- Award ID(s):
- 1734030
- NSF-PAR ID:
- 10099881
- Date Published:
- Journal Name:
- eLife
- Volume:
- 8
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.more » « less
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Contents Summary I. Introduction II. MT arrays in plant cells III. γ‐Tubulin and MT nucleation IV. MT nucleation sites or flexible MTOCs in plant cells V. MT‐dependent MT nucleation VI. Generating new MTs for spindle assembly VII. Generation of MTs for phragmoplast expansion during cytokinesis VIII. MT generation for the cortical MT array IX. MT nucleation: looking forward Acknowledgements References Summary Cytoskeletal microtubules (
MT s) have a multitude of functions including intracellular distribution of molecules and organelles, cell morphogenesis, as well as segregation of the genetic material and separation of the cytoplasm during cell division among eukaryotic organisms. In response to internal and external cues, eukaryotic cells remodel theirMT network in a regulated manner in order to assemble physiologically important arrays for cell growth, cell proliferation, or for cells to cope with biotic or abiotic stresses. Nucleation of newMT s is a critical step forMT remodeling. Although many key factors contributing toMT nucleation and organization are well conserved in different kingdoms, the centrosome, representing the most prominent microtubule organizing centers (MTOC s), disappeared during plant evolution as angiosperms lack the structure. Instead, flexibleMTOC s may emerge on the plasma membrane, the nuclear envelope, and even organelles depending on types of cells and organisms and/or physiological conditions.MT ‐dependentMT nucleation is particularly noticeable in plant cells because it accounts for the primary source ofMT generation for assembling spindle, phragmoplast, and cortical arrays when the γ‐tubulin ring complex is anchored and activated by the augmin complex. It is intriguing what proteins are associated with plant‐specificMTOC s and how plant cells activate or inactivateMT nucleation activities in spatiotemporally regulated manners. -
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