Figure S1. In support of Figs. 1 and 2 :... | Rockefeller University Press

Figure S1.

$In support of Figs. 1 and 2: Measurement of concentration fold-change of Plk4 in overexpression lines. (A) Insertion of a fluorescent tag into Drosophila Plk4 endogenous locus. Schematic representation of the WT dmPlk4 locus (WT) and of the dmPlk4 locus after successful tag integration by homologous recombination (HR). A donor plasmid carrying the mNeonGreen reporter and a small linker (dark green) flanked by 1 kbp homology arms was used for homologous recombination. The UTRs are shown in gray, and the coding sequences are depicted in orange. The arrows indicate the position of the screening primers dmPLK4 5′UTR 3 FW and dmPLK4 1exon Rev, which are located outside the homology arms. The same strategy was used for mNeonGreen and GFP tags, generating two lines that were used at different parts of this paper. The GFP knock-in was used as WT control in measurement of Plk4 expression level. The inset shows the integration of a fluorescent tag into Plk4 endogenous locus (HR Plk4) by Western blot, causing a migration shift of the PCR product in the agarose gel compared with the untagged Plk4 locus (WT Plk4). (B) Western blot analysis of Plk4 concentration for endogenous expression and for overexpression constructs. Two (out of four) representative Western blots are shown. We emphasize that the detection of endogenous Plk4 with a Western blot approach is extremely challenging. In fact, most studies so far have only detected Plk4 by means of affinity-\ tag or fluorescent reporter and/or under an overexpression scenario. We were able to visualize the endogenous Plk4 tagged with mEGFP using αGFP antibody. Plk4 overexpression was visualized with a GFP–Plk4 overexpression (o.e.) construct, whose extract shows similar centriole biogenesis results as the nontagged Plk4 overexpression construct used in most parts of this work. We also made extract from flies overexpressing nondegradable (ND) Plk4, which accumulates in embryos and serves as a positive control (pUASp–ND-Plk4–EGFP; Cunha-Ferreira et al., 2013). WT embryos (w1118) were loaded as negative control as they do not have GFP-tagged protein. The black arrowhead points at GFP-tagged Plk4 constructs, while the white arrowhead points at an unspecific signal also present in WT embryos. We register 3.2 ± 1.9 times higher Plk4 concentration in the extract of embryos overexpressing Plk4 as compared with the WT (n = 4). Inter-experiment variability is largely due to systematic errors of Western blot quantification, but also due to the endogenous concentration of Plk4 being near the detection limit. Despite the variability, this quantitation is in line with our dilution results (Fig. 4 B); at 1/5 dilution of the Plk4-overexpressing extract, we detect very few de novo events, suggesting that with further dilution, the kinetics converges toward WT conditions where de novo events are not observed. Note that while the V32 driver for protein expression used in our experiments normally leads to high levels of protein expression, pUAS–GFP–Plk4 is likely being down-regulated through targeted degradation, in contrast to pUAS–ND-Plk4–GFP, which accumulates to higher levels. Ab, antibody; Chr., chromosome.$

In support of Figs. 1 and 2: Measurement of concentration fold-change of Plk4 in overexpression lines. (A) Insertion of a fluorescent tag into Drosophila Plk4 endogenous locus. Schematic representation of the WT dmPlk4 locus (WT) and of the dmPlk4 locus after successful tag integration by homologous recombination (HR). A donor plasmid carrying the mNeonGreen reporter and a small linker (dark green) flanked by 1 kbp homology arms was used for homologous recombination. The UTRs are shown in gray, and the coding sequences are depicted in orange. The arrows indicate the position of the screening primers dmPLK4 5′UTR 3 FW and dmPLK4 1exon Rev, which are located outside the homology arms. The same strategy was used for mNeonGreen and GFP tags, generating two lines that were used at different parts of this paper. The GFP knock-in was used as WT control in measurement of Plk4 expression level. The inset shows the integration of a fluorescent tag into Plk4 endogenous locus (HR Plk4) by Western blot, causing a migration shift of the PCR product in the agarose gel compared with the untagged Plk4 locus (WT Plk4). (B) Western blot analysis of Plk4 concentration for endogenous expression and for overexpression constructs. Two (out of four) representative Western blots are shown. We emphasize that the detection of endogenous Plk4 with a Western blot approach is extremely challenging. In fact, most studies so far have only detected Plk4 by means of affinity-\ tag or fluorescent reporter and/or under an overexpression scenario. We were able to visualize the endogenous Plk4 tagged with mEGFP using αGFP antibody. Plk4 overexpression was visualized with a GFP–Plk4 overexpression (o.e.) construct, whose extract shows similar centriole biogenesis results as the nontagged Plk4 overexpression construct used in most parts of this work. We also made extract from flies overexpressing nondegradable (ND) Plk4, which accumulates in embryos and serves as a positive control (pUASp–ND-Plk4–EGFP; Cunha-Ferreira et al., 2013). WT embryos (w¹¹¹⁸) were loaded as negative control as they do not have GFP-tagged protein. The black arrowhead points at GFP-tagged Plk4 constructs, while the white arrowhead points at an unspecific signal also present in WT embryos. We register 3.2 ± 1.9 times higher Plk4 concentration in the extract of embryos overexpressing Plk4 as compared with the WT (n = 4). Inter-experiment variability is largely due to systematic errors of Western blot quantification, but also due to the endogenous concentration of Plk4 being near the detection limit. Despite the variability, this quantitation is in line with our dilution results (Fig. 4 B); at 1/5 dilution of the Plk4-overexpressing extract, we detect very few de novo events, suggesting that with further dilution, the kinetics converges toward WT conditions where de novo events are not observed. Note that while the V32 driver for protein expression used in our experiments normally leads to high levels of protein expression, pUAS–GFP–Plk4 is likely being down-regulated through targeted degradation, in contrast to pUAS–ND-Plk4–GFP, which accumulates to higher levels. Ab, antibody; Chr., chromosome.