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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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Localization of Pericentrin at mouse olfactory cilia.(A–C) Micrographs of a cryostat section through an adult mouse olfactory epithelium double labeled with the MmPeriC1 antiserum against Pcnt (A and A', green) and an antibody against Cen3 (B and B', red), as a marker for the ciliary apparatus. (A) Pcnt is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of the other cells in the mouse olfactory epithelium. (A') The higher magnification shows a punctate staining for Pcnt inside the olfactory knobs of the receptor neurons. (B) Cen3, like Pcnt, is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of the other cells in the mouse olfactory epithelium. (B') The higher magnification shows that Cen3 is preferentially localized at the ciliary surface of the olfactory knobs of the receptor neurons. (C) Merge of the stainings for Pcnt (green) and Cen3 (red) combined with a DAPI nuclear staining (blue). (C') The higher magnification of the merged signals shows a partial colocalization of Pcnt and Cen3 at the ciliary apparatus of the olfactory knobs of the receptor neurons. (D) Schematic drawing of an olfactory receptor neuron and a higher-power view of its olfactory knob with the localization of Pcnt (green) at the basal body complexes (BBCs) and of Cen3 (red) at the transition zones (TZs) of the BBCs of the olfactory cilia (OC). (E–G) Micrographs of a section through an adult mouse olfactory epithelium double labeled for Pcnt (E, green) and ac. tubulin (F, red) as a marker for the axoneme of olfactory cilia. (E) Pcnt is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of other cells in the mouse olfactory epithelium. (F) Ac. tubulin, like Pcnt, is localized in the ciliary region of the olfactory receptor neurons. (G) The merge of the stainings combined with a DAPI nuclear stain (blue) shows a partial colocalization of Pcnt and ac. tubulin in the ciliary region of the olfactory receptor neurons. C: centriole; BB: basal body. Scale bars: 20 µm (C G), 5 µm (C').
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pone-0026496-g003: Localization of Pericentrin at mouse olfactory cilia.(A–C) Micrographs of a cryostat section through an adult mouse olfactory epithelium double labeled with the MmPeriC1 antiserum against Pcnt (A and A', green) and an antibody against Cen3 (B and B', red), as a marker for the ciliary apparatus. (A) Pcnt is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of the other cells in the mouse olfactory epithelium. (A') The higher magnification shows a punctate staining for Pcnt inside the olfactory knobs of the receptor neurons. (B) Cen3, like Pcnt, is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of the other cells in the mouse olfactory epithelium. (B') The higher magnification shows that Cen3 is preferentially localized at the ciliary surface of the olfactory knobs of the receptor neurons. (C) Merge of the stainings for Pcnt (green) and Cen3 (red) combined with a DAPI nuclear staining (blue). (C') The higher magnification of the merged signals shows a partial colocalization of Pcnt and Cen3 at the ciliary apparatus of the olfactory knobs of the receptor neurons. (D) Schematic drawing of an olfactory receptor neuron and a higher-power view of its olfactory knob with the localization of Pcnt (green) at the basal body complexes (BBCs) and of Cen3 (red) at the transition zones (TZs) of the BBCs of the olfactory cilia (OC). (E–G) Micrographs of a section through an adult mouse olfactory epithelium double labeled for Pcnt (E, green) and ac. tubulin (F, red) as a marker for the axoneme of olfactory cilia. (E) Pcnt is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of other cells in the mouse olfactory epithelium. (F) Ac. tubulin, like Pcnt, is localized in the ciliary region of the olfactory receptor neurons. (G) The merge of the stainings combined with a DAPI nuclear stain (blue) shows a partial colocalization of Pcnt and ac. tubulin in the ciliary region of the olfactory receptor neurons. C: centriole; BB: basal body. Scale bars: 20 µm (C G), 5 µm (C').

Mentions: D-PLP is the orthologue protein of Pcnt in Drosophila, and D-PLP-mutant flies were shown to display malformed sensory cilia in the mechanosensory and chemosensory neurons [14], [29]. Ciliated sensory neurons are also present in mammals, e.g. the receptor neurons of the olfactory epithelium, which are specialized in odorant detection. In 2009 Miyoshi and co-workers showed that splice variants of Pcnt are essential for the assembly of the chemosensory cilia in the olfactory receptor neurons. To verify the localization of Pcnt in the olfactory receptor neurons of wild type mice, we stained cryostat sections of the olfactory epithelium with the MmPeriC1 antiserum. To show the exact localization of Pcnt at the olfactory cilium of the receptor neurons, we used the Cen3 antibody as a marker of the transition zone of the ciliary axoneme and the BBC [19], [21]. Pcnt was partially colocalized with Cen3, which is consistent with the position of the BBCs in the dendritic knob of the olfactory receptor neurons in the wild type mouse (Fig. 3A–D). The localization of Pcnt at the BBCs below the ciliary layer is best seen in the high power views shown in the insets in Figure 3 (Fig. 3A'–C'). Additional double labeling experiments using ac. tubulin to mark the complete ciliary axoneme confirmed the localization of Pcnt at the olfactory cilium (Fig. 3E–G). In addition to Pcnt labeling in the receptor neurons, Pcnt was present at the centrosomes of the other cell types in the mouse olfactory epithelium (Fig. 3).


