Microtubules stable

The microtubule-associated proteins MAP2 and tau both have two separate functional regions (Lewis et al., 1989). One is the microtubule-binding site, which nucleates microtubule assembly and controls the rate of elongation (by slowing the rate of assembly). The second functional domain shared by MAP2 and tau is a short C-terminal a-helical sequence that can cross-link microtubules into bundles by self-interaction. This domain has some of the properties of a leucine zipper. Likely it is responsible for the organization of microtubules into dense stable parallel arrays in axons and dendrites (Lewis et al., 1989). 

An intracellular fibrous system exists of filaments with an axial periodicity of 21 nm and a diameter of 8-10 nm that is intermediate between that of microfilaments (6 nm) and microtubules (23 nm). Four classes of intermediate filaments are found, as indicated in Table 49-13. They are all elongated, fibrous molecules, with a central rod domain, an amino terminal head, and a carboxyl terminal tail. They form a structure like a rope, and the mature filaments are composed of tetramers packed together in a helical manner. They are important structural components of cells, and most are relatively stable components of the cytoskeleton, not undergoing rapid assembly and disassembly and not... 

Although intermediate filaments are not universally associated with the cytoskeleton, neutrophils possess intermediate filaments of the vimentin type. Vimentin is a rod-shaped molecule of relative molecular mass 57 kDa that readily polymerises under physiological conditions to produce stable filaments 10-12 nm in diameter. Intermediate filaments are more robust than microfilaments and microtubules, and in neutrophils they form an open network of single filaments in the perinuclear space. 

Taxol i is a naturally occurring substance isolated from the Pacific yew tree Taxus brevifolia), which has been approved for clinical treatment of cancer patients. Taxol enhances polymerization of tubuhn and the consequent formation of stable microtubules, inhibiting cellular mitosis. 

Biain adenosinetriphosphatase This enzymatic activity is persistendy associated with brain micFotubules even after multiple cycles of warm-induced microtubule assembly, centrifugation to separate protomer and polymer, cold-induced disassembly, and subsequent centrifugation to remove cold-stable aggregates (White et aL, 1980). The enzyme hydrolyzes boA GTP and ATP, and recent work by Tominaga and Kaziro (1982) indicates that there are two distinct ATP-ases, one that is of low M, (around 33,000) and tubulin dependent in the presence of calcium ion, and the other of larger size and associated with membrane vesicles... 

Finally, one should recognize that determinations of the critical concentration depend wholly on the validity of the equilibrium or steady-state assumptions. If a stable end point for prdtomer-polymer coexistence is not attained, then kinetic factors affect the observed behavior. With the well recognized tendency of tubulin to lose its ability to engage in assembly reactions upon storage even at low temperature, and with the presence of various nucleotide hydrolases and transphosphorylases in microtubule protein, such kinetic effects are a serious problem. 

Free protein monomers of intermediate filaments rarely occur in the cytoplasm, in contrast to microfilaments and microtubules. Their polymerization leads to stable polymers that have no polarity. 

The principal cytoskeletal proteins in non-muscle cells are actin, tubulin, and the components of intermediate filaments. Actin can exist either as monomers ( G-actin ) or polymerized into 70 A diameter double filament ( F-actin ). Polymerized actin usually is localized at the margins of the cells, linked by other proteins to the cell membrane. In contrast, tubulin forms hollow filaments, approximately 250 A in diameter, that are distributed within a cell in association, generally, with cell organelles. Stabilized microtubule structures are found in the flagella and cilia of eucaryotic cells however, in other instances - examples being the mitotic apparatus and the cytoskeletal elements arising in directed cell locomotion - the microtubules are temporal entities. Intermediate filaments, which are composed of keratin-like proteins, are approximately 100 A thick and form stable structural elements that impart rigidity, for example, to nerve axons and epithelial cells. 

The threshold concentration of monomer that must be exceeded for any observable polymer formation in a self-assembling system. In the context of Oosawa s condensation-equilibrium model for protein polymerization, the cooperativity of nucleation and the intrinsic thermodynamic instability of nuclei contribute to the sudden onset of polymer formation as the monomer concentration reaches and exceeds the critical concentration. Condensation-equilibrium processes that exhibit critical concentration behavior in vitro include F-actin formation from G-actin, microtubule self-assembly from tubulin, and fibril formation from amyloid P protein. Critical concentration behavior will also occur in indefinite isodesmic polymerization reactions that involve a stable template. One example is the elongation of microtubules from centrosomes, basal bodies, or axonemes. 

Figure 1. Top Turbidity, measured at 350 nm, as a function of microtubule polymer mass concentration (expressed as mg/mL polymerized tubulin). Tubulin solutions of varying concentrations were polymerized until they reached stable plateau values in a Cary 118C spectrophotometer. Each sample was then transferred to an ultracentrifuge tube, and microtubules were pelleted, separated from the unpolymerized tubulin in the supernatant fraction, and then resuspended for protein concentration determination. The corresponding turbidity and polymer mass concentrations are plotted here. Bottom Time-course of tubulin polymerization assayed by turbidity.Repro-duced from MacNeal and Purich with permission from the American Society for Biochemistry and Molecular Biology.

Johnson and Borisy first showed that the lag phase in the plot of turbidity (i.e., polymer weight concentration) versus time accounted for only 5—10% of the entire amplitude obtained upon completion of the polymerization process. By fitting the elongation phase to a single exponential process, these investigators arrived at the correct conclusion that microtubule number concentration becomes relatively stable within the first minutes... 

Benzimidazoles are highly selective. Oomycete fungi are insensitive to the benzimidazoles, as are higher plants. The reason for the distinct differences in sensitivity is unknown but probably depends on single structural differences of the microtubule binding site. Resistance of this type, if stable, spreads rapidly and results in catastrophic disease control... 

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... 

Isolated microtubules always contain small amounts of larger 300-kDa microtubule-associated proteins (MAPS).330 These elongated molecules may in part lie in the grooves between the tubulin subunits and in part be extended outward to form a low-density layer around the tubule.283 309 Nerve cells that contain stable microtubules have associated stabilizing proteins.331 A family of proteins formed by differential splicing of mRNA are known as tau. The tau proteins are prominent components of the cytoskeleton of neurons. They not only interact with microtubules but also undergo reversible phosphorylation. Hyperphosphorylated tau is the primary component of the paired helical filaments found in the brains of persons with Alzheimer disease.330... 

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