RESUMEN
Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , División Celular , Ciclina B1 , Microtúbulos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras , Ciclina B1/genética , Ciclina B1/metabolismo , Microtúbulos/metabolismo , Mitosis/genéticaRESUMEN
To ensure an even segregation of chromosomes during somatic cell division, eukaryotes rely on mitotic spindles. Here, we measured prime characteristics of the Arabidopsis mitotic spindle and built a three-dimensional dynamic model using Cytosim. We identified the cell-cycle regulator CYCLIN-DEPENDENT KINASE B1 (CDKB1) together with its cyclin partner CYCB3;1 as key regulators of spindle morphology in Arabidopsis. We found that the augmin component ENDOSPERM DEFECTIVE1 (EDE1) is a substrate of the CDKB1;1-CYCB3;1 complex. A non-phosphorylatable mutant rescue of ede1 resembled the spindle phenotypes of cycb3;1 and cdkb1 mutants and the protein associated less efficiently with spindle microtubules. Accordingly, reducing the level of augmin in simulations recapitulated the phenotypes observed in the mutants. Our findings emphasize the importance of cell-cycle-dependent phospho-control of the mitotic spindle in plant cells and support the validity of our model as a framework for the exploration of mechanisms controlling the organization of the eukaryotic spindle.
RESUMEN
A fair amount of research on microtubules since their discovery in 1963 has focused on their dynamic tips. In contrast, the microtubule lattice was long believed to be highly regular and static, and consequently received far less attention. Yet, as it turned out, the microtubule lattice is neither as regular, nor as static as previously believed: structural studies uncovered the remarkable wealth of different conformations the lattice can accommodate. In the last decade, the microtubule lattice was shown to be labile and to spontaneously undergo renovation, a phenomenon that is intimately linked to structural defects and was called "microtubule self-repair". Following this breakthrough discovery, further recent research provided a deeper understanding of the lattice self-repair mechanism, which we review here. Instrumental to these discoveries were in vitro microtubule reconstitution assays, in which microtubules are grown from the minimal components required for their dynamics. In this review, we propose a shift from the term "lattice self-repair" to "lattice dynamics", since this phenomenon is an inherent property of microtubules and can happen without microtubule damage. We focus on how in vitro microtubule reconstitution assays helped us learn (1) which types of structural variations microtubules display, (2) how these structural variations influence lattice dynamics and microtubule damage caused by mechanical stress, (3) how lattice dynamics impact tip dynamics, and (4) how microtubule-associated proteins (MAPs) can play a role in structuring the lattice. Finally, we discuss the unanswered questions about lattice dynamics and how technical advances will help us tackle these questions.