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Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP.

Conduit PT, Wainman A, Novak ZA, Weil TT, Raff JW - Elife (2015)

Bottom Line: In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl.We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4.Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Centrosomes have many important functions and comprise a 'mother' and 'daughter' centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic 'S-CAP' complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes.

No MeSH data available.


Two-colour-3D-SIM FRAP reveals that Sas-4-mCherry is recruited only to growing daughter centrioles, while PCM proteins are recruited only around mother centrioles.(A) 3D-SIM images show how during S-phase two adjacent Sas-4-GFP foci can be resolved at an individual centrosome (t = −20 s), and how the Sas-4-GFP fluorescence signal recovers only as a single foci after photobleaching (t = 120 s to t = 280 s). Time in seconds before and after photobleaching (t = 0 s) is shown in the top right of each panel. (B) Two-colour 3D-SIM images show how Sas-4-GFP fluorescence recovers relative to Asl-mCherry fluorescence (which surrounds the mother centriole). The Sas-4-GFP is shown in greyscale; insets (yellow dashed lines) display the overlay of Sas-4-GFP (green) and Asl-mCherry (red). Note how after photobleaching the Sas-4-GFP fluorescence recovers outside of the Asl-mCherry toroid. (C) Complementary images of a control centrosome adjacent to the one shown in (B) where the Sas-4-GFP signal was not photobleached. The t = 0 s panel is a widefield image (see ‘Materials and methods’). (D, E) Schematic (D) and box-plot (E) show how the average position of the post-bleached Sas-4-GFP signal is similar to the position of the daughter, but not the mother, centriole's prebleached Sas-4-GFP signal, relative to the Asl-mCherry signal. Boxes in E extend from the 25th-75th percentiles, whiskers extend from min to max values, lines in boxes are the median values, ‘+’ in boxes are the mean values; n = 25 centrosomes from 4 embryos. **** indicates where p < 0.0001; n.s. indicates where p = 0.09, and is therefore not significant. (F) 3D-SIM images show how Sas-4-mCherry (red) recovers relative to recovering Spd-2-GFP (green). An overlay of Sas-4-mCherry and Spd-2-GFP fluorescence is shown in the main panels; insets (yellow dashed lines) display the Sas-4-mCherry signal (greyscale). Note how after photobleaching the Sas-4-mCherry recovers outside of the hollow created by the recovering Spd-2-GFP (t = 300 s). (G) Complementary images of a control centrosome adjacent to the one in (F) where the Sas-4-mCherry and Spd-2-GFP signals were not photobleached. The t = 0 s image is a widefield image, as in (C).DOI:http://dx.doi.org/10.7554/eLife.08483.01010.7554/eLife.08483.011Figure 3—source data 1.Measuring the spatial offset between recovering Sas-4-GFP and Asl-mCherry at super resolution.This flie includes the raw data used to calculate the offset between the centroids of the two Sas-4-GFP foci and the single Asl-mCherry foci prior to photobleaching and the offset between the centroids of the single Sas-4-GFP foci and the single Asl-mCherry foci 5 min post-photobleaching. The data is used to plot the graph shown in Figure 3E.DOI:http://dx.doi.org/10.7554/eLife.08483.011
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fig3: Two-colour-3D-SIM FRAP reveals that Sas-4-mCherry is recruited only to growing daughter centrioles, while PCM proteins are recruited only around mother centrioles.(A) 3D-SIM images show how during S-phase two adjacent Sas-4-GFP foci can be resolved at an individual centrosome (t = −20 s), and how the Sas-4-GFP fluorescence signal recovers only as a single foci after photobleaching (t = 120 s to t = 280 s). Time in seconds before and after photobleaching (t = 0 s) is shown in the top right of each panel. (B) Two-colour 3D-SIM images show how Sas-4-GFP fluorescence recovers relative to Asl-mCherry fluorescence (which surrounds the mother centriole). The Sas-4-GFP is shown in greyscale; insets (yellow dashed lines) display the overlay of Sas-4-GFP (green) and Asl-mCherry (red). Note how after photobleaching the Sas-4-GFP fluorescence recovers outside of the Asl-mCherry toroid. (C) Complementary images of a control centrosome adjacent to the one shown in (B) where the Sas-4-GFP signal was not photobleached. The t = 0 s panel is a widefield image (see ‘Materials and methods’). (D, E) Schematic (D) and box-plot (E) show how the average position of the post-bleached Sas-4-GFP signal is similar to the position of the daughter, but not the mother, centriole's prebleached Sas-4-GFP signal, relative to the Asl-mCherry signal. Boxes in E extend from the 25th-75th percentiles, whiskers extend from min to max values, lines in boxes are the median values, ‘+’ in boxes are the mean values; n = 25 centrosomes from 4 embryos. **** indicates where p < 0.0001; n.s. indicates where p = 0.09, and is therefore not significant. (F) 3D-SIM images show how Sas-4-mCherry (red) recovers relative to recovering Spd-2-GFP (green). An overlay of Sas-4-mCherry and Spd-2-GFP fluorescence is shown in the main panels; insets (yellow dashed lines) display the Sas-4-mCherry signal (greyscale). Note how after photobleaching the Sas-4-mCherry recovers outside of the hollow created by the recovering Spd-2-GFP (t = 300 s). (G) Complementary images of a control centrosome adjacent to the one in (F) where the Sas-4-mCherry and Spd-2-GFP signals were not photobleached. The t = 0 s image is a widefield image, as in (C).DOI:http://dx.doi.org/10.7554/eLife.08483.01010.7554/eLife.08483.011Figure 3—source data 1.Measuring the spatial offset between recovering Sas-4-GFP and Asl-mCherry at super resolution.This flie includes the raw data used to calculate the offset between the centroids of the two Sas-4-GFP foci and the single Asl-mCherry foci prior to photobleaching and the offset between the centroids of the single Sas-4-GFP foci and the single Asl-mCherry foci 5 min post-photobleaching. The data is used to plot the graph shown in Figure 3E.DOI:http://dx.doi.org/10.7554/eLife.08483.011

