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The centrosomal protein pericentrin identified at the basal body complex of the connecting cilium in mouse photoreceptors.

Mühlhans J, Brandstätter JH, Giessl A - PLoS ONE (2011)

Bottom Line: Here, Pcnt colocalized with the whole protein machinery responsible for transport processes between the two compartments.Surprisingly, photoreceptors expressed a small Pcnt splice transcript - most likely a modified variant of Pcnt S - which was not present in receptor neurons of the olfactory epithelium.The individual patchwork of different Pcnt splice transcripts seems to reflect the complexity of Pcnt function, an assumption corroborated by the heterogeneous clinical manifestations associated with mutations in the Pcnt gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, Erlangen, Germany.

ABSTRACT

Background: Pericentrin (Pcnt), a conserved protein of the pericentriolar material, serves as a multifunctional scaffold for numerous proteins and plays an important role in microtubule organization. Recent studies indicate that Pcnt mutations are associated with a range of diseases including primordial dwarfism and ciliopathies. To date, three Pcnt splice variants from orthologous genes in mice and humans are known.

Principal findings: We generated a specific Pcnt antiserum detecting all known Pcnt splice variants and examined the cellular and subcellular distribution of Pcnt in ciliated tissues of the mouse, the olfactory epithelium and the retina. For the first time, we identified Pcnt and its centrosomal interaction partners at the basal body complex of mouse retinal photoreceptors. Photoreceptors are morphologically and functionally subdivided into the light sensitive outer segment and the inner segment comprising the metabolic function of the cell. The two compartments are linked via a modified, specialized, non-motile cilium, the connecting cilium. Here, Pcnt colocalized with the whole protein machinery responsible for transport processes between the two compartments. Surprisingly, photoreceptors expressed a small Pcnt splice transcript - most likely a modified variant of Pcnt S - which was not present in receptor neurons of the olfactory epithelium.

Conclusions: Our findings suggest distinct functional roles of several Pcnt variants in different ciliated tissues and sensory neurons, like the olfactory epithelium and the retina of the mouse. The individual patchwork of different Pcnt splice transcripts seems to reflect the complexity of Pcnt function, an assumption corroborated by the heterogeneous clinical manifestations associated with mutations in the Pcnt gene.

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Colocalization of various Pericentrin interaction partners at the basal body complex of mouse photoreceptors.(A) Scheme of a vertebrate rod photoreceptor. (B–F) Micrographs of a vertical cryostat section through an adult mouse retina double labeled for CG-NAP (B, green) and Cen3 (C, red) as a marker for the ciliary apparatus (the connecting cilium (CC) and the basal body complex (BBC)) and the centrosomes. (B) CG-NAP and (C) Cen3 are localized in the ciliary region of the photoreceptors and at the centrosomes of the other retinal cells. (D) The merge of the stainings with the additional DAPI nuclear staining (blue) shows the partial colocalization of CG-NAP and Cen3 in the ciliary region of the photoreceptors and the centrosomes of the other retinal cells. (E–F) High power views showing the localization of CG-NAP at the BBC of the connecting cilium (E) and at the centrosomes of the other retinal cells (F). (G) Scheme of the ciliary apparatus. (H–K) High power views showing the localization of the Pcnt interaction partners PCM1 (H, green), DISC1 (I, green), IFT20 (J, green), and PC2 (K, green) at the BBC of the connecting cilium of the photoreceptor. Cen3 (red) marks the whole cilium with the ciliary apparatus. RPE: retinal pigment epithel; OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; BB: basal body; CC: connecting cilium; C: centriole; CP: calycal process; MTs: microtubules; N: nucleus; S: synapse. Scale bars: 10 µm (B), 1 µm (E, F, J, K, H, I).
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pone-0026496-g006: Colocalization of various Pericentrin interaction partners at the basal body complex of mouse photoreceptors.(A) Scheme of a vertebrate rod photoreceptor. (B–F) Micrographs of a vertical cryostat section through an adult mouse retina double labeled for CG-NAP (B, green) and Cen3 (C, red) as a marker for the ciliary apparatus (the connecting cilium (CC) and the basal body complex (BBC)) and the centrosomes. (B) CG-NAP and (C) Cen3 are localized in the ciliary region of the photoreceptors and at the centrosomes of the other retinal cells. (D) The merge of the stainings with the additional DAPI nuclear staining (blue) shows the partial colocalization of CG-NAP and Cen3 in the ciliary region of the photoreceptors and the centrosomes of the other retinal cells. (E–F) High power views showing the localization of CG-NAP at the BBC of the connecting cilium (E) and at the centrosomes of the other retinal cells (F). (G) Scheme of the ciliary apparatus. (H–K) High power views showing the localization of the Pcnt interaction partners PCM1 (H, green), DISC1 (I, green), IFT20 (J, green), and PC2 (K, green) at the BBC of the connecting cilium of the photoreceptor. Cen3 (red) marks the whole cilium with the ciliary apparatus. RPE: retinal pigment epithel; OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; BB: basal body; CC: connecting cilium; C: centriole; CP: calycal process; MTs: microtubules; N: nucleus; S: synapse. Scale bars: 10 µm (B), 1 µm (E, F, J, K, H, I).

