Abstract
Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation.
Highlights
Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown
The major regulator of MT nucleation in vivo is the g-tubulin complex (g-TuC; in higher eukaryotes, called g-tubulin ring complex, or g-TuRC)[1,3,4,5,6], and understanding the regulation of g-TuC-dependent MT nucleation is critical to understanding MT organization from a mechanistic perspective
Possibly redundant, mechanisms have been suggested to contribute to cell cycle regulation of g-TuC localization, and many of these involve phosphorylation of structural proteins within the centrosome by cell cycle-dependent protein kinases such as CDK1, Plk[1] and Aurora A
Summary
Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the g-tubulin complex and recruits the g-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells This provides a molecular-level understanding of how phosphorylation ‘switches off’ microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation. Mechanisms that switch off MT nucleation may be of particular importance in cytoskeletal rearrangements that accompany cell differentiation—including muscle, neuronal and epithelial cell development[16,17,18,19,20,21] In many of these instances, noncentrosomal MTOCs (for example, Golgi apparatus, nuclear envelope (NE), or regions of plasma membrane) can nucleate MTs alongside, or instead of, the centrosome. During cytokinesis, MTOCs are redistributed to the contractile actomyosin ring, to form a post-anaphase array of MTs24
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