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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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Reorganization of the centrosome structure in interphase. (A) SIM images of WT embryos stained for the indicated proteins. The presence (arrows) and absence (arrowheads) of γTub within Cnn flares is shown. (B) Mean radial intensity distribution of centrosome proteins in mitosis (left) and interphase (right) calculated from line scans derived from n = 30–110 centrosomes (broken lines in A). Shaded areas show the centriole (C, blue), PCM (P, orange), and flare (F, brown) zones as defined by the outer edges (OE) of Asl, γTub, and Cnn, respectively (see Materials and methods). The asterisk denotes satellite or flare measurement. (B′) Diagram of centrosome zones at mitosis (left) and interphase (right). (C) Confocal projections of the indicated proteins assayed for localization to the C, P, and F zones; +, present; −, absent; and +/−, low or variable levels; *, protein detected by GFP transgene. See Fig. S1 C for contrast-enhanced versions of Sas4, Bld10, Plk4, Polo, and Spd2. Open arrowheads show low localization of protein to the flare zone; closed arrowheads show Polo extending into the PCM zone. The brown arrowhead highlights the strong localization of PLP to the flare zone. (D) SIM image of a WT interphase centrosome with a Cnn flare (bracket); arrows show PLP at the centriole (blue) and satellites (brown). Line scan (broken line, D′) shows representative distribution relative to the centriole center. Bars: (A and D) 2.5 µm; (C) 1 µm.
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fig2: Reorganization of the centrosome structure in interphase. (A) SIM images of WT embryos stained for the indicated proteins. The presence (arrows) and absence (arrowheads) of γTub within Cnn flares is shown. (B) Mean radial intensity distribution of centrosome proteins in mitosis (left) and interphase (right) calculated from line scans derived from n = 30–110 centrosomes (broken lines in A). Shaded areas show the centriole (C, blue), PCM (P, orange), and flare (F, brown) zones as defined by the outer edges (OE) of Asl, γTub, and Cnn, respectively (see Materials and methods). The asterisk denotes satellite or flare measurement. (B′) Diagram of centrosome zones at mitosis (left) and interphase (right). (C) Confocal projections of the indicated proteins assayed for localization to the C, P, and F zones; +, present; −, absent; and +/−, low or variable levels; *, protein detected by GFP transgene. See Fig. S1 C for contrast-enhanced versions of Sas4, Bld10, Plk4, Polo, and Spd2. Open arrowheads show low localization of protein to the flare zone; closed arrowheads show Polo extending into the PCM zone. The brown arrowhead highlights the strong localization of PLP to the flare zone. (D) SIM image of a WT interphase centrosome with a Cnn flare (bracket); arrows show PLP at the centriole (blue) and satellites (brown). Line scan (broken line, D′) shows representative distribution relative to the centriole center. Bars: (A and D) 2.5 µm; (C) 1 µm.

Mentions: As expected, we found that the distributions of centriole proteins, such as SAS6 and Asterless (Asl), remain constant in mitosis and interphase (Fig. 2, A and B). These data were used to define a centriole zone with an ∼200-nm radius (Fig. 2 B, blue shading; and Fig. S1, A and B). In contrast, the PCM zone, defined by γTub, expands from a radius of 450 nm in mitosis to 600 nm in interphase (Fig. 2 B, orange shading; and Fig. S1, A and B). Thus, the outer edge of the PCM zone expands as embryos enter interphase. The major structural change to the interphase centrosome is the addition of extensive Cnn flares, which protrude well beyond the PCM zone. We term this zone the interphase flare zone (Fig. 2 B, brown shading; and Fig. S1, A and B). Notably, these interphase-specific flares extend ∼1,380 nm, with some reaching >2 µm, more than twice as far as the PCM zone (Figs. 2 B and S1 A). In sum, our analysis of embryonic centrosome organization shows that interphase centrosomes form a distinct flare zone in addition to the centriole and PCM zones (Fig. 2 B′). Moreover, our imaging (Fig. 1 C) reveals that the mitotic Cnn bolus appears to unfold to provide the source of interphase Cnn flares.