Mentions: A centrosomal interaction partner of Pcnt is CG-NAP, a giant coiled-coil protein, which is found at the centrosome throughout the cell cycle and at the Golgi apparatus during interphase [2]. The double labeling experiments with antibodies against CG-NAP and Cen3 showed a partial colocalization of the two proteins at the ciliary region of the photoreceptors and at the centrosomes of the other cells in the retina (Fig. 6A–F). As seen in the higher power view of the photoreceptor ciliary region (Fig. 6E), CG-NAP is present at the BBC of the connecting cilium, the site where Pcnt is also found (Fig. 4, Fig. 5, Fig. S1). At the other retinal cells, CG-NAP is found at the centrosomes and at parts of the Golgi apparatus (Fig. 6F) [2]. Other known centrosomal interaction partners of Pcnt, which we detected at the BBC of the photoreceptor's connecting cilium, were the proteins PCM1 [25], [26], [30] (Fig. 6H) and DISC1 (disrupted-in-schizophrenia 1) [31], [32] (Fig. 6I). DISC1 is also known to be involved in the regulation of the formation and/or the maintenance of primary cilia and in establishing the integration of dopamine receptors to the ciliary surface [33], [34].


The centrosomal protein pericentrin identified at the basal body complex of the connecting cilium in mouse photoreceptors.

Mühlhans J, Brandstätter JH, Giessl A - PLoS ONE (2011)

