[
International Worm Meeting,
2021]
Centrioles are microtubule-based cylindrical structures that recruit a proteinaceous matrix called pericentriolar matrix (PCM). Together these two entities constitute a centrosome that serves as a microtubule-organizing center. In mitotic cells, centrosome form the poles of the spindle and in some terminally differentiated cells, centrioles serve as basal bodies to template sensory or motile cilia. Maintaining proper centriole number is critically important for spindle assembly and cilia function and is achieved through a highly regulated duplication process that occurs once per cell cycle. Too many or too few centrioles manifest in various human diseases such as cancer, primordial dwarfism and primary microcephaly. Studies in C. elegans have identified the conserved proteins - SAS-7, SPD-2, ZYG-1, SAS-6, SAS-5 and SAS-4 as indispensable for centriole biogenesis. The kinase ZYG-1, a homolog of human Plk4, is considered the master regulator of centriole assembly but the identity of its critical substrates and the cascade that triggers centriole assembly in vivo are not completely understood. A major hurdle in addressing this and other questions is the difficulty in obtaining functional recombinant centriole proteins. Using a novel method, we were successful in obtaining sufficient amounts of recombinant full-length proteins from E. coli. Using these proteins, we could show that ZYG-1 phosphorylates SAS-5 extensively in vitro and that most of phosphorylated residues in SAS-5 were conserved in nematodes. Interestingly, a few of these residues formed a part of a larger highly conserved 25 amino acid motif. Surprisingly, we found that phosphorylation of the residues in this motif modulate phosphorylation elsewhere in SAS-5. We also show that this motif is required for both ZYG-1 and SAS-4 to bind SAS-5. Using phosphomimetic mutations within the motif, we find that a specific array of mutations dictates the choice of binding partner. Our results indicate that phosphorylation of this region aids in establishing a mutually exclusive interaction pattern of SAS-5 with either ZYG-1 or SAS-4. We have also used CRISPR/Cas9 to systematically analyze the effect of knocking out phosphorylation sites in endogenous SAS-5 and have found that while many of these mutation impact centriole assembly, none of them complete block this process. Our work suggests that ZYG-1 does not regulate SAS-5 through a single phosphorylation event but rather controls multiple functions of SAS-5 that collectively are required to ensure the formation of a new centriole.
[
International Worm Meeting,
2019]
Centrioles are microtubule-based organelles that aid in bipolar spindle formation and serve as basal bodies for nucleating cilia. Centriole number is dependent upon a tightly controlled duplication event in dividing cells that happens only once each cell cycle and misregulation of this process has been linked to diseases such as cancer and primary microcephaly. Studies in C. elegans have identified a set of five core conserved centriole proteins: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. The overall theme of centriole assembly is conserved across genera, with only minor variations. Although general details of the assembly process have been worked out, several aspects are still not completely understood. The factors that trigger centriole duplication, the critical substrates of the kinase ZYG-1, and the importance of substrate phosphorylation are some of the intense areas of research. Unfortunately, difficulty in recombinant expression and purification of centriolar proteins has hampered in vitro studies designed to address these issues. Here we report that we have managed to express abundant levels of the proteins in E. coli. Although each protein was initially insoluble, we could efficiently refold them in vitro. Based on circular dichroism measurements and functional assays the proteins had refolded properly and are stable on long-term storage. Of particular interest, we discovered an in vitro interaction between ZYG-1 and SAS-5 and mapped the regions necessary for their binding. We found that ZYG-1 could phosphorylate SAS-5. In vitro, SAS-5 was phosphorylated both in the N- and C-terminal regions, a distinctly different pattern than those observed in mammalian and fly SAS-5 homologs. Most of the residues phosphorylated in SAS-5 were found to be conserved among Caenorhabditis species and more distantly related nematodes. In the N-terminus, these Ser/Thr residues formed the part of a larger conserved motif. Surprisingly, this motif is essential for both SAS-4 and ZYG-1-binding to SAS-5 in vitro. Specific phospho-mimetic mutants of SAS-5 display inverse relationships with respect to SAS-4 and ZYG-1 binding in vitro. CRISPR/Cas9-mediated mutagenesis of a few of these residues resulted in embryonic lethality, suggesting that these residues may play a critical role in centriole assembly. We hypothesize that distinct phosphorylations in SAS-5 may result in altered specificity in vivo, which may be important both for centriole biogenesis and regulation.
Smith, Amy, Sankaralingam, Prabhu, Kropp, Peter, O'Connell, Kevin, Bowerman, Bruce, Guargliardo, Sarah, Bergwell, Mary, Gentry, Lindsey, Liu, Yan, Iyer, Jyoti
[
International Worm Meeting,
2021]
Centrioles are submicron-scale, barrel-shaped organelles typically found in pairs, and play important roles in ciliogenesis and bipolar spindle assembly. In general, successful execution of centriole-dependent processes is highly reliant on the ability of the cell to stringently control centriole number. This in turn is mainly achieved through the precise duplication of centrioles during each S phase. Aberrations in centriole duplication disrupt spindle assembly and cilia based signaling and have been linked to cancer, primary microcephaly and a variety of growth disorders. Studies aimed at understanding how centriole duplication is controlled have mainly focused on the post-translation regulation of two key components of this pathway: the master regulatory kinase ZYG-1/Plk4 and the scaffold component SAS-6. In contrast, how transcriptional control mechanisms might contribute to this process have not been well explored. Here we show that the chromatin remodeling protein CHD-1 contributes to the regulation of centriole duplication in the C. elegans embryo. Specifically, we find that loss of CHD-1 or inactivation of its ATPase activity can restore embryonic viability and centriole duplication to a strain expressing insufficient ZYG-1 activity. Interestingly, loss of CHD-1 is associated with increases in the levels of two ZYG-1-binding partners: SPD-2, the centriole receptor for ZYG-1 and SAS-6. Finally, we explore transcriptional regulatory networks governing centriole duplication and find that CHD-1 and a second transcription factor, EFL-1/DPL-1 cooperate to down regulate expression of CDK-2, which in term promotes SAS-6 protein levels. Disruption of this transcriptional regulatory network results in the production of extra centrioles.