can be seen in live material. This allows analysis of unstained fixed preparations and selection of the most suitable ones for immunostaining. Remarkably, the Y loops, which are usually faint and labile in living preparations, become clearly apparent after this type of fixation. Moreover, this fixation protocol results in excellent microtubule preservation for immunostaining with antitubulin antibodies. The main disadvantage of this technique is a poor preservation of chromosome structure. In most instances, the chromosomes do not exhibit a distinct morphology and tend to coalesce into one or more masses of chromatin. 

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