Microtubule nucleation is the event that initiates de novo formation of microtubules. These filaments of the cytoskeleton typically form through the polymerization of α- and β-tubulin dimers, the basic building blocks of the microtubule, which initially interact to nucleate a seed from which the filament can elongate.
Microtubule nucleation occurs spontaneously in vitro with solutions of purified tubulin giving rise to full-length polymers. The tubulin dimers that make up the polymers have an intrinsic capacity to self-aggregate and assemble into cylindrical tubes provided there is an adequate supply of GTP. The kinetics barriers of such a process however mean that the rate at which microtubules spontaneously nucleate is relatively low.
In vivo, cells get around this kinetic barrier by employing various proteins to aid and facilitate microtubule nucleation. The primary pathway by which microtubule nucleation is assisted requires the action of a third type of tubulin, γ-tubulin, which is distinct from the α and β subunits which compose the microtubules themselves. The γ-tubulin combines with several other associated proteins to form a conical structure known as the γ-tubulin ring complex (γ-TuRC). This complex, with its 13-fold symmetry, acts as a scaffold or template for α/β tubulin dimers during the nucleation process, speeding up the assembly of the ring of 13 protofilaments which make up the growing microtubule. The γ-TuRC also acts as a cap of the (−) end while the microtubule continues growth from its (+) end. This cap provides both stability and protection to the microtubule (-) end from enzymes which could lead to its depolymerization, while also inhibiting (-) end growth.
The γ-TuRC is typically found as the core functional unit in a microtubule organizing center (MTOC), such as the centrosome in animal cells or the spindle pole bodies in fungi and algae. The γ-TuRCs in the centrosome nucleate an array of microtubules in interphase which extend their (+)-ends radially outwards into the cytoplasm towards the periphery of the cell. Among its other functions, this radial array is used by microtubule-based motor proteins to transport various cargoes such as vesicles to the plasma membrane. In animal cells undergoing mitosis, a similar radial array is generated from two MTOCs called the spindle poles which produce the bipolar mitotic spindle. Some cells however, such as those of higher plants and oocytes, lack distinct MTOCs and microtubules are nucleated via a non-centrosomal pathway. Other cells, such as neurons, skeletal muscle cells, and epithelial cells, which do have MTOCs, possess arrays of microtubules not associated with a centrosome. These non-centrosomal microtubule arrays can take on various geometries such as those which give rise to the long, slender shape of myotubes, the fine protrusions of an axon, or the strongly polarized domains of an epithelial cell. It is thought that the microtubules in these arrays are generated first by the γ-TuRCs, then transported via motor proteins or treadmilling to their desired location, and finally stabilized in the needed configuration through the action of various anchoring and cross-linking proteins.
In the cortical array of plants as well as in the axons of neurons it is believed that microtubules are nucleated from existing microtubules via the action of severing enzymes such as katanin. Akin to the action of cofilin in generating actin filament arrays, the severing of microtubules by MAPs creates new (+) ends from which microtubules can grow. In this fashion dynamic arrays of microtubules can be generated without the aid of the γ-TuRC.
Recent studies using Xenopus egg extracts have identified a novel form of microtubule nucleation generating fan-like branching arrays in which new microtubules are grown at an angle off of older microtubules. It is suspected that involved in this process are non-centrosomal γ-TuRCs which bind to the sides of existing microtubules through the augmin complex. This method of microtubule-dependent microtubule nucleation leads to a rapid amplification in microtubule number and creates daughter microtubules which have the same polarity as the mother microtubules they branch off of. It is postulated that such a method could be important in the generation of the mitotic spindle.
Though the γ-TuRC is the primary protein cells turn to when faced with the task of nucleating microtubules, it is not the only protein to be postulated to act as a nucleation factor. Several other MAPs have been shown to assist the γ-TuRC with the nucleation process, while others have been shown to be able to nucleate microtubules independently of γ-TuRC. In the branching nucleation described above, the addition of TPX2 to the egg extracts led to a dramatic increase in nucleation events, while in other studies the protein XMAP215 has been shown in vitro to nucleate microtubule asters with its depletion in vivo reducing the nucleation potential of centrosomes. The microtubule-binding protein doublecortin has also been shown in vitro to nucleate microtubules, acting by binding to the side rather than the end of growing microtubules. Thus a family of proteins acting as nucleation factors may be present in cells, lowering, through various mechanisms, the energetic cost of nucleating microtubules.
Several proteins are involved in the formation of the γ-TuRC and the temporal and spatial control of microtubule nucleation. These include for example coiled-coil proteins with structural functions and regulatory proteins, such as components of the Ran cycle. NEDD1 recruits the γ-TuRC to the centrosome by binding to γ-tubulin.
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