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Renal cystic disease proteins play critical roles in the organization of the olfactory epithelium.

Pluznick JL, Rodriguez-Gil DJ, Hull M, Mistry K, Gattone V, Johnson CA, Weatherbee S, Greer CA, Caplan MJ - PLoS ONE (2011)

Bottom Line: We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs.Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture.Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale School of Medicine New Haven, Connecticut, United States of America.

ABSTRACT
It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases - polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) - localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (G(olf), AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia.

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Swollen knobs and a decreased number of cilia are detected in mutant                                MKS3 mutant rat olfactory epithelium.Representative olfactory epithelium images from normal (A, B) and MKS3                            mutant (C, D) rats. Mutant sections showed enlarged OSN knobs, some of                            which exhibited irregular shapes (e.g., to the left in C), with knobs                            frequently appearing to be “detached” from the OE surface.                            At this magnification, coronally sectioned OSN cilia could be                            distinguished from microvilli as a result of their different sizes.                            Control sections showed multiple easily identifiable cilia (arrowheads,                            only shown in A), while mutant sections showed a decreased number                            (arrowheads in D; none could be recognized in C). Scale bar                             = 1 µm: shown in (D) for A–D.
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pone-0019694-g005: Swollen knobs and a decreased number of cilia are detected in mutant MKS3 mutant rat olfactory epithelium.Representative olfactory epithelium images from normal (A, B) and MKS3 mutant (C, D) rats. Mutant sections showed enlarged OSN knobs, some of which exhibited irregular shapes (e.g., to the left in C), with knobs frequently appearing to be “detached” from the OE surface. At this magnification, coronally sectioned OSN cilia could be distinguished from microvilli as a result of their different sizes. Control sections showed multiple easily identifiable cilia (arrowheads, only shown in A), while mutant sections showed a decreased number (arrowheads in D; none could be recognized in C). Scale bar  = 1 µm: shown in (D) for A–D.

Mentions: To further characterize these anomalies, we used electron microscopy to study the ultrastructure of the OE. Figure 5 shows the general structure of the surface of the OE in control (top) and MKS3 mutant (bottom) animals. In agreement with our confocal observations, cystic animals showed dendritic knobs that were swollen with abnormal shapes (e.g