TY  - GEN
T1  - Tracking of centriole inheritance in C. elegans
AU  - Erpf, Anna C.
AU  - Mikeladze-Dvali, Tamara
DO  - 10.17912/micropub.biology.000256
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000256/
AB  - The nematode C. elegans possesses a relatively small subset of centrosome proteins and hence, has emerged as an important model system in elucidating mechanisms of centrosome biogenesis and dynamics. The most basic factors of the centrosome assembly pathway were discovered and characterized in the worm. The centrosome consists of a pair of centrioles, surrounded by the pericentriolar material, and its duplication is strictly coupled to the cell cycle. In worms, the centriole duplication pathway comprises the protein SPD-2Cep192, which recruits the kinase ZYG-1PLK4 to centrioles to initiate centriole assembly (O’Connell et al., 2001; Kemp et al., 2004; Pelletier et al., 2004). ZYG-1PLK4 in turn, recruits SAS-6hsSAS6, which in complex with SAS-5STIL triggers the formation of the central tube (Dammermann et al., 2004; Delattre et al., 2004; Leidel et al., 2005; Kitagawa et al., 2009; Qiao et al. 2012; Hilbert et al., 2013; Lettman et al., 2013; Rogala et al., 2015). Subsequently, the coiled-coil protein SAS-4CPAP is stably incorporated into the centriole wall and assembles singlet microtubules around the forming centriole (Kirkham et al., 2003; Leidel and Gönczy, 2003, Balestra et al., 2015). SAS-7Cep295 is required for paddlewheel structure formation and recruits SPD-2Cep192 for a new round of centriole formation in the following cell cycle (Chang et al., 2016; Saurya et al., 2016; Sugioka et al., 2017). Due to the nature of the duplication of centrioles, there is always one older and one younger centriole present in a centriolar pair. Hence, in the subsequent cell division, daughter cells will inherit centrosomes carrying mother centrioles of different ages. Studies in Drosophila melanogaster and mammals have shown that mother centrosome and daughter centrosome can be segregated in a non-random manner during stem cell divisions and that this segregation pattern correlates with the fates of the daughter cells (Yamashita et al., 2007; Wang et al., 2009; Conduit et al., 2010; Januschke et al., 2011). Due to the invariant cell lineage of C. elegans and the possibility to follow individual cell fates, tracking centriole inheritance in regard to their age in worms could provide valuable information about the impact of centriole age on differentiation (Sulston and Schierenberg, 1983). However, factors that localize to one of the two centrioles in an age-dependent manner have not been identified in C. elegans to date, making it impossible to distinguish older from younger centrosomes in worms. To overcome the limitation of following age-related centrosome inheritance in C. elegans, we generated a strain in which centrioles are labeled with a photo-switchable marker. Once centrioles are photo-converted, they can be tracked over several cell cycles, and centrosome age can be distinguished after the second round of duplication (Figure 1A). The photo-switchable fluorescent protein Dendra originates from the octocoral Dendronephthya sp. The protein can be irreversibly converted from a green-to-red fluorescence state by exposure to visible blue or ultraviolet light. In this study, we made use of the bright and fast-maturing Dendra2 version of the protein, which was fused to the centriolar protein SAS-4CPAP and expressed under endogenous regulatory sequences (Figure 1 B, Gurskaya et al., 2006, Ihara et al., 2011). SAS-4CPAP is stably incorporated into centrioles and shows no cytoplasmic exchange once centrioles are formed (Dammermann et al., 2004, Balestra et al., 2015). We did not notice any significant difference in embryonic lethality of the strain at 25°C. On average we found 1.4% embryonic lethality for sas-4p::dendra2::sas-4 (n=1322 embryos); in comparison to 1.1% for a wild-type control strain (n=1692 embryos). In this study, we show that the older and younger centrosome can be successfully distinguished within cells of different lineages in C. elegans (Figure 1 C).
PY  - 2020
JO  - microPublication Biology
ER  -