Colocalization of various Pericentrin interaction partners at the basal body complex of mouse photoreceptors.(A) Scheme of a vertebrate rod photoreceptor. (B–F) Micrographs of a vertical cryostat section through an adult mouse retina double labeled for CG-NAP (B, green) and Cen3 (C, red) as a marker for the ciliary apparatus (the connecting cilium (CC) and the basal body complex (BBC)) and the centrosomes. (B) CG-NAP and (C) Cen3 are localized in the ciliary region of the photoreceptors and at the centrosomes of the other retinal cells. (D) The merge of the stainings with the additional DAPI nuclear staining (blue) shows the partial colocalization of CG-NAP and Cen3 in the ciliary region of the photoreceptors and the centrosomes of the other retinal cells. (E–F) High power views showing the localization of CG-NAP at the BBC of the connecting cilium (E) and at the centrosomes of the other retinal cells (F). (G) Scheme of the ciliary apparatus. (H–K) High power views showing the localization of the Pcnt interaction partners PCM1 (H, green), DISC1 (I, green), IFT20 (J, green), and PC2 (K, green) at the BBC of the connecting cilium of the photoreceptor. Cen3 (red) marks the whole cilium with the ciliary apparatus. RPE: retinal pigment epithel; OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; BB: basal body; CC: connecting cilium; C: centriole; CP: calycal process; MTs: microtubules; N: nucleus; S: synapse. Scale bars: 10 µm (B), 1 µm (E, F, J, K, H, I).
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pone-0026496-g006: Colocalization of various Pericentrin interaction partners at the basal body complex of mouse photoreceptors.(A) Scheme of a vertebrate rod photoreceptor. (B–F) Micrographs of a vertical cryostat section through an adult mouse retina double labeled for CG-NAP (B, green) and Cen3 (C, red) as a marker for the ciliary apparatus (the connecting cilium (CC) and the basal body complex (BBC)) and the centrosomes. (B) CG-NAP and (C) Cen3 are localized in the ciliary region of the photoreceptors and at the centrosomes of the other retinal cells. (D) The merge of the stainings with the additional DAPI nuclear staining (blue) shows the partial colocalization of CG-NAP and Cen3 in the ciliary region of the photoreceptors and the centrosomes of the other retinal cells. (E–F) High power views showing the localization of CG-NAP at the BBC of the connecting cilium (E) and at the centrosomes of the other retinal cells (F). (G) Scheme of the ciliary apparatus. (H–K) High power views showing the localization of the Pcnt interaction partners PCM1 (H, green), DISC1 (I, green), IFT20 (J, green), and PC2 (K, green) at the BBC of the connecting cilium of the photoreceptor. Cen3 (red) marks the whole cilium with the ciliary apparatus. RPE: retinal pigment epithel; OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; BB: basal body; CC: connecting cilium; C: centriole; CP: calycal process; MTs: microtubules; N: nucleus; S: synapse. Scale bars: 10 µm (B), 1 µm (E, F, J, K, H, I).
Mentions: A centrosomal interaction partner of Pcnt is CG-NAP, a giant coiled-coil protein, which is found at the centrosome throughout the cell cycle and at the Golgi apparatus during interphase [2]. The double labeling experiments with antibodies against CG-NAP and Cen3 showed a partial colocalization of the two proteins at the ciliary region of the photoreceptors and at the centrosomes of the other cells in the retina (Fig. 6A–F). As seen in the higher power view of the photoreceptor ciliary region (Fig. 6E), CG-NAP is present at the BBC of the connecting cilium, the site where Pcnt is also found (Fig. 4, Fig. 5, Fig. S1). At the other retinal cells, CG-NAP is found at the centrosomes and at parts of the Golgi apparatus (Fig. 6F) [2]. Other known centrosomal interaction partners of Pcnt, which we detected at the BBC of the photoreceptor's connecting cilium, were the proteins PCM1 [25], [26], [30] (Fig. 6H) and DISC1 (disrupted-in-schizophrenia 1) [31], [32] (Fig. 6I). DISC1 is also known to be involved in the regulation of the formation and/or the maintenance of primary cilia and in establishing the integration of dopamine receptors to the ciliary surface [33], [34].

Bottom Line: Here, Pcnt colocalized with the whole protein machinery responsible for transport processes between the two compartments.Surprisingly, photoreceptors expressed a small Pcnt splice transcript - most likely a modified variant of Pcnt S - which was not present in receptor neurons of the olfactory epithelium.The individual patchwork of different Pcnt splice transcripts seems to reflect the complexity of Pcnt function, an assumption corroborated by the heterogeneous clinical manifestations associated with mutations in the Pcnt gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, Erlangen, Germany.

ABSTRACT

Background: Pericentrin (Pcnt), a conserved protein of the pericentriolar material, serves as a multifunctional scaffold for numerous proteins and plays an important role in microtubule organization. Recent studies indicate that Pcnt mutations are associated with a range of diseases including primordial dwarfism and ciliopathies. To date, three Pcnt splice variants from orthologous genes in mice and humans are known.

Principal findings: We generated a specific Pcnt antiserum detecting all known Pcnt splice variants and examined the cellular and subcellular distribution of Pcnt in ciliated tissues of the mouse, the olfactory epithelium and the retina. For the first time, we identified Pcnt and its centrosomal interaction partners at the basal body complex of mouse retinal photoreceptors. Photoreceptors are morphologically and functionally subdivided into the light sensitive outer segment and the inner segment comprising the metabolic function of the cell. The two compartments are linked via a modified, specialized, non-motile cilium, the connecting cilium. Here, Pcnt colocalized with the whole protein machinery responsible for transport processes between the two compartments. Surprisingly, photoreceptors expressed a small Pcnt splice transcript - most likely a modified variant of Pcnt S - which was not present in receptor neurons of the olfactory epithelium.

Conclusions: Our findings suggest distinct functional roles of several Pcnt variants in different ciliated tissues and sensory neurons, like the olfactory epithelium and the retina of the mouse. The individual patchwork of different Pcnt splice transcripts seems to reflect the complexity of Pcnt function, an assumption corroborated by the heterogeneous clinical manifestations associated with mutations in the Pcnt gene.

Show MeSH
Related in: MedlinePlus