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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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Immunoelectron microscopic localization of Pericentrin in mouse photoreceptors.(A) Scheme of a mouse rod photoreceptor with the tip of the photoreceptor’s light-sensitive outer segment (OS) enclosed by the retinal pigment epithelium (RPE). (B) Scheme of the ciliary apparatus. The photosensitive OS is linked by the connecting cilium (CC) to the metabolic inner segment (IS), which includes the basal body complex (BBC) in its apical region. (C–H) Electron micrographs showing the ultrastructural localization of Pcnt in the photoreceptor ciliary region. Longitudinal (C, D, G, H) and cross sections (E, F) through the ciliary region show the presence of Pcnt at the basal bodies (BB) (C, D, G) but not in other parts of the CC (F, H). In the apical region of the IS, Pcnt is located at the centriole (C) (F, G, H) at the BBC (E) but not in the cytoplasm of the calycal process (CP) (C, D, H). N: nucleus; CR: ciliary rootlet. Scale bars: 0.5 µm (B, C, D, H), 0.2 µm (E, F, G).
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pone-0026496-g005: Immunoelectron microscopic localization of Pericentrin in mouse photoreceptors.(A) Scheme of a mouse rod photoreceptor with the tip of the photoreceptor’s light-sensitive outer segment (OS) enclosed by the retinal pigment epithelium (RPE). (B) Scheme of the ciliary apparatus. The photosensitive OS is linked by the connecting cilium (CC) to the metabolic inner segment (IS), which includes the basal body complex (BBC) in its apical region. (C–H) Electron micrographs showing the ultrastructural localization of Pcnt in the photoreceptor ciliary region. Longitudinal (C, D, G, H) and cross sections (E, F) through the ciliary region show the presence of Pcnt at the basal bodies (BB) (C, D, G) but not in other parts of the CC (F, H). In the apical region of the IS, Pcnt is located at the centriole (C) (F, G, H) at the BBC (E) but not in the cytoplasm of the calycal process (CP) (C, D, H). N: nucleus; CR: ciliary rootlet. Scale bars: 0.5 µm (B, C, D, H), 0.2 µm (E, F, G).

Mentions: To confirm the Pcnt localization at the BBC of the photoreceptor connecting cilium, we performed an immunoelectron microscopical analysis using the MmPeriC1 antiserum (Fig. 5). Figure 5A shows a schematic illustration of a rod photoreceptor and an enlarged view of the ciliary region between the inner and the outer segment of the photoreceptor is shown in Figure 5B. Employing the method of preembedding immunoelectron microscopy [24], we successfully localized Pcnt at the region of the BBC (Fig. 5C–H). Pcnt was present at the surface of the microtubules of the BB and its centriole (Fig. 5C, D, G, H). Furthermore Pcnt staining surrounded the BBC, where the PCM is located (Fig. 5E, G). No labeling for Pcnt was found inside the connecting cilium (Fig. 5F). Our immunoelectron microscopical examination also confirmed the localization of Pcnt at the centrioles of the centrosomes of non-photoreceptor cells in the retina (data not shown).


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)

Immunoelectron microscopic localization of Pericentrin in mouse photoreceptors.(A) Scheme of a mouse rod photoreceptor with the tip of the photoreceptor’s light-sensitive outer segment (OS) enclosed by the retinal pigment epithelium (RPE). (B) Scheme of the ciliary apparatus. The photosensitive OS is linked by the connecting cilium (CC) to the metabolic inner segment (IS), which includes the basal body complex (BBC) in its apical region. (C–H) Electron micrographs showing the ultrastructural localization of Pcnt in the photoreceptor ciliary region. Longitudinal (C, D, G, H) and cross sections (E, F) through the ciliary region show the presence of Pcnt at the basal bodies (BB) (C, D, G) but not in other parts of the CC (F, H). In the apical region of the IS, Pcnt is located at the centriole (C) (F, G, H) at the BBC (E) but not in the cytoplasm of the calycal process (CP) (C, D, H). N: nucleus; CR: ciliary rootlet. Scale bars: 0.5 µm (B, C, D, H), 0.2 µm (E, F, G).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198765&req=5

pone-0026496-g005: Immunoelectron microscopic localization of Pericentrin in mouse photoreceptors.(A) Scheme of a mouse rod photoreceptor with the tip of the photoreceptor’s light-sensitive outer segment (OS) enclosed by the retinal pigment epithelium (RPE). (B) Scheme of the ciliary apparatus. The photosensitive OS is linked by the connecting cilium (CC) to the metabolic inner segment (IS), which includes the basal body complex (BBC) in its apical region. (C–H) Electron micrographs showing the ultrastructural localization of Pcnt in the photoreceptor ciliary region. Longitudinal (C, D, G, H) and cross sections (E, F) through the ciliary region show the presence of Pcnt at the basal bodies (BB) (C, D, G) but not in other parts of the CC (F, H). In the apical region of the IS, Pcnt is located at the centriole (C) (F, G, H) at the BBC (E) but not in the cytoplasm of the calycal process (CP) (C, D, H). N: nucleus; CR: ciliary rootlet. Scale bars: 0.5 µm (B, C, D, H), 0.2 µm (E, F, G).
Mentions: To confirm the Pcnt localization at the BBC of the photoreceptor connecting cilium, we performed an immunoelectron microscopical analysis using the MmPeriC1 antiserum (Fig. 5). Figure 5A shows a schematic illustration of a rod photoreceptor and an enlarged view of the ciliary region between the inner and the outer segment of the photoreceptor is shown in Figure 5B. Employing the method of preembedding immunoelectron microscopy [24], we successfully localized Pcnt at the region of the BBC (Fig. 5C–H). Pcnt was present at the surface of the microtubules of the BB and its centriole (Fig. 5C, D, G, H). Furthermore Pcnt staining surrounded the BBC, where the PCM is located (Fig. 5E, G). No labeling for Pcnt was found inside the connecting cilium (Fig. 5F). Our immunoelectron microscopical examination also confirmed the localization of Pcnt at the centrioles of the centrosomes of non-photoreceptor cells in the retina (data not shown).

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