Microtubule assembly, nucleation

Any highly cooperative chemical or physical process that serves to generate structures that then grow by linear accretion or elongation. Nucleation is an important phase in indefinite polymerization processes (e.g., actin filament and microtubule assembly). Nucleation is typically of a high kinetic order,... 

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). 

Although the self-assembly of polymeric structures can involve nucleation and elongation steps (See Actin Assembly Kinetics Microtubule Assembly Kinetics), one can simplify the assembly process through what is known as seeded assembly. At an initial monomer concentration [M], seeded assembly is induced by the addition of pre-assembled polymeric structures consequently the polymer number concentration must remain constant. The rate of monomer incorporation into indefinite length polymers can be written as follows ... 

ACTIN ASSEMBLY KINETICS BIOCHEMICAL SELF-ASSEMBLY BIOMINERALIZATION PRION PLAQUE FORMATION Nucleation as a highly cooperative process, MICROTUBULE ASSEMBLY KINETICS... 

Erickson, H.P., and Pantaloni, D. "The role of subunit entropy in co-operative assembly. Nucleation of microtubules and other two-dimensional polymers". Biophys. ]. 34, 293-309 (1981). 

Microtubule assembly in cells differs in some ways from assembly in vitro. In cells, nucleation of microtubules requires a third type of tubulin, which is called y-tubulin, that functions in concert with other proteins in the form of a y-tubulin ring complex. In most animal cells, the y-tubuIin ring complex is located at the pericentriolar region of the microtubule organizing center (or centrosome) where it nucleates microtubule assembly at the minus ends (7). The y-tubulin does not become incorporated into the microtubule, but rather it only localizes to the minus ends. Assembly of tubulin to form microtubules during the early stages of polymerization in vitro can be considered a pseudo first-order reaction. A steady state is eventually attained in which both the soluble tubulin concentration and the microtubule polymer mass attain stable plateaus (8). The critical concentration at apparent equilibrium (actually a steady state, see below) is the concentration of soluble tubulin in apparent equilibrium with the microtubule polymers. 

Figure 3 A typical microtubule assembly reaction is initiated by warming a solution of ice-cold tubulin dimers to 37°C in the presence of GTP. Tubulin dimers (adjacent white and gray circles) slowly form nucleating seeds (heptameric tubulin aggregate), which catalyze a rapid phase of microtubule elongation (growing microtubule) enroute to a steady state condition of microtubule formation and destruction. The assembly reaction is monitored by measuring the change in absorbance at 350 nm. In vitro incubation of microtubules with 2,5-HD or in vivo exposure of animals to 2,5-HD followed by tubulin purification yields pyrrolylated tubulin with altered assembly behavior. 2,5-HD-modified tubulin quickly forms numerous seeds, resulting in more rapid assembly into greater numbers of shorter microtubules compared to the control.

Sioussat Boekelheide (41) explored the biochemical nature of the 2,5-HD effect on tubulin in greater detail. Isolated bovine brain tubulin was treated with 100 mM 2,5-HD for 16 h at 37°C and subjected to three cycles of microtubule assembly with the assembly temperature of each cycle progressively lower, making microtubule assembly progressively restrictive. This procedure effectively concentrated a component of 2,5-HD-treated tubulin with strong nucleating features. 

Shu HB, Joshi HC. 1995. Gamma-tubulin can both nucleate microtubule assembly and self-assemble into novel tubular structures in mammalian cells. J. Cell Biol. 130 1137 17... 

This complex is thought to nucleate microtubule assembly by... 

TuRC in nucleating microtubule assembly. A model of y-TuRC based on electron microscopy shows y-tubulin in contact with the (—) end of a microtubule (Figure 20-15b). 

Because microtubule assembly Is nucleated from MTOCs, the (—) end of most microtubules Is adjacent to the MTOC and the (+) end Is distal (see Figure 20-14). 

Microtubules within a cell appear to be arranged In specific arrays. What cellular structure Is responsible for determining the arrangement of microtubules within a cell How many of these structures are found In a typical cell Describe how such structures serve to nucleate microtubule assembly. 

T) is shown in the bottom trace. In this experiment the solution conditions were adjusted to obtain a better separation of prenucleation events and assembly (e.g., lower pH and salt concentration, see [11]). Following the temperature jump there is a pronounced undershoot (best visible in the top curve of Fig. 4) which appears to level out before the onset of the rise due to microtubule assembly. The undershoot follows nearly linearly the temperature rise in other words, the observed rate does not reflect the intrinsic rate of oligomer dissolution, but rather a rapid temperature-dependent equilibrium. By contrast, microtubule assembly follows slowly, well after the temperature has reached 37 °C (for a more detailed analysis of this phase see [4, 5]. Other experiments show that the pre-nucleation phase occurs even in conditions where microtubule formation is inhibited, for example, in the absence of GTP, or... 

The polymer self-assembly theory of Oosawa and Kasai (1962) provides valuable insights into the nature of the nucleation process. The polymerization nucleus is considered to form by the accretion of protomers, but the process is highly cooperative and unfavorable. Indeed, this is strongly suggested by the observation that thousands of actin or tubulin protomers are found in F-actin and microtubule structures if nucleation of self-assembly were readily accomplished and highly favorable, the consequence would be that many more fibers of shorter polymer length would be observed. The Oosawa kinetic theory for nucleation permits one to obtain information about the size of the polymerization nucleus if two basic assumptions can be satisfied in the experimental system. First, the rate of nuclei formation is assumed to be proportional to the loth power of the protomer concentration with io representing the number of protomers required to create the nucleus. Second, the treat-... 

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