Formation of a new centriole adjacent to a pre-existing centriole occurs only once per cell cycle. Despite being crucial for genome integrity, the mechanisms controlling centriole biogenesis remain elusive. Here, we identify RBM14 as a novel suppressor of assembly of centriolar protein complexes. Depletion of RBM14 in human cells induces ectopic formation of centriolar protein complexes through function of the STIL/CPAP complex. Intriguingly, the formation of such structures seems not to require the cartwheel structure that normally acts as a scaffold for centriole formation, whereas they can retain pericentriolar material and microtubule nucleation activity. Moreover, we find that, upon RBM14 depletion, a part of the ectopic centriolar protein complexes in turn assemble into structures more akin to centrioles, presumably by incorporating HsSAS-6, a cartwheel component, and cause multipolar spindle formation. We further demonstrate that such structures assemble in the cytoplasm even in the presence of pre-existing centrioles. This study sheds light on the possibility that ectopic formation of aberrant structures related to centrioles may contribute to genome instability and tumorigenesis.
At the onset of procentriole formation, a structure called the cartwheel is formed adjacent to the pre-existing centriole. SAS-6 proteins are thought to constitute the hub of the cartwheel structure. However, the exact function of the cartwheel in the process of centriole formation has not been well characterized. In this study, we focused on the functions of human SAS-6 (HsSAS-6, also known as SASS6). By using an in vitro reconstitution system with recombinant HsSAS-6, we first observed its conserved molecular property of forming the central part of the cartwheel structure. Furthermore, we uncovered critical functions of HsSAS-6 by using a combination of an auxin-inducible HsSAS-6-degron (AID) system and super-resolution microscopy in human cells. Our results demonstrate that the HsSAS-6 is required not only for the initiation of centriole formation, but also for the stabilization of centriole intermediates. Moreover, after procentriole formation, HsSAS-6 is necessary for limiting Plk4 accumulation at the centrioles and thereby suppressing the formation of initiation sites that would otherwise promote the development of extra procentrioles. Overall, these findings illustrate the conserved and fundamental functions of the cartwheel in centriole duplication.
Highlights d Microtubule arrays at the mid half-spindle are compliant and fluid-like d Microtubule arrays near the pole and the equator are robust against force d Spindle length change occurs via filament sliding at the mid half-spindle d Kinesin-5 and dynein are required to achieve the spindle's mechanical heterogeneity
As cells exit mitosis, the decision to commit to the next cell cycle is made during G1. Not only DNA replication, but also centriole duplication is initiated as cells enter the S-phase. The kinase NEK7 is required for the timely regulation of G1 progression, S-phase entry, and procentriole formation.
SummaryCentriole duplication occurs once per cell cycle through the assembly of daughter centrioles on the side wall of pre-existing centrioles. Little is known about the molecules involved in the assembly of new centrioles. Here, we identify CRC70 as a Chlamydomonas protein with an important role in the accumulation of centriole proteins at the site of assembly. CRC70 contains a highly conserved ,50-aminoacid sequence shared by mammalian Cep70 and preferentially localizes to immature centrioles (the procentrioles). This localization is maintained in the mutant bld10, in which centriole formation is blocked before the assembly of centriolar microtubules. RNA interference (RNAi)-mediated knockdown of CRC70 produces flagella-less cells and inhibits the recruitment of other centriole components, such as SAS-6 and Bld10p to the centriole. Overexpression of CRC70 induces an accumulation of these proteins in discrete spots in the cytoplasm. Overexpression of EGFP-tagged CRC70 in mouse NIH3T3 cells causes the formation of structures apparently related to centrioles. These findings suggest that CRC70 is a member of a conserved protein family and functions as a scaffold for the assembly of the centriole precursor.
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