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)

Localization of Pericentrin at mouse olfactory cilia.(A–C) Micrographs of a cryostat section through an adult mouse olfactory epithelium double labeled with the MmPeriC1 antiserum against Pcnt (A and A', green) and an antibody against Cen3 (B and B', red), as a marker for the ciliary apparatus. (A) Pcnt is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of the other cells in the mouse olfactory epithelium. (A') The higher magnification shows a punctate staining for Pcnt inside the olfactory knobs of the receptor neurons. (B) Cen3, like Pcnt, is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of the other cells in the mouse olfactory epithelium. (B') The higher magnification shows that Cen3 is preferentially localized at the ciliary surface of the olfactory knobs of the receptor neurons. (C) Merge of the stainings for Pcnt (green) and Cen3 (red) combined with a DAPI nuclear staining (blue). (C') The higher magnification of the merged signals shows a partial colocalization of Pcnt and Cen3 at the ciliary apparatus of the olfactory knobs of the receptor neurons. (D) Schematic drawing of an olfactory receptor neuron and a higher-power view of its olfactory knob with the localization of Pcnt (green) at the basal body complexes (BBCs) and of Cen3 (red) at the transition zones (TZs) of the BBCs of the olfactory cilia (OC). (E–G) Micrographs of a section through an adult mouse olfactory epithelium double labeled for Pcnt (E, green) and ac. tubulin (F, red) as a marker for the axoneme of olfactory cilia. (E) Pcnt is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of other cells in the mouse olfactory epithelium. (F) Ac. tubulin, like Pcnt, is localized in the ciliary region of the olfactory receptor neurons. (G) The merge of the stainings combined with a DAPI nuclear stain (blue) shows a partial colocalization of Pcnt and ac. tubulin in the ciliary region of the olfactory receptor neurons. C: centriole; BB: basal body. Scale bars: 20 µm (C G), 5 µm (C').
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198765&req=5

pone-0026496-g003: Localization of Pericentrin at mouse olfactory cilia.(A–C) Micrographs of a cryostat section through an adult mouse olfactory epithelium double labeled with the MmPeriC1 antiserum against Pcnt (A and A', green) and an antibody against Cen3 (B and B', red), as a marker for the ciliary apparatus. (A) Pcnt is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of the other cells in the mouse olfactory epithelium. (A') The higher magnification shows a punctate staining for Pcnt inside the olfactory knobs of the receptor neurons. (B) Cen3, like Pcnt, is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of the other cells in the mouse olfactory epithelium. (B') The higher magnification shows that Cen3 is preferentially localized at the ciliary surface of the olfactory knobs of the receptor neurons. (C) Merge of the stainings for Pcnt (green) and Cen3 (red) combined with a DAPI nuclear staining (blue). (C') The higher magnification of the merged signals shows a partial colocalization of Pcnt and Cen3 at the ciliary apparatus of the olfactory knobs of the receptor neurons. (D) Schematic drawing of an olfactory receptor neuron and a higher-power view of its olfactory knob with the localization of Pcnt (green) at the basal body complexes (BBCs) and of Cen3 (red) at the transition zones (TZs) of the BBCs of the olfactory cilia (OC). (E–G) Micrographs of a section through an adult mouse olfactory epithelium double labeled for Pcnt (E, green) and ac. tubulin (F, red) as a marker for the axoneme of olfactory cilia. (E) Pcnt is localized in the ciliary region of the olfactory receptor neurons and at the centrosomes of other cells in the mouse olfactory epithelium. (F) Ac. tubulin, like Pcnt, is localized in the ciliary region of the olfactory receptor neurons. (G) The merge of the stainings combined with a DAPI nuclear stain (blue) shows a partial colocalization of Pcnt and ac. tubulin in the ciliary region of the olfactory receptor neurons. C: centriole; BB: basal body. Scale bars: 20 µm (C G), 5 µm (C').
Mentions: D-PLP is the orthologue protein of Pcnt in Drosophila, and D-PLP-mutant flies were shown to display malformed sensory cilia in the mechanosensory and chemosensory neurons [14], [29]. Ciliated sensory neurons are also present in mammals, e.g. the receptor neurons of the olfactory epithelium, which are specialized in odorant detection. In 2009 Miyoshi and co-workers showed that splice variants of Pcnt are essential for the assembly of the chemosensory cilia in the olfactory receptor neurons. To verify the localization of Pcnt in the olfactory receptor neurons of wild type mice, we stained cryostat sections of the olfactory epithelium with the MmPeriC1 antiserum. To show the exact localization of Pcnt at the olfactory cilium of the receptor neurons, we used the Cen3 antibody as a marker of the transition zone of the ciliary axoneme and the BBC [19], [21]. Pcnt was partially colocalized with Cen3, which is consistent with the position of the BBCs in the dendritic knob of the olfactory receptor neurons in the wild type mouse (Fig. 3A–D). The localization of Pcnt at the BBCs below the ciliary layer is best seen in the high power views shown in the insets in Figure 3 (Fig. 3A'–C'). Additional double labeling experiments using ac. tubulin to mark the complete ciliary axoneme confirmed the localization of Pcnt at the olfactory cilium (Fig. 3E–G). In addition to Pcnt labeling in the receptor neurons, Pcnt was present at the centrosomes of the other cell types in the mouse olfactory epithelium (Fig. 3).

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