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Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.

Lerit DA, Jordan HA, Poulton JS, Fagerstrom CJ, Galletta BJ, Peifer M, Rusan NM - J. Cell Biol. (2015)

Bottom Line: Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes.A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle.Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.

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PLP is required for MT organization. (A and B) WT and plp− mitotic (A) and interphase (B) embryos stained for the indicated proteins. (A’) Acentriolar Cnn particle (inset) organizes MTs. Boxes in B show radial MT array, enlarged in the insets on the right. (C and D) Live GFP-MT in embryos. Broken circles show MTOC inactivation. Arrows show nuclear collisions and the arrowhead shows an orthogonal spindle. (E) Centrosome separation (closed arrowhead) and detachment (open arrowhead) defects are quantified in F. (G) Centrosome and nucleus positioning defects with two nuclei (asterisk) and more than two centrosomes (numerals) per pseudo-cell are quantified in H and F, and show mean ± SD (error bars). ***, P < 0.0001. Data shown are from a single representative experiment out of two repeats. Bars: (A and B, main panels) 5 µm; (A′ and B, right) 1 µm; (C–G) 10 µm.
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fig6: PLP is required for MT organization. (A and B) WT and plp− mitotic (A) and interphase (B) embryos stained for the indicated proteins. (A’) Acentriolar Cnn particle (inset) organizes MTs. Boxes in B show radial MT array, enlarged in the insets on the right. (C and D) Live GFP-MT in embryos. Broken circles show MTOC inactivation. Arrows show nuclear collisions and the arrowhead shows an orthogonal spindle. (E) Centrosome separation (closed arrowhead) and detachment (open arrowhead) defects are quantified in F. (G) Centrosome and nucleus positioning defects with two nuclei (asterisk) and more than two centrosomes (numerals) per pseudo-cell are quantified in H and F, and show mean ± SD (error bars). ***, P < 0.0001. Data shown are from a single representative experiment out of two repeats. Bars: (A and B, main panels) 5 µm; (A′ and B, right) 1 µm; (C–G) 10 µm.

Mentions: Expanded localization of Pcnt to centrosomes has previously been correlated with MT organization in mouse epithelial cells (Mogensen et al., 1997), and knockdown of Pcnt is associated with MT disorganization in cultured cells (Zimmerman et al., 2004), indicating that Pcnt contributes to proper MT organization. While immunodepletion experiments previously suggested that PLP plays a role in MT nucleation (Kawaguchi and Zheng, 2004), mutant analysis within NBs indicates that plp− cells are efficient MT nucleators (Martinez-Campos et al., 2004; Lerit and Rusan, 2013; Singh et al., 2014). Indeed, similar to cnn− mutants (Megraw et al., 1999; Vaizel-Ohayon and Schejter, 1999), we found that embryonic plp− centrosomes maintain robust MTOC activity comparable to WT (Fig. 6 A). In addition, the cytoplasmic Cnn particles found in plp− mutants serve as a platform for MT organization independent of the centrosome (80%; n = 33/40 of cytoplasmic foci; Fig. 6 A′), which is consistent with ectopic cytoplasmic Cnn foci organizing MT asters within unfertilized eggs (Conduit et al., 2014a). In interphase, it was difficult to determine if these cytoplasmic particles altered the density of the MT network, but there is a clear reduction in radial MT symmetry in plp− embryos, as evident by MT crossovers and more randomized MTs (55%; n = 11/20 embryos; Fig. 6 B). Similarly, disorganized astral MTs were recently reported in a novel Pcnt mouse model (Chen et al., 2014), which suggests that MT organization is a conserved function of PLP. Live imaging of MTs shows that plp− mutants (n = 3/5 embryos) display MTOC inactivation followed by failed and abortive spindle formation (Fig. 6 C, broken line), indicating that PLP is required to maintain MTOC activity throughout the cell cycle. Further, loss of PLP also disrupts mitotic spindle orientation (Fig. 6 D), and collisions of neighboring nuclei result in uneven nuclear spacing and association of more than two MTOCs with a single nucleus (Fig. 6 C, arrows; and Video 8).


Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.

