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

Characterization of the Pericentrin antiserum MmPeriC1.(A) Scheme of the known and published Pcnt splice variants: Pcnt B (360, accession number (AN): NP_032813 or BAF36559), Pcnt A (AN: partial, AAO24322.1) and Pcnt S (250, BAF36560). The 280 amino-acid epitope against which the MmPeriC1 antiserum was generated is indicated in grey. (B-D) Triple labeling of Pcnt (B, green), ac. tubulin (C, red), and DAPI (D merge, blue) in NIH 3T3 mouse fibroblasts. Pcnt colocalizes partially with ac. tubulin at the BBC (arrowheads) of primary cilia (PC). (E) High resolution 3D reconstruction demonstrates that Pcnt (green) ensheaths the BBC of the PC (red, ac. tubulin). (F, G) Western blot analysis using the MmPeriC1 antiserum. (F) The antiserum detects a 360 kDa and a 240–250 kDa protein band. (G) Preincubation of the MmPeriC1 antiserum with the antigen (recombinant fusion protein) blocks the immunodetection of the two bands. (H) Preadsorption of the MmPeriC1 antiserum with the antigen results in the absence of Pcnt staining at the BBC at primary cilia labeled with ac. tubulin. Scale bar: 5 µm (D, H).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198765&req=5

pone-0026496-g001: Characterization of the Pericentrin antiserum MmPeriC1.(A) Scheme of the known and published Pcnt splice variants: Pcnt B (360, accession number (AN): NP_032813 or BAF36559), Pcnt A (AN: partial, AAO24322.1) and Pcnt S (250, BAF36560). The 280 amino-acid epitope against which the MmPeriC1 antiserum was generated is indicated in grey. (B-D) Triple labeling of Pcnt (B, green), ac. tubulin (C, red), and DAPI (D merge, blue) in NIH 3T3 mouse fibroblasts. Pcnt colocalizes partially with ac. tubulin at the BBC (arrowheads) of primary cilia (PC). (E) High resolution 3D reconstruction demonstrates that Pcnt (green) ensheaths the BBC of the PC (red, ac. tubulin). (F, G) Western blot analysis using the MmPeriC1 antiserum. (F) The antiserum detects a 360 kDa and a 240–250 kDa protein band. (G) Preincubation of the MmPeriC1 antiserum with the antigen (recombinant fusion protein) blocks the immunodetection of the two bands. (H) Preadsorption of the MmPeriC1 antiserum with the antigen results in the absence of Pcnt staining at the BBC at primary cilia labeled with ac. tubulin. Scale bar: 5 µm (D, H).

Mentions: To study the localization of Pcnt in the olfactory epithelium and the retina of the mouse, we first generated and affinity purified a specific polyclonal antiserum against a recombinant expressed part of murine Pcnt (Fig. 1A, Fig. S3A). The Pcnt antiserum, MmPeriC1, should detect all to date known Pcnt splice variants because of the immunogenic region against which the MmPeriC1 antiserum is directed (Fig. 1A). To validate the specificity of the MmPeriC1 antiserum, we examined the localization of Pcnt in cultured NIH 3T3 mouse fibroblasts, a cell system in which the formation of primary cilia can be induced by starvation. Famished NIH 3T3 mouse fibroblasts stop dividing, they get arrested in the G0 phase and primary cilia assembly occurs. Triple labeling and high resolution immunofluorescence imaging of induced primary cilia in resting NIH 3T3 mouse fibroblasts demonstrated that Pcnt partially overlapped with acetylated tubulin (ac. tubulin), a marker for the ciliary axoneme and the BBC at the base of primary cilia (Fig. 1B-E). Double labeling experiments for Pcnt and centrin3 (Cen3), which is present at the transition zone of the ciliary axoneme and the BBC [19], gave comparable results (data not shown). The BBC consists of the basal body (BB) and its centriole where the pericentriolar material (PCM) is localized. High resolution 3D reconstructions of the cilia and their BBCs demonstrated that Pcnt surrounds the BBC of primary cilia (Fig. 1E). In Western blot analyses of extracts of NIH 3T3 mouse fibroblasts, the MmPeriC1 antiserum recognized two protein bands at approximately 360 and 240–250 kDa corresponding to the predicted size of the holo protein Pcnt B (360) and of the Pcnt splice variants A and/or S (250) [1] (Fig. 1A, F). Preadsorption of the MmPeriC1 antiserum with an excess of the antigen used for immunization completely blocked the immunodetection of the two protein bands in the Western blot (Fig. 1G) and resulted in the absence of Pcnt staining at the BBC of primary cilia of NIH 3T3 mouse fibroblasts (Fig. 1H). Pcnt expression was additionally examined using a polyclonal Pcnt pAb (Covance) which was raised against an epitope common to all three isoforms. The comparison of Western blot analyses with the MmPeriC1 and the Covance Pcnt antiserum using various tissue extracts showed for both antisera the same Pcnt bands (Fig. S3) [7], [15]. These results demonstrate the specificity of the affinity purified MmPeriC1 antiserum. In our study we did not use the anti-Pcnt monoclonal antibody (Pcnt mAb; BD Biosciences), because Endoh-Yamagami and co-workers clearly showed that this antibody does not detect all Pcnt splice variants [7].