Mentions: In order to directly test if Sas-4 molecules are only recruited to growing daughter centrioles, we turned to 3D-Structured Illumination Microscopy (3D-SIM), which has approximately twice the spatial resolution of standard confocal microscopy. Using 3D-SIM in living embryos, we could clearly distinguish two adjacent Sas-4-GFP foci at individual centrosomes during S-phase (Figure 3A, t = −20 s), presumably representing mother-daughter centriole pairs. We combined 3D-SIM with FRAP (Conduit et al., 2014b) and found that the Sas-4-GFP signal only recovered at a single foci (Figure 3A, t = 120 s to t = 280 s). We confirmed that this recovery occurred at the daughter centriole by performing a two-colour-3D-SIM-FRAP experiment in embryos co-expressing Sas-4-GFP and Asl-mCherry, as Asl forms a toroid around only the mother centrioles (Figure 3B, t = −30 s) (Novak et al., 2014). Strikingly, the recovering Sas-4-GFP fluorescence always lay outside of the Asl-mCherry toroid (Figure 3B, t = 300 s), whereas control unbleached centrosomes still contained two Sas-4-GFP foci, one of which lay inside the Asl-mCherry toroid (Figure 3C, t = 300 s). This suggested that new Sas-4 molecules are recruited only to the daughter centrioles. However, during acquisition centrosomes move in the x-y plane, and given that the green and red channels are acquired sequentially on this particular imaging system it is possible for the green and red signals to become misaligned. To be sure the recovering Sas-4-GFP signal represented the daughter centriole, rather than a mis-positioned mother centriole, we therefore measured the distance between the centre of the Asl-mCherry signal and the centres of the pre- and post-bleached Sas-4-GFP signals (Figure 3D; Figure 3—source data 1). This revealed that the average position of the post-bleached Sas-4-GFP signal closely matched the position of the daughter Sas-4-GFP signal, but not the mother Sas-4-GFP signal (Figure 3E), confirming that Sas-4 molecules are recruited only to daughter centrioles. As Asl molecules are known to turn over at the mother centriole at this stage in the cycle (Novak et al., 2014), their recruitment cannot occur as part of Sas-4 dependent S-CAP complexes.10.7554/eLife.08483.010Figure 3.Two-colour-3D-SIM FRAP reveals that Sas-4-mCherry is recruited only to growing daughter centrioles, while PCM proteins are recruited only around mother centrioles.


Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP.

Conduit PT, Wainman A, Novak ZA, Weil TT, Raff JW - Elife (2015)

Two-colour-3D-SIM FRAP reveals that Sas-4-mCherry is recruited only to growing daughter centrioles, while PCM proteins are recruited only around mother centrioles.(A) 3D-SIM images show how during S-phase two adjacent Sas-4-GFP foci can be resolved at an individual centrosome (t = −20 s), and how the Sas-4-GFP fluorescence signal recovers only as a single foci after photobleaching (t = 120 s to t = 280 s). Time in seconds before and after photobleaching (t = 0 s) is shown in the top right of each panel. (B) Two-colour 3D-SIM images show how Sas-4-GFP fluorescence recovers relative to Asl-mCherry fluorescence (which surrounds the mother centriole). The Sas-4-GFP is shown in greyscale; insets (yellow dashed lines) display the overlay of Sas-4-GFP (green) and Asl-mCherry (red). Note how after photobleaching the Sas-4-GFP fluorescence recovers outside of the Asl-mCherry toroid. (C) Complementary images of a control centrosome adjacent to the one shown in (B) where the Sas-4-GFP signal was not photobleached. The t = 0 s panel is a widefield image (see ‘Materials and methods’). (D, E) Schematic (D) and box-plot (E) show how the average position of the post-bleached Sas-4-GFP signal is similar to the position of the daughter, but not the mother, centriole's prebleached Sas-4-GFP signal, relative to the Asl-mCherry signal. Boxes in E extend from the 25th-75th percentiles, whiskers extend from min to max values, lines in boxes are the median values, ‘+’ in boxes are the mean values; n = 25 centrosomes from 4 embryos. **** indicates where p < 0.0001; n.s. indicates where p = 0.09, and is therefore not significant. (F) 3D-SIM images show how Sas-4-mCherry (red) recovers relative to recovering Spd-2-GFP (green). An overlay of Sas-4-mCherry and Spd-2-GFP fluorescence is shown in the main panels; insets (yellow dashed lines) display the Sas-4-mCherry signal (greyscale). Note how after photobleaching the Sas-4-mCherry recovers outside of the hollow created by the recovering Spd-2-GFP (t = 300 s). (G) Complementary images of a control centrosome adjacent to the one in (F) where the Sas-4-mCherry and Spd-2-GFP signals were not photobleached. The t = 0 s image is a widefield image, as in (C).DOI:http://dx.doi.org/10.7554/eLife.08483.01010.7554/eLife.08483.011Figure 3—source data 1.Measuring the spatial offset between recovering Sas-4-GFP and Asl-mCherry at super resolution.This flie includes the raw data used to calculate the offset between the centroids of the two Sas-4-GFP foci and the single Asl-mCherry foci prior to photobleaching and the offset between the centroids of the single Sas-4-GFP foci and the single Asl-mCherry foci 5 min post-photobleaching. The data is used to plot the graph shown in Figure 3E.DOI:http://dx.doi.org/10.7554/eLife.08483.011
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fig3: Two-colour-3D-SIM FRAP reveals that Sas-4-mCherry is recruited only to growing daughter centrioles, while PCM proteins are recruited only around mother centrioles.(A) 3D-SIM images show how during S-phase two adjacent Sas-4-GFP foci can be resolved at an individual centrosome (t = −20 s), and how the Sas-4-GFP fluorescence signal recovers only as a single foci after photobleaching (t = 120 s to t = 280 s). Time in seconds before and after photobleaching (t = 0 s) is shown in the top right of each panel. (B) Two-colour 3D-SIM images show how Sas-4-GFP fluorescence recovers relative to Asl-mCherry fluorescence (which surrounds the mother centriole). The Sas-4-GFP is shown in greyscale; insets (yellow dashed lines) display the overlay of Sas-4-GFP (green) and Asl-mCherry (red). Note how after photobleaching the Sas-4-GFP fluorescence recovers outside of the Asl-mCherry toroid. (C) Complementary images of a control centrosome adjacent to the one shown in (B) where the Sas-4-GFP signal was not photobleached. The t = 0 s panel is a widefield image (see ‘Materials and methods’). (D, E) Schematic (D) and box-plot (E) show how the average position of the post-bleached Sas-4-GFP signal is similar to the position of the daughter, but not the mother, centriole's prebleached Sas-4-GFP signal, relative to the Asl-mCherry