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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 organizes the Cnn scaffold. (A and B) Live Cnn-mCherry in WT (A) and plp− (B) embryos in interphase. Released particles (arrows), extending flare (arrowheads), and disrupted PCM (bracket) are shown. Time is given in seconds. (C and D) Embryos stained for Cnn. (E) Cnn flare length in interphase embryos; n > 80 centrosomes. Mean ± SD is indicated. (F) Cytoplasmic Cnn particles in a 100-µm2 area (interphase embryos: WT n = 87, plp−n = 18, PLPΔ5n = 24, cnnB4n = 21; mitotic embryos: WT n = 40, plp−n = 18, PLPΔ5n = 9, cnnB4n = 15). Data are mean ± SD (error bars). ***, P < 0.001; ****, P < 0.0001 by a Student’s two-tailed t test relative to WT. Data shown are from a single representative experiment out of two or more repeats. Bars: (A and B) 2.5 µm; (C and D) 5 µm.
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fig5: PLP organizes the Cnn scaffold. (A and B) Live Cnn-mCherry in WT (A) and plp− (B) embryos in interphase. Released particles (arrows), extending flare (arrowheads), and disrupted PCM (bracket) are shown. Time is given in seconds. (C and D) Embryos stained for Cnn. (E) Cnn flare length in interphase embryos; n > 80 centrosomes. Mean ± SD is indicated. (F) Cytoplasmic Cnn particles in a 100-µm2 area (interphase embryos: WT n = 87, plp−n = 18, PLPΔ5n = 24, cnnB4n = 21; mitotic embryos: WT n = 40, plp−n = 18, PLPΔ5n = 9, cnnB4n = 15). Data are mean ± SD (error bars). ***, P < 0.001; ****, P < 0.0001 by a Student’s two-tailed t test relative to WT. Data shown are from a single representative experiment out of two or more repeats. Bars: (A and B) 2.5 µm; (C and D) 5 µm.

Mentions: To date, a functional role for PLP has not been examined in the Drosophila embryo. However, a recently reported mouse model shows that Pcnt organizes Cep215 and is required for cardiovascular and neural development (Chen et al., 2014). To investigate whether PLP regulates centrosome size or function, and whether this regulation is important for early Drosophila development, we generated plp− embryos (Fig. S3 A). Loss of PLP leads to 100% lethality; 32% die as embryos and 68% as first instar larva (see Materials and methods), which indicates that PLP is essential for viability. To determine if PLP is required to maintain the Cnn interphase lattice, we visualized Cnn in live wild-type (WT) and plp− embryos. Compared with WT embryos expressing Cnn-GFP (Fig. 5 A and Video 6), plp− centrosomes show Cnn disorganization and dispersal (Fig. 5 B). In plp− embryos, Cnn appears to ooze away from the centrosome, forming extended flares that eject an increased number of cytoplasmic particles. In addition, large voids interrupt the normally uniform distribution of Cnn within the PCM zone (Fig. 5 B, bracket).


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 organizes the Cnn scaffold. (A and B) Live Cnn-mCherry in WT (A) and plp− (B) embryos in interphase. Released particles (arrows), extending flare (arrowheads), and disrupted PCM (bracket) are shown. Time is given in seconds. (C and D) Embryos stained for Cnn. (E) Cnn flare length in interphase embryos; n > 80 centrosomes. Mean ± SD is indicated. (F) Cytoplasmic Cnn particles in a 100-µm2 area (interphase embryos: WT n = 87, plp−n = 18, PLPΔ5n = 24, cnnB4n = 21; mitotic embryos: WT n = 40, plp−n = 18, PLPΔ5n = 9, cnnB4n = 15). Data are mean ± SD (error bars). ***, P < 0.001; ****, P < 0.0001 by a Student’s two-tailed t test relative to WT. Data shown are from a single representative experiment out of two or more repeats. Bars: (A and B) 2.5 µm; (C and D) 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: PLP organizes the Cnn scaffold. (A and B) Live Cnn-mCherry in WT (A) and plp− (B) embryos in interphase. Released particles (arrows), extending flare (arrowheads), and disrupted PCM (bracket) are shown. Time is given in seconds. (C and D) Embryos stained for Cnn. (E) Cnn flare length in interphase embryos; n > 80 centrosomes. Mean ± SD is indicated. (F) Cytoplasmic Cnn particles in a 100-µm2 area (interphase embryos: WT n = 87, plp−n = 18, PLPΔ5n = 24, cnnB4n = 21; mitotic embryos: WT n = 40, plp−n = 18, PLPΔ5n = 9, cnnB4n = 15). Data are mean ± SD (error bars). ***, P < 0.001; ****, P < 0.0001 by a Student’s two-tailed t test relative to WT. Data shown are from a single representative experiment out of two or more repeats. Bars: (A and B) 2.5 µm; (C and D) 5 µm.
Mentions: To date, a functional role for PLP has not been examined in the Drosophila embryo. However, a recently reported mouse model shows that Pcnt organizes Cep215 and is required for cardiovascular and neural development (Chen et al., 2014). To investigate whether PLP regulates centrosome size or function, and whether this regulation is important for early Drosophila development, we generated plp− embryos (Fig. S3 A). Loss of PLP leads to 100% lethality; 32% die as embryos and 68% as first instar larva (see Materials and methods), which indicates that PLP is essential for viability. To determine if PLP is required to maintain the Cnn interphase lattice, we visualized Cnn in live wild-type (WT) and plp− embryos. Compared with WT embryos expressing Cnn-GFP (Fig. 5 A and Video 6), plp− centrosomes show Cnn disorganization and dispersal (Fig. 5 B). In plp− embryos, Cnn appears to ooze away from the centrosome, forming extended flares that eject an increased number of cytoplasmic particles. In addition, large voids interrupt the normally uniform distribution of Cnn within the PCM zone (Fig. 5 B, bracket).

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