Lerit DA, Jordan HA, Poulton JS, Fagerstrom CJ, Galletta BJ, Peifer M, Rusan NM - J. Cell Biol. (2015)

PLP is required for MT organization. (A and B) WT and plp− mitotic (A) and interphase (B) embryos stained for the indicated proteins. (A’) Acentriolar Cnn particle (inset) organizes MTs. Boxes in B show radial MT array, enlarged in the insets on the right. (C and D) Live GFP-MT in embryos. Broken circles show MTOC inactivation. Arrows show nuclear collisions and the arrowhead shows an orthogonal spindle. (E) Centrosome separation (closed arrowhead) and detachment (open arrowhead) defects are quantified in F. (G) Centrosome and nucleus positioning defects with two nuclei (asterisk) and more than two centrosomes (numerals) per pseudo-cell are quantified in H and F, and show mean ± SD (error bars). ***, P < 0.0001. Data shown are from a single representative experiment out of two repeats. Bars: (A and B, main panels) 5 µm; (A′ and B, right) 1 µm; (C–G) 10 µm.
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Related In: Results  -  Collection

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fig6: PLP is required for MT organization. (A and B) WT and plp− mitotic (A) and interphase (B) embryos stained for the indicated proteins. (A’) Acentriolar Cnn particle (inset) organizes MTs. Boxes in B show radial MT array, enlarged in the insets on the right. (C and D) Live GFP-MT in embryos. Broken circles show MTOC inactivation. Arrows show nuclear collisions and the arrowhead shows an orthogonal spindle. (E) Centrosome separation (closed arrowhead) and detachment (open arrowhead) defects are quantified in F. (G) Centrosome and nucleus positioning defects with two nuclei (asterisk) and more than two centrosomes (numerals) per pseudo-cell are quantified in H and F, and show mean ± SD (error bars). ***, P < 0.0001. Data shown are from a single representative experiment out of two repeats. Bars: (A and B, main panels) 5 µm; (A′ and B, right) 1 µm; (C–G) 10 µm.
Mentions: Expanded localization of Pcnt to centrosomes has previously been correlated with MT organization in mouse epithelial cells (Mogensen et al., 1997), and knockdown of Pcnt is associated with MT disorganization in cultured cells (Zimmerman et al., 2004), indicating that Pcnt contributes to proper MT organization. While immunodepletion experiments previously suggested that PLP plays a role in MT nucleation (Kawaguchi and Zheng, 2004), mutant analysis within NBs indicates that plp− cells are efficient MT nucleators (Martinez-Campos et al., 2004; Lerit and Rusan, 2013; Singh et al., 2014). Indeed, similar to cnn− mutants (Megraw et al., 1999; Vaizel-Ohayon and Schejter, 1999), we found that embryonic plp− centrosomes maintain robust MTOC activity comparable to WT (Fig. 6 A). In addition, the cytoplasmic Cnn particles found in plp− mutants serve as a platform for MT organization independent of the centrosome (80%; n = 33/40 of cytoplasmic foci; Fig. 6 A′), which is consistent with ectopic cytoplasmic Cnn foci organizing MT asters within unfertilized eggs (Conduit et al., 2014a). In interphase, it was difficult to determine if these cytoplasmic particles altered the density of the MT network, but there is a clear reduction in radial MT symmetry in plp− embryos, as evident by MT crossovers and more randomized MTs (55%; n = 11/20 embryos; Fig. 6 B). Similarly, disorganized astral MTs were recently reported in a novel Pcnt mouse model (Chen et al., 2014), which suggests that MT organization is a conserved function of PLP. Live imaging of MTs shows that plp− mutants (n = 3/5 embryos) display MTOC inactivation followed by failed and abortive spindle formation (Fig. 6 C, broken line), indicating that PLP is required to maintain MTOC activity throughout the cell cycle. Further, loss of PLP also disrupts mitotic spindle orientation (Fig. 6 D), and collisions of neighboring nuclei result in uneven nuclear spacing and association of more than two MTOCs with a single nucleus (Fig. 6 C, arrows; and Video 8).

Bottom Line: Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes.A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle.Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.

Show MeSH
Related in: MedlinePlus