Abstract
The formation of kinetochores shortly before each cell division is a prerequisite for proper chromosome segregation. The synchronous mitoses of Drosophila syncytial embryos have provided an ideal in vivo system to follow kinetochore assembly kinetics and so address the question of how kinetochore formation is regulated. We found that the nuclear exclusion of the Spc105/KNL1 protein during interphase prevents precocious assembly of the Mis12 complex. The nuclear import of Spc105 in early prophase and its immediate association with the Mis12 complex on centromeres are thus the first steps in kinetochore assembly. The cumulative kinetochore levels of Spc105 and Mis12 complex then determine the rate of Ndc80 complex recruitment commencing only after nuclear envelope breakdown. The carboxy-terminal part of Spc105 directs its nuclear import and is sufficient for the assembly of all core kinetochore components and CENP-C, when localized ectopically to centrosomes. Super-resolution microscopy shows that carboxy-terminus of Spc105 lies at the junction of the Mis12 and Ndc80 complexes on stretched kinetochores. Our study thus indicates that physical accessibility of kinetochore components plays a crucial role in the regulation of Drosophila kinetochore assembly and leads us to a model in which Spc105 is a licensing factor for its onset.
Highlights
The kinetochore is essential for chromosome segregation and has been highly conserved in structure, despite the divergence of its subunits in primary sequence [1,2,3,4]
Knowledge of the precise timing and kinetics of recruitment of individual KMN components to centromeres would greatly contribute to the understanding of mechanisms of kinetochore assembly and may identify key steps in coordinating this process
We generated transgenic flies that would express the KMN components, Mis12, Nsl1, Mitch/Spc25, Nuf2 or Spc105, each tagged with green fluorescent protein (GFP), and filmed the behaviour of these kinetochore components in the synchronous syncytial nuclear division cycles
Summary
The kinetochore is essential for chromosome segregation and has been highly conserved in structure, despite the divergence of its subunits in primary sequence [1,2,3,4]. One end of the complex interacts directly with the centromere [7,8], whereas the other forms a docking platform both for the Ndc complex and Spc105. The heterotetrameric Ndc complex comprises Ndc, Nuf, Spc and Spc, and has a long coiled-coil structure with globular domains at the two ends. The head domains of Spc and Spc interact directly with the Mis complex [10]. The tightly interacting Nuf and Ndc calponinhomology domains and the unstructured N-terminal tail of Ndc form the microtubule-binding interface [11]. Its C-terminal part interacts with the Mis complex [4], and the amino-terminal part is responsible for binding Bub and BubR1 [12] and PP1 phosphatase [13]
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