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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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Expression of Pericentrin splice variants in separated mouse photoreceptors.(A–D) Illustration of the method of laser microdissection to separate parts of the photoreceptor cell layer or of the residual retinal layers. (E) RT-PCR made with cDNA from dissected parts of the photoreceptor cell layer (PhRL) and from dissected parts of the rest of the retina (rest retina) using specific primer sets for Pcnt S, Pcnt B and β-actin as a positive control. All three primer sets and the used reagents were tested by a negative control without cDNA (control). Pcnt S is expressed in the PhRL and in the rest of the retina, but the signal in the PhRL is slightly stronger than in the rest of the retina. Pcnt B is expressed in the PhRL and in the rest of the retina, but the signal in the PhRL is slightly weaker than in the rest of the retina. The β-actin positive control demonstrates the approximate amount and the quality of the used cDNA. (F) In Western blot analysis of extracts of dissected parts of the PhRL two specific bands with a molecular weight of approximately 360 kDa and 225 kDa were detected. The two bands represent the two Pcnt splice variants of the photoreceptors. The smaller 225 kDa Pcnt form, most likely a variant of Pcnt S, is much stronger expressed than the larger Pcnt B 360 kDa form.
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pone-0026496-g008: Expression of Pericentrin splice variants in separated mouse photoreceptors.(A–D) Illustration of the method of laser microdissection to separate parts of the photoreceptor cell layer or of the residual retinal layers. (E) RT-PCR made with cDNA from dissected parts of the photoreceptor cell layer (PhRL) and from dissected parts of the rest of the retina (rest retina) using specific primer sets for Pcnt S, Pcnt B and β-actin as a positive control. All three primer sets and the used reagents were tested by a negative control without cDNA (control). Pcnt S is expressed in the PhRL and in the rest of the retina, but the signal in the PhRL is slightly stronger than in the rest of the retina. Pcnt B is expressed in the PhRL and in the rest of the retina, but the signal in the PhRL is slightly weaker than in the rest of the retina. The β-actin positive control demonstrates the approximate amount and the quality of the used cDNA. (F) In Western blot analysis of extracts of dissected parts of the PhRL two specific bands with a molecular weight of approximately 360 kDa and 225 kDa were detected. The two bands represent the two Pcnt splice variants of the photoreceptors. The smaller 225 kDa Pcnt form, most likely a variant of Pcnt S, is much stronger expressed than the larger Pcnt B 360 kDa form.

Mentions: To examine the expression of Pcnt in mouse photoreceptors in more detail, we first double labeled isolated mouse photoreceptors for Pcnt and Opsin, a marker for the photoreceptor outer segment (Fig. S2A–D). The two Pcnt immunoreactive puncta present at the base of the ciliary region of an isolated photoreceptor clearly identify Pcnt as a component of the BBC and the PCM of the connecting cilium (Fig. S2A–D). Knowing that two Pcnt splice variants are present in whole retinal tissue (Fig. 7A–B), we next analyzed the specific expression pattern of Pcnt in photoreceptors (Fig. 8A–F). As there are no splice variant specific Pcnt antibodies available to distinguish between the transcripts immunocytochemically, we first separated parts of the photoreceptor layer from the rest of the retina with the method of laser microdissection, followed by RT-PCR and Western blot analysis (Fig. 8A–F). The Pcnt splice variants found in whole retinal tissue (Fig. 7A–B) were also present in the photoreceptors (Fig. 8E–F). We performed RT-PCR analysis with cDNA from dissected parts of the photoreceptor cell layer and from dissected parts of the rest of the retina using highly specific primer sets for the Pcnt splice variants Pcnt S and Pcnt B. The experiments revealed that Pcnt S is slightly stronger expressed in the photoreceptor cell layer than in the rest of the retina (Fig. 8E). However, Pcnt B is slightly weaker expressed in the photoreceptor cell layer than in the rest of the retina (Fig. 8E).


