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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 different mouse tissues.(A) RT-PCR made with cDNA from retina, olfactory epithelium (olf. epith.) and NIH 3T3 mouse fibroblasts 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 retina and in NIH 3T3 mouse fibroblasts, but not in the olfactory epithelium. Pcnt B is expressed in all three tested probes. The β-actin positive control demonstrates the approximate amount and the quality of the used cDNA. (B) Western blot analysis of protein extracts of retina, olfactory epithelium and NIH 3T3 mouse fibroblasts using the MmPeriC1 antiserum. A 360 kDa protein band is detected in all three samples. A second protein band with varying molecular weight – approximately 250 kDa in olfactory epithelium and NIH 3T3 mouse fibroblasts, approximately 225 kDa in retina – suggests the existence of different Pcnt variants in different tissues. (C) Preadsorption control of the MmPeric1 antiserum with the respective antigen in saturating concentrations blocks the detection of the protein bands in all three tissue samples.
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pone-0026496-g007: Expression of Pericentrin splice variants in different mouse tissues.(A) RT-PCR made with cDNA from retina, olfactory epithelium (olf. epith.) and NIH 3T3 mouse fibroblasts 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 retina and in NIH 3T3 mouse fibroblasts, but not in the olfactory epithelium. Pcnt B is expressed in all three tested probes. The β-actin positive control demonstrates the approximate amount and the quality of the used cDNA. (B) Western blot analysis of protein extracts of retina, olfactory epithelium and NIH 3T3 mouse fibroblasts using the MmPeriC1 antiserum. A 360 kDa protein band is detected in all three samples. A second protein band with varying molecular weight – approximately 250 kDa in olfactory epithelium and NIH 3T3 mouse fibroblasts, approximately 225 kDa in retina – suggests the existence of different Pcnt variants in different tissues. (C) Preadsorption control of the MmPeric1 antiserum with the respective antigen in saturating concentrations blocks the detection of the protein bands in all three tissue samples.

Mentions: The Western blot analysis of NIH 3T3 mouse fibroblasts using the MmPeriC1 antiserum suggested the expression of two Pcnt splice variants (Fig. 1F). In a next step, we therefore analyzed the retina and the olfactory epithelium for the presence of Pcnt splice variants. We performed RT-PCR analysis with cDNA from the retina, the olfactory epithelium and NIH 3T3 mouse fibroblasts using highly specific primer sets for the Pcnt splice variants Pcnt S and Pcnt B (Fig. 7A). The experiments revealed that Pcnt S is expressed in the retina and in NIH 3T3 mouse fibroblasts, but not in the olfactory epithelium. However, Pcnt B is expressed in all three tested probes (Fig. 7A). In our analysis concerning Pcnt A, we found discrepancies in the sequence compared to the published sequence (GenBank/EBI/DDBJ accession number Pcnt A partial, AAO24322.1). For this reason we were not able to create a set of primers for which we could guarantee absolute specificity for the splice variant called Pcnt A and decided to concentrate only on Pcnt S and Pcnt B on mRNA-level.


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 different mouse tissues.(A) RT-PCR made with cDNA from retina, olfactory epithelium (olf. epith.) and NIH 3T3 mouse fibroblasts 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 retina and in NIH 3T3 mouse fibroblasts, but not in the olfactory epithelium. Pcnt B is expressed in all three tested probes. The β-actin positive control demonstrates the approximate amount and the quality of the used cDNA. (B) Western blot analysis of protein extracts of retina, olfactory epithelium and NIH 3T3 mouse fibroblasts using the MmPeriC1 antiserum. A 360 kDa protein band is detected in all three samples. A second protein band with varying molecular weight – approximately 250 kDa in olfactory epithelium and NIH 3T3 mouse fibroblasts, approximately 225 kDa in retina – suggests the existence of different Pcnt variants in different tissues. (C) Preadsorption control of the MmPeric1 antiserum with the respective antigen in saturating concentrations blocks the detection of the protein bands in all three tissue samples.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026496-g007: Expression of Pericentrin splice variants in different mouse tissues.(A) RT-PCR made with cDNA from retina, olfactory epithelium (olf. epith.) and NIH 3T3 mouse fibroblasts 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 retina and in NIH 3T3 mouse fibroblasts, but not in the olfactory epithelium. Pcnt B is expressed in all three tested probes. The β-actin positive control demonstrates the approximate amount and the quality of the used cDNA. (B) Western blot analysis of protein extracts of retina, olfactory epithelium and NIH 3T3 mouse fibroblasts using the MmPeriC1 antiserum. A 360 kDa protein band is detected in all three samples. A second protein band with varying molecular weight – approximately 250 kDa in olfactory epithelium and NIH 3T3 mouse fibroblasts, approximately 225 kDa in retina – suggests the existence of different Pcnt variants in different tissues. (C) Preadsorption control of the MmPeric1 antiserum with the respective antigen in saturating concentrations blocks the detection of the protein bands in all three tissue samples.
Mentions: The Western blot analysis of NIH 3T3 mouse fibroblasts using the MmPeriC1 antiserum suggested the expression of two Pcnt splice variants (Fig. 1F). In a next step, we therefore analyzed the retina and the olfactory epithelium for the presence of Pcnt splice variants. We performed RT-PCR analysis with cDNA from the retina, the olfactory epithelium and NIH 3T3 mouse fibroblasts using highly specific primer sets for the Pcnt splice variants Pcnt S and Pcnt B (Fig. 7A). The experiments revealed that Pcnt S is expressed in the retina and in NIH 3T3 mouse fibroblasts, but not in the olfactory epithelium. However, Pcnt B is expressed in all three tested probes (Fig. 7A). In our analysis concerning Pcnt A, we found discrepancies in the sequence compared to the published sequence (GenBank/EBI/DDBJ accession number Pcnt A partial, AAO24322.1). For this reason we were not able to create a set of primers for which we could guarantee absolute specificity for the splice variant called Pcnt A and decided to concentrate only on Pcnt S and Pcnt B on mRNA-level.

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