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)

Reorganization of the centrosome structure in interphase. (A) SIM images of WT embryos stained for the indicated proteins. The presence (arrows) and absence (arrowheads) of γTub within Cnn flares is shown. (B) Mean radial intensity distribution of centrosome proteins in mitosis (left) and interphase (right) calculated from line scans derived from n = 30–110 centrosomes (broken lines in A). Shaded areas show the centriole (C, blue), PCM (P, orange), and flare (F, brown) zones as defined by the outer edges (OE) of Asl, γTub, and Cnn, respectively (see Materials and methods). The asterisk denotes satellite or flare measurement. (B′) Diagram of centrosome zones at mitosis (left) and interphase (right). (C) Confocal projections of the indicated proteins assayed for localization to the C, P, and F zones; +, present; −, absent; and +/−, low or variable levels; *, protein detected by GFP transgene. See Fig. S1 C for contrast-enhanced versions of Sas4, Bld10, Plk4, Polo, and Spd2. Open arrowheads show low localization of protein to the flare zone; closed arrowheads show Polo extending into the PCM zone. The brown arrowhead highlights the strong localization of PLP to the flare zone. (D) SIM image of a WT interphase centrosome with a Cnn flare (bracket); arrows show PLP at the centriole (blue) and satellites (brown). Line scan (broken line, D′) shows representative distribution relative to the centriole center. Bars: (A and D) 2.5 µm; (C) 1 µm.
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fig2: Reorganization of the centrosome structure in interphase. (A) SIM images of WT embryos stained for the indicated proteins. The presence (arrows) and absence (arrowheads) of γTub within Cnn flares is shown. (B) Mean radial intensity distribution of centrosome proteins in mitosis (left) and interphase (right) calculated from line scans derived from n = 30–110 centrosomes (broken lines in A). Shaded areas show the centriole (C, blue), PCM (P, orange), and flare (F, brown) zones as defined by the outer edges (OE) of Asl, γTub, and Cnn, respectively (see Materials and methods). The asterisk denotes satellite or flare measurement. (B′) Diagram of centrosome zones at mitosis (left) and interphase (right). (C) Confocal projections of the indicated proteins assayed for localization to the C, P, and F zones; +, present; −, absent; and +/−, low or variable levels; *, protein detected by GFP transgene. See Fig. S1 C for contrast-enhanced versions of Sas4, Bld10, Plk4, Polo, and Spd2. Open arrowheads show low localization of protein to the flare zone; closed arrowheads show Polo extending into the PCM zone. The brown arrowhead highlights the strong localization of PLP to the flare zone. (D) SIM image of a WT interphase centrosome with a Cnn flare (bracket); arrows show PLP at the centriole (blue) and satellites (brown). Line scan (broken line, D′) shows representative distribution relative to the centriole center. Bars: (A and D) 2.5 µm; (C) 1 µm.
Mentions: As expected, we found that the distributions of centriole proteins, such as SAS6 and Asterless (Asl), remain constant in mitosis and interphase (Fig. 2, A and B). These data were used to define a centriole zone with an ∼200-nm radius (Fig. 2 B, blue shading; and Fig. S1, A and B). In contrast, the PCM zone, defined by γTub, expands from a radius of 450 nm in mitosis to 600 nm in interphase (Fig. 2 B, orange shading; and Fig. S1, A and B). Thus, the outer edge of the PCM zone expands as embryos enter interphase. The major structural change to the interphase centrosome is the addition of extensive Cnn flares, which protrude well beyond the PCM zone. We term this zone the interphase flare zone (Fig. 2 B, brown shading; and Fig. S1, A and B). Notably, these interphase-specific flares extend ∼1,380 nm, with some reaching >2 µm, more than twice as far as the PCM zone (Figs. 2 B and S1 A). In sum, our analysis of embryonic centrosome organization shows that interphase centrosomes form a distinct flare zone in addition to the centriole and PCM zones (Fig. 2 B′). Moreover, our imaging (Fig. 1 C) reveals that the mitotic Cnn bolus appears to unfold to provide the source of interphase Cnn flares.

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