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)

Expression of Pericentrin splice variants in separated mouse photoreceptors.(A–D) Illustration of the method of laser microdissection to separate parts of the photoreceptor cell layer or of the residual retinal layers. (E) RT-PCR made with cDNA from dissected parts of the photoreceptor cell layer (PhRL) and from dissected parts of the rest of the retina (rest retina) using specific primer sets for Pcnt S, Pcnt B and β-actin as a positive control. All three primer sets and the used reagents were tested by a negative control without cDNA (control). Pcnt S is expressed in the PhRL and in the rest of the retina, but the signal in the PhRL is slightly stronger than in the rest of the retina. Pcnt B is expressed in the PhRL and in the rest of the retina, but the signal in the PhRL is slightly weaker than in the rest of the retina. The β-actin positive control demonstrates the approximate amount and the quality of the used cDNA. (F) In Western blot analysis of extracts of dissected parts of the PhRL two specific bands with a molecular weight of approximately 360 kDa and 225 kDa were detected. The two bands represent the two Pcnt splice variants of the photoreceptors. The smaller 225 kDa Pcnt form, most likely a variant of Pcnt S, is much stronger expressed than the larger Pcnt B 360 kDa form.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198765&req=5

pone-0026496-g008: Expression of Pericentrin splice variants in separated mouse photoreceptors.(A–D) Illustration of the method of laser microdissection to separate parts of the photoreceptor cell layer or of the residual retinal layers. (E) RT-PCR made with cDNA from dissected parts of the photoreceptor cell layer (PhRL) and from dissected parts of the rest of the retina (rest retina) using specific primer sets for Pcnt S, Pcnt B and β-actin as a positive control. All three primer sets and the used reagents were tested by a negative control without cDNA (control). Pcnt S is expressed in the PhRL and in the rest of the retina, but the signal in the PhRL is slightly stronger than in the rest of the retina. Pcnt B is expressed in the PhRL and in the rest of the retina, but the signal in the PhRL is slightly weaker than in the rest of the retina. The β-actin positive control demonstrates the approximate amount and the quality of the used cDNA. (F) In Western blot analysis of extracts of dissected parts of the PhRL two specific bands with a molecular weight of approximately 360 kDa and 225 kDa were detected. The two bands represent the two Pcnt splice variants of the photoreceptors. The smaller 225 kDa Pcnt form, most likely a variant of Pcnt S, is much stronger expressed than the larger Pcnt B 360 kDa form.
Mentions: To examine the expression of Pcnt in mouse photoreceptors in more detail, we first double labeled isolated mouse photoreceptors for Pcnt and Opsin, a marker for the photoreceptor outer segment (Fig. S2A–D). The two Pcnt immunoreactive puncta present at the base of the ciliary region of an isolated photoreceptor clearly identify Pcnt as a component of the BBC and the PCM of the connecting cilium (Fig. S2A–D). Knowing that two Pcnt splice variants are present in whole retinal tissue (Fig. 7A–B), we next analyzed the specific expression pattern of Pcnt in photoreceptors (Fig. 8A–F). As there are no splice variant specific Pcnt antibodies available to distinguish between the transcripts immunocytochemically, we first separated parts of the photoreceptor layer from the rest of the retina with the method of laser microdissection, followed by RT-PCR and Western blot analysis (Fig. 8A–F). The Pcnt splice variants found in whole retinal tissue (Fig. 7A–B) were also present in the photoreceptors (Fig. 8E–F). We performed RT-PCR analysis with cDNA from dissected parts of the photoreceptor cell layer and from dissected parts of the rest of the retina using highly specific primer sets for the Pcnt splice variants Pcnt S and Pcnt B. The experiments revealed that Pcnt S is slightly stronger expressed in the photoreceptor cell layer than in the rest of the retina (Fig. 8E). However, Pcnt B is slightly weaker expressed in the photoreceptor cell layer than in the rest of the retina (Fig. 8E).

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