signal. Boxes in E extend from the 25th-75th percentiles, whiskers extend from min to max values, lines in boxes are the median values, ‘+’ in boxes are the mean values; n = 25 centrosomes from 4 embryos. **** indicates where p < 0.0001; n.s. indicates where p = 0.09, and is therefore not significant. (F) 3D-SIM images show how Sas-4-mCherry (red) recovers relative to recovering Spd-2-GFP (green). An overlay of Sas-4-mCherry and Spd-2-GFP fluorescence is shown in the main panels; insets (yellow dashed lines) display the Sas-4-mCherry signal (greyscale). Note how after photobleaching the Sas-4-mCherry recovers outside of the hollow created by the recovering Spd-2-GFP (t = 300 s). (G) Complementary images of a control centrosome adjacent to the one in (F) where the Sas-4-mCherry and Spd-2-GFP signals were not photobleached. The t = 0 s image is a widefield image, as in (C).DOI:http://dx.doi.org/10.7554/eLife.08483.01010.7554/eLife.08483.011Figure 3—source data 1.Measuring the spatial offset between recovering Sas-4-GFP and Asl-mCherry at super resolution.This flie includes the raw data used to calculate the offset between the centroids of the two Sas-4-GFP foci and the single Asl-mCherry foci prior to photobleaching and the offset between the centroids of the single Sas-4-GFP foci and the single Asl-mCherry foci 5 min post-photobleaching. The data is used to plot the graph shown in Figure 3E.DOI:http://dx.doi.org/10.7554/eLife.08483.011
Mentions: In order to directly test if Sas-4 molecules are only recruited to growing daughter centrioles, we turned to 3D-Structured Illumination Microscopy (3D-SIM), which has approximately twice the spatial resolution of standard confocal microscopy. Using 3D-SIM in living embryos, we could clearly distinguish two adjacent Sas-4-GFP foci at individual centrosomes during S-phase (Figure 3A, t = −20 s), presumably representing mother-daughter centriole pairs. We combined 3D-SIM with FRAP (Conduit et al., 2014b) and found that the Sas-4-GFP signal only recovered at a single foci (Figure 3A, t = 120 s to t = 280 s). We confirmed that this recovery occurred at the daughter centriole by performing a two-colour-3D-SIM-FRAP experiment in embryos co-expressing Sas-4-GFP and Asl-mCherry, as Asl forms a toroid around only the mother centrioles (Figure 3B, t = −30 s) (Novak et al., 2014). Strikingly, the recovering Sas-4-GFP fluorescence always lay outside of the Asl-mCherry toroid (Figure 3B, t = 300 s), whereas control unbleached centrosomes still contained two Sas-4-GFP foci, one of which lay inside the Asl-mCherry toroid (Figure 3C, t = 300 s). This suggested that new Sas-4 molecules are recruited only to the daughter centrioles. However, during acquisition centrosomes move in the x-y plane, and given that the green and red channels are acquired sequentially on this particular imaging system it is possible for the green and red signals to become misaligned. To be sure the recovering Sas-4-GFP signal represented the daughter centriole, rather than a mis-positioned mother centriole, we therefore measured the distance between the centre of the Asl-mCherry signal and the centres of the pre- and post-bleached Sas-4-GFP signals (Figure 3D; Figure 3—source data 1). This revealed that the average position of the post-bleached Sas-4-GFP signal closely matched the position of the daughter Sas-4-GFP signal, but not the mother Sas-4-GFP signal (Figure 3E), confirming that Sas-4 molecules are recruited only to daughter centrioles. As Asl molecules are known to turn over at the mother centriole at this stage in the cycle (Novak et al., 2014), their recruitment cannot occur as part of Sas-4 dependent S-CAP complexes.10.7554/eLife.08483.010Figure 3.Two-colour-3D-SIM FRAP reveals that Sas-4-mCherry is recruited only to growing daughter centrioles, while PCM proteins are recruited only around mother centrioles.

Bottom Line: In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl.We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4.Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Centrosomes have many important functions and comprise a 'mother' and 'daughter' centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic 'S-CAP' complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes.

No MeSH data available.