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)

Characterization of the Pericentrin antiserum MmPeriC1.(A) Scheme of the known and published Pcnt splice variants: Pcnt B (360, accession number (AN): NP_032813 or BAF36559), Pcnt A (AN: partial, AAO24322.1) and Pcnt S (250, BAF36560). The 280 amino-acid epitope against which the MmPeriC1 antiserum was generated is indicated in grey. (B-D) Triple labeling of Pcnt (B, green), ac. tubulin (C, red), and DAPI (D merge, blue) in NIH 3T3 mouse fibroblasts. Pcnt colocalizes partially with ac. tubulin at the BBC (arrowheads) of primary cilia (PC). (E) High resolution 3D reconstruction demonstrates that Pcnt (green) ensheaths the BBC of the PC (red, ac. tubulin). (F, G) Western blot analysis using the MmPeriC1 antiserum. (F) The antiserum detects a 360 kDa and a 240–250 kDa protein band. (G) Preincubation of the MmPeriC1 antiserum with the antigen (recombinant fusion protein) blocks the immunodetection of the two bands. (H) Preadsorption of the MmPeriC1 antiserum with the antigen results in the absence of Pcnt staining at the BBC at primary cilia labeled with ac. tubulin. Scale bar: 5 µm (D, H).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026496-g001: Characterization of the Pericentrin antiserum MmPeriC1.(A) Scheme of the known and published Pcnt splice variants: Pcnt B (360, accession number (AN): NP_032813 or BAF36559), Pcnt A (AN: partial, AAO24322.1) and Pcnt S (250, BAF36560). The 280 amino-acid epitope against which the MmPeriC1 antiserum was generated is indicated in grey. (B-D) Triple labeling of Pcnt (B, green), ac. tubulin (C, red), and DAPI (D merge, blue) in NIH 3T3 mouse fibroblasts. Pcnt colocalizes partially with ac. tubulin at the BBC (arrowheads) of primary cilia (PC). (E) High resolution 3D reconstruction demonstrates that Pcnt (green) ensheaths the BBC of the PC (red, ac. tubulin). (F, G) Western blot analysis using the MmPeriC1 antiserum. (F) The antiserum detects a 360 kDa and a 240–250 kDa protein band. (G) Preincubation of the MmPeriC1 antiserum with the antigen (recombinant fusion protein) blocks the immunodetection of the two bands. (H) Preadsorption of the MmPeriC1 antiserum with the antigen results in the absence of Pcnt staining at the BBC at primary cilia labeled with ac. tubulin. Scale bar: 5 µm (D, H).
Mentions: To study the localization of Pcnt in the olfactory epithelium and the retina of the mouse, we first generated and affinity purified a specific polyclonal antiserum against a recombinant expressed part of murine Pcnt (Fig. 1A, Fig. S3A). The Pcnt antiserum, MmPeriC1, should detect all to date known Pcnt splice variants because of the immunogenic region against which the MmPeriC1 antiserum is directed (Fig. 1A). To validate the specificity of the MmPeriC1 antiserum, we examined the localization of Pcnt in cultured NIH 3T3 mouse fibroblasts, a cell system in which the formation of primary cilia can be induced by starvation. Famished NIH 3T3 mouse fibroblasts stop dividing, they get arrested in the G0 phase and primary cilia assembly occurs. Triple labeling and high resolution immunofluorescence imaging of induced primary cilia in resting NIH 3T3 mouse fibroblasts demonstrated that Pcnt partially overlapped with acetylated tubulin (ac. tubulin), a marker for the ciliary axoneme and the BBC at the base of primary cilia (Fig. 1B-E). Double labeling experiments for Pcnt and centrin3 (Cen3), which is present at the transition zone of the ciliary axoneme and the BBC [19], gave comparable results (data not shown). The BBC consists of the basal body (BB) and its centriole where the pericentriolar material (PCM) is localized. High resolution 3D reconstructions of the cilia and their BBCs demonstrated that Pcnt surrounds the BBC of primary cilia (Fig. 1E). In Western blot analyses of extracts of NIH 3T3 mouse fibroblasts, the MmPeriC1 antiserum recognized two protein bands at approximately 360 and 240–250 kDa corresponding to the predicted size of the holo protein Pcnt B (360) and of the Pcnt splice variants A and/or S (250) [1] (Fig. 1A, F). Preadsorption of the MmPeriC1 antiserum with an excess of the antigen used for immunization completely blocked the immunodetection of the two protein bands in the Western blot (Fig. 1G) and resulted in the absence of Pcnt staining at the BBC of primary cilia of NIH 3T3 mouse fibroblasts (Fig. 1H). Pcnt expression was additionally examined using a polyclonal Pcnt pAb (Covance) which was raised against an epitope common to all three isoforms. The comparison of Western blot analyses with the MmPeriC1 and the Covance Pcnt antiserum using various tissue extracts showed for both antisera the same Pcnt bands (Fig. S3) [7], [15]. These results demonstrate the specificity of the affinity purified MmPeriC1 antiserum. In our study we did not use the anti-Pcnt monoclonal antibody (Pcnt mAb; BD Biosciences), because Endoh-Yamagami and co-workers clearly showed that this antibody does not detect all Pcnt splice variants [7].

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