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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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Rearrangement of Cnn at the mitotic-to-interphase transition. (A) Live Cnn-GFP and H2A-RFP in a pseudo-cell (broken line) of a WT embryo through three cell cycles. Arrows show the enlarged regions below (Cnn, white). Cnn flares (red arrowheads) and separating daughter centrosomes (white arrowheads) in interphase are indicated. Bars in top panels, 5 µm. (B and C) SIM images of mitotic (B) and interphase (C) embryos stained for Cnn. Centrosomes (red boxes) are magnified to the right as projections (B′ and C′) and single optical sections (B″ and C″) through the centrosome center. Interphase flare (red arrowhead) and centriole position (white arrows) are shown. (B‴ and C‴) Cytoplasmic regions (orange boxes) show particles (open arrowheads) and a particle release event (orange arrow). (D) Live Cnn-GFP at mitotic exit. Released particles (orange) and unfolding flares (red) are shown. Time is given in minutes:seconds.
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fig1: Rearrangement of Cnn at the mitotic-to-interphase transition. (A) Live Cnn-GFP and H2A-RFP in a pseudo-cell (broken line) of a WT embryo through three cell cycles. Arrows show the enlarged regions below (Cnn, white). Cnn flares (red arrowheads) and separating daughter centrosomes (white arrowheads) in interphase are indicated. Bars in top panels, 5 µm. (B and C) SIM images of mitotic (B) and interphase (C) embryos stained for Cnn. Centrosomes (red boxes) are magnified to the right as projections (B′ and C′) and single optical sections (B″ and C″) through the centrosome center. Interphase flare (red arrowhead) and centriole position (white arrows) are shown. (B‴ and C‴) Cytoplasmic regions (orange boxes) show particles (open arrowheads) and a particle release event (orange arrow). (D) Live Cnn-GFP at mitotic exit. Released particles (orange) and unfolding flares (red) are shown. Time is given in minutes:seconds.

Mentions: To study Cnn during the rapid early divisions, it is critical to precisely track the timing of its reorganization. By imaging recombineered Cnn in embryos coexpressing the nuclear marker H2A-RFP, we find that Cnn is a compact structure during mitosis (Fig. 1 A and Video 1). Upon mitotic exit and throughout interphase, Cnn becomes expansive and particulate, ultimately extending reticulated flares that greatly increase centrosome volume and eject particles into the cytoplasm (Fig. 1 A, 4:06). These findings are in agreement with a seminal study of Cnn dynamics (Megraw et al., 2002). To monitor the conspicuous changes to centrosome size and shape, we visualized Cnn distribution by SIM. SIM resolves mitotic Cnn as a hollow sphere with a discernable strand-like substructure (Fig. 1, B–B″) with few cytoplasmic particles (Fig. 1 B‴). In contrast, interphase centrosomes form a more elaborate shape with extended flares of uneven thickness and length (Fig. 1, C–C″). These flares are sites of Cnn particle release (Fig. 1 C‴), which appear analogous to mammalian centriole satellites (Rattner, 1992; Balczon et al., 1994; Zhang and Megraw, 2007), as both are ejected from the centrosome and bidirectionally move during interphase (Kubo et al., 1999; Megraw et al., 2002). In addition, SIM resolves Cnn particles as rings, similar to the structure of mammalian centriole satellites revealed by EM (de-Thé, 1964; Kubo et al., 1999). Although mammalian centriole satellites facilitate protein trafficking to and from centrosomes and cilia, the overall functional significance of mammalian satellites requires further study (Mahjoub and Tsou, 2013).


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)

Rearrangement of Cnn at the mitotic-to-interphase transition. (A) Live Cnn-GFP and H2A-RFP in a pseudo-cell (broken line) of a WT embryo through three cell cycles. Arrows show the enlarged regions below (Cnn, white). Cnn flares (red arrowheads) and separating daughter centrosomes (white arrowheads) in interphase are indicated. Bars in top panels, 5 µm. (B and C) SIM images of mitotic (B) and interphase (C) embryos stained for Cnn. Centrosomes (red boxes) are magnified to the right as projections (B′ and C′) and single optical sections (B″ and C″) through the centrosome center. Interphase flare (red arrowhead) and centriole position (white arrows) are shown. (B‴ and C‴) Cytoplasmic regions (orange boxes) show particles (open arrowheads) and a particle release event (orange arrow). (D) Live Cnn-GFP at mitotic exit. Released particles (orange) and unfolding flares (red) are shown. Time is given in minutes:seconds.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Rearrangement of Cnn at the mitotic-to-interphase transition. (A) Live Cnn-GFP and H2A-RFP in a pseudo-cell (broken line) of a WT embryo through three cell cycles. Arrows show the enlarged regions below (Cnn, white). Cnn flares (red arrowheads) and separating daughter centrosomes (white arrowheads) in interphase are indicated. Bars in top panels, 5 µm. (B and C) SIM images of mitotic (B) and interphase (C) embryos stained for Cnn. Centrosomes (red boxes) are magnified to the right as projections (B′ and C′) and single optical sections (B″ and C″) through the centrosome center. Interphase flare (red arrowhead) and centriole position (white arrows) are shown. (B‴ and C‴) Cytoplasmic regions (orange boxes) show particles (open arrowheads) and a particle release event (orange arrow). (D) Live Cnn-GFP at mitotic exit. Released particles (orange) and unfolding flares (red) are shown. Time is given in minutes:seconds.
Mentions: To study Cnn during the rapid early divisions, it is critical to precisely track the timing of its reorganization. By imaging recombineered Cnn in embryos coexpressing the nuclear marker H2A-RFP, we find that Cnn is a compact structure during mitosis (Fig. 1 A and Video 1). Upon mitotic exit and throughout interphase, Cnn becomes expansive and particulate, ultimately extending reticulated flares that greatly increase centrosome volume and eject particles into the cytoplasm (Fig. 1 A, 4:06). These findings are in agreement with a seminal study of Cnn dynamics (Megraw et al., 2002). To monitor the conspicuous changes to centrosome size and shape, we visualized Cnn distribution by SIM. SIM resolves mitotic Cnn as a hollow sphere with a discernable strand-like substructure (Fig. 1, B–B″) with few cytoplasmic particles (Fig. 1 B‴). In contrast, interphase centrosomes form a more elaborate shape with extended flares of uneven thickness and length (Fig. 1, C–C″). These flares are sites of Cnn particle release (Fig. 1 C‴), which appear analogous to mammalian centriole satellites (Rattner, 1992; Balczon et al., 1994; Zhang and Megraw, 2007), as both are ejected from the centrosome and bidirectionally move during interphase (Kubo et al., 1999; Megraw et al., 2002). In addition, SIM resolves Cnn particles as rings, similar to the structure of mammalian centriole satellites revealed by EM (de-Thé, 1964; Kubo et al., 1999). Although mammalian centriole satellites facilitate protein trafficking to and from centrosomes and cilia, the overall functional significance of mammalian satellites requires further study (Mahjoub and Tsou, 2013).

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