Limits...
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.

Show MeSH

Related in: MedlinePlus

Localization of Pericentrin at connecting cilia and at centrosomes in the mouse retina.(A–D) Micrographs of a vertical cryostat section through an adult mouse retina double labeled for Pcnt (A, green) and Cen3 (B, red). (C, D) As seen in the merge of the stainings with the additional labeling of the cell nuclei with DAPI, Pcnt and Cen3 colocalize at the ciliary region of the photoreceptors and at the centrosomes of the other retinal cells. (E) Preadsorption control of the MmPeriC1 antiserum with its antigenic peptide results in the absence of Pcnt labeling. (F–M) Subcellular localization of Pcnt (F, J, green) and Cen3 (G, K, red) at the BBC of the photoreceptor connecting cilia (F–H) and at the centrosomes of non-photoreceptor cells in the INL (J–L). (H') Higher power view showing the partial colocalization of Pcnt and Cen3 at the BBC. (L) In non-photoreceptor cells, Pcnt and Cen3 colocalize at the centrioles of the centrosomes. (I, M) Schemes summarizing the subcellular localization of Pcnt and Cen3 at the connecting cilium of photoreceptors (I) and at the centrosomes of non-photoreceptor cells (M). OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer; BB: basal body; C: centriole; CC: connecting cilium; CP: calycal process; MTs: microtubules; N: nucleus. Scale bars: 20 µm (D, F), 2 µm (I), 1 µm (I'), 2 µm (M).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198765&req=5

pone-0026496-g004: Localization of Pericentrin at connecting cilia and at centrosomes in the mouse retina.(A–D) Micrographs of a vertical cryostat section through an adult mouse retina double labeled for Pcnt (A, green) and Cen3 (B, red). (C, D) As seen in the merge of the stainings with the additional labeling of the cell nuclei with DAPI, Pcnt and Cen3 colocalize at the ciliary region of the photoreceptors and at the centrosomes of the other retinal cells. (E) Preadsorption control of the MmPeriC1 antiserum with its antigenic peptide results in the absence of Pcnt labeling. (F–M) Subcellular localization of Pcnt (F, J, green) and Cen3 (G, K, red) at the BBC of the photoreceptor connecting cilia (F–H) and at the centrosomes of non-photoreceptor cells in the INL (J–L). (H') Higher power view showing the partial colocalization of Pcnt and Cen3 at the BBC. (L) In non-photoreceptor cells, Pcnt and Cen3 colocalize at the centrioles of the centrosomes. (I, M) Schemes summarizing the subcellular localization of Pcnt and Cen3 at the connecting cilium of photoreceptors (I) and at the centrosomes of non-photoreceptor cells (M). OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer; BB: basal body; C: centriole; CC: connecting cilium; CP: calycal process; MTs: microtubules; N: nucleus. Scale bars: 20 µm (D, F), 2 µm (I), 1 µm (I'), 2 µm (M).

Mentions: To determine the distribution of Pcnt in the mouse retina, cryostat sections through whole unfixed eyes were analyzed with immunofluorescence microscopy using the MmPeriC1 antiserum. Punctate staining for Pcnt was present in the region between the inner (IS) and the outer segments (OS) of the photoreceptors, in the outer nuclear layer (ONL), the inner nuclear layer (INL), and the ganglion cell layer (GCL) (Fig. 4A–D). Double labeling with the Cen3 antibody, which stained the whole photoreceptor connecting cilium corresponding to the transition zone of a prototypic cilium [19], showed a colocalization of Pcnt and Cen3 at the ciliary region of the photoreceptors and at the centrosomes of the other cells in the retina (Fig. 4C, D). High magnification imaging further revealed a partial colocalization of Pcnt and Cen3 at the BBC of the photoreceptor's connecting cilium (Fig. 4F–I, Fig. S1A–D), and, like in other primary cilia (Fig. 1E), Pcnt is present at the PCM, which ensheaths the BBC (Fig. 4I, Fig. S1). Additional high resolution 3D reconstructions of the connecting cilium stained with Pcnt and Cen3 (Fig. S1A–D) and with Pcnt and ac. tubulin (Fig. S1F–I) demonstrate that Pcnt surrounds the BBC of the connecting cilium in photoreceptors (Fig. S1). These results assign Pcnt as a component of the BBC and the PCM of the connecting cilium in photoreceptors, the only intracellular bridge between the metabolic inner and the light sensitive outer segment of the photoreceptor. In the cells of the INL and the GCL, Pcnt localized to the mother and daughter centrioles and to the PCM of the centrosomes. Here Pcnt colocalized with Cen3 (Fig. 4J–M, Fig. S1E) and other centrosomal markers (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)

Localization of Pericentrin at connecting cilia and at centrosomes in the mouse retina.(A–D) Micrographs of a vertical cryostat section through an adult mouse retina double labeled for Pcnt (A, green) and Cen3 (B, red). (C, D) As seen in the merge of the stainings with the additional labeling of the cell nuclei with DAPI, Pcnt and Cen3 colocalize at the ciliary region of the photoreceptors and at the centrosomes of the other retinal cells. (E) Preadsorption control of the MmPeriC1 antiserum with its antigenic peptide results in the absence of Pcnt labeling. (F–M) Subcellular localization of Pcnt (F, J, green) and Cen3 (G, K, red) at the BBC of the photoreceptor connecting cilia (F–H) and at the centrosomes of non-photoreceptor cells in the INL (J–L). (H') Higher power view showing the partial colocalization of Pcnt and Cen3 at the BBC. (L) In non-photoreceptor cells, Pcnt and Cen3 colocalize at the centrioles of the centrosomes. (I, M) Schemes summarizing the subcellular localization of Pcnt and Cen3 at the connecting cilium of photoreceptors (I) and at the centrosomes of non-photoreceptor cells (M). OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer; BB: basal body; C: centriole; CC: connecting cilium; CP: calycal process; MTs: microtubules; N: nucleus. Scale bars: 20 µm (D, F), 2 µm (I), 1 µm (I'), 2 µm (M).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198765&req=5

pone-0026496-g004: Localization of Pericentrin at connecting cilia and at centrosomes in the mouse retina.(A–D) Micrographs of a vertical cryostat section through an adult mouse retina double labeled for Pcnt (A, green) and Cen3 (B, red). (C, D) As seen in the merge of the stainings with the additional labeling of the cell nuclei with DAPI, Pcnt and Cen3 colocalize at the ciliary region of the photoreceptors and at the centrosomes of the other retinal cells. (E) Preadsorption control of the MmPeriC1 antiserum with its antigenic peptide results in the absence of Pcnt labeling. (F–M) Subcellular localization of Pcnt (F, J, green) and Cen3 (G, K, red) at the BBC of the photoreceptor connecting cilia (F–H) and at the centrosomes of non-photoreceptor cells in the INL (J–L). (H') Higher power view showing the partial colocalization of Pcnt and Cen3 at the BBC. (L) In non-photoreceptor cells, Pcnt and Cen3 colocalize at the centrioles of the centrosomes. (I, M) Schemes summarizing the subcellular localization of Pcnt and Cen3 at the connecting cilium of photoreceptors (I) and at the centrosomes of non-photoreceptor cells (M). OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer; BB: basal body; C: centriole; CC: connecting cilium; CP: calycal process; MTs: microtubules; N: nucleus. Scale bars: 20 µm (D, F), 2 µm (I), 1 µm (I'), 2 µm (M).
Mentions: To determine the distribution of Pcnt in the mouse retina, cryostat sections through whole unfixed eyes were analyzed with immunofluorescence microscopy using the MmPeriC1 antiserum. Punctate staining for Pcnt was present in the region between the inner (IS) and the outer segments (OS) of the photoreceptors, in the outer nuclear layer (ONL), the inner nuclear layer (INL), and the ganglion cell layer (GCL) (Fig. 4A–D). Double labeling with the Cen3 antibody, which stained the whole photoreceptor connecting cilium corresponding to the transition zone of a prototypic cilium [19], showed a colocalization of Pcnt and Cen3 at the ciliary region of the photoreceptors and at the centrosomes of the other cells in the retina (Fig. 4C, D). High magnification imaging further revealed a partial colocalization of Pcnt and Cen3 at the BBC of the photoreceptor's connecting cilium (Fig. 4F–I, Fig. S1A–D), and, like in other primary cilia (Fig. 1E), Pcnt is present at the PCM, which ensheaths the BBC (Fig. 4I, Fig. S1). Additional high resolution 3D reconstructions of the connecting cilium stained with Pcnt and Cen3 (Fig. S1A–D) and with Pcnt and ac. tubulin (Fig. S1F–I) demonstrate that Pcnt surrounds the BBC of the connecting cilium in photoreceptors (Fig. S1). These results assign Pcnt as a component of the BBC and the PCM of the connecting cilium in photoreceptors, the only intracellular bridge between the metabolic inner and the light sensitive outer segment of the photoreceptor. In the cells of the INL and the GCL, Pcnt localized to the mother and daughter centrioles and to the PCM of the centrosomes. Here Pcnt colocalized with Cen3 (Fig. 4J–M, Fig. S1E) and other centrosomal markers (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