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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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Identification of two sites of direct interaction between Cnn and PLP. (A) ClustalW multiple sequence alignment of the Cnn CM2 motif; similar (yellow) and identical (green) residues are shown. The asterisk shows an invariant arginine mutated in cnnB4 mutants. (B) Graphic showing PLP and Cnn truncations used in Y2H. Two distinct interaction sites are shown. (C) Y2H assays for growth (left) and interaction (right; Materials and methods). (D) Graphic showing truncations of Cnn-F3 used for interaction refinement. The asterisk shows the R1141H mutation that mimics the cnnB4 mutation.
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fig8: Identification of two sites of direct interaction between Cnn and PLP. (A) ClustalW multiple sequence alignment of the Cnn CM2 motif; similar (yellow) and identical (green) residues are shown. The asterisk shows an invariant arginine mutated in cnnB4 mutants. (B) Graphic showing PLP and Cnn truncations used in Y2H. Two distinct interaction sites are shown. (C) Y2H assays for growth (left) and interaction (right; Materials and methods). (D) Graphic showing truncations of Cnn-F3 used for interaction refinement. The asterisk shows the R1141H mutation that mimics the cnnB4 mutation.

Mentions: Our collective work strongly suggests that Cnn and PLP function within a complex to regulate centrosome size and activity. To date, a direct interaction between Cep215/Cnn and Pcnt/PLP has not been reported. Immunoprecipitation experiments from mammalian and Drosophila extracts, however, indicate that these proteins comprise a biochemical complex through a conserved motif located in the C terminus of Cep215/Cnn (Fig. 8 A), termed Conserved Motif 2 (CM2; Kao and Megraw, 2009; Buchman et al., 2010; Conduit et al., 2010; Wang et al., 2010; Gopalakrishnan et al., 2011; Kim and Rhee, 2014). To test whether Cnn and PLP interact directly, we truncated Cnn and PLP proteins into a series of fragments (Fig. 8 B) and conducted yeast two-hybrid (Y2H) analysis (Fig. 8 C). These studies identified two sites likely to mediate direct interaction. One occurs between Cnn fragment 1 (Cnn-F1) and PLP fragment 5 (PLP-F5; Fig. 8, B and C). A second was detected between Cnn fragment 3 (Cnn-F3) and PLP fragment 2 (PLP-F2; Fig. 8, B and C). Previous work indicates that CM2 interacts directly with Calmodulin (Wang et al., 2010) and Centrocortin (Cen; Kao and Megraw, 2009), which suggests that this conserved motif mediates several protein interactions. Further Y2H analysis confirms that the CM2 domain within Cnn-F3 is necessary and sufficient to mediate the direct interaction with PLP-F2 (Fig. 8, C and D).


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)

Identification of two sites of direct interaction between Cnn and PLP. (A) ClustalW multiple sequence alignment of the Cnn CM2 motif; similar (yellow) and identical (green) residues are shown. The asterisk shows an invariant arginine mutated in cnnB4 mutants. (B) Graphic showing PLP and Cnn truncations used in Y2H. Two distinct interaction sites are shown. (C) Y2H assays for growth (left) and interaction (right; Materials and methods). (D) Graphic showing truncations of Cnn-F3 used for interaction refinement. The asterisk shows the R1141H mutation that mimics the cnnB4 mutation.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494003&req=5

fig8: Identification of two sites of direct interaction between Cnn and PLP. (A) ClustalW multiple sequence alignment of the Cnn CM2 motif; similar (yellow) and identical (green) residues are shown. The asterisk shows an invariant arginine mutated in cnnB4 mutants. (B) Graphic showing PLP and Cnn truncations used in Y2H. Two distinct interaction sites are shown. (C) Y2H assays for growth (left) and interaction (right; Materials and methods). (D) Graphic showing truncations of Cnn-F3 used for interaction refinement. The asterisk shows the R1141H mutation that mimics the cnnB4 mutation.
Mentions: Our collective work strongly suggests that Cnn and PLP function within a complex to regulate centrosome size and activity. To date, a direct interaction between Cep215/Cnn and Pcnt/PLP has not been reported. Immunoprecipitation experiments from mammalian and Drosophila extracts, however, indicate that these proteins comprise a biochemical complex through a conserved motif located in the C terminus of Cep215/Cnn (Fig. 8 A), termed Conserved Motif 2 (CM2; Kao and Megraw, 2009; Buchman et al., 2010; Conduit et al., 2010; Wang et al., 2010; Gopalakrishnan et al., 2011; Kim and Rhee, 2014). To test whether Cnn and PLP interact directly, we truncated Cnn and PLP proteins into a series of fragments (Fig. 8 B) and conducted yeast two-hybrid (Y2H) analysis (Fig. 8 C). These studies identified two sites likely to mediate direct interaction. One occurs between Cnn fragment 1 (Cnn-F1) and PLP fragment 5 (PLP-F5; Fig. 8, B and C). A second was detected between Cnn fragment 3 (Cnn-F3) and PLP fragment 2 (PLP-F2; Fig. 8, B and C). Previous work indicates that CM2 interacts directly with Calmodulin (Wang et al., 2010) and Centrocortin (Cen; Kao and Megraw, 2009), which suggests that this conserved motif mediates several protein interactions. Further Y2H analysis confirms that the CM2 domain within Cnn-F3 is necessary and sufficient to mediate the direct interaction with PLP-F2 (Fig. 8, C and D).

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