Abstract
Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.
Highlights
Most eukaryotes harbor microtubule-based cylindrical organelles called centrioles that exhibit a striking ninefold radial symmetry, and which are crucial for a wide range of cellular functions
Characterization of such purified SAS-5FL using circular dichroism (CD) revealed the presence of protein aggregates (Figure 1—figure supplement 2A,B), a conclusion supported by size-exclusion chromatography multi-angle light scattering (SEC-MALS, Figure 1—figure supplement 2C) and negative-stain electron microscopy (Figure 1—figure supplement 2D)
Excising this region from untagged SAS-5FL (SAS-5Δ282–295) or replacing it with a Gly-Ser-Ala-rich flexible linker of equal length (SAS-5FLEX) resulted in proteins with CD spectra characteristic of mainly α-helical proteins (Figure 1B). These results suggest that the predicted β-strand between residues 282–295 of SAS-5 drives formation of large protein aggregates
Summary
Most eukaryotes harbor microtubule-based cylindrical organelles called centrioles that exhibit a striking ninefold radial symmetry, and which are crucial for a wide range of cellular functions (reviewed in Gonczy, 2012; Agircan et al, 2014). Centrioles are usually found near the plasma membrane where they organize the formation of flagella and cilia, whereas in proliferating cells centrioles typically reside adjacent to the nucleus, where they recruit pericentriolar material to form the centrosome, the major microtubule organizing center of animal cells. Centrosomes play a major role in directing cellular architecture during interphase and bipolar spindle assembly during mitosis. Centriole numbers are tightly regulated, with centriole duplication occurring only once per cell cycle, in concert with replication of the genetic material (reviewed in Firat-Karalar and Stearns, 2014). Abnormalities in centriole formation can impair cell signaling and motility owing to defective cilia or flagella, as well as cause spindle positioning defects and genome instability due to aberrations in centrosome numbers and/or sizes. It is not surprising that centriolar defects are at the root of multiple medical conditions, including primary microcephaly, male sterility and possibly cancer (reviewed in Nigg and Raff, 2009; Arquint et al, 2014; Chavali et al, 2014; Godinho and Pellman, 2014; and Nachury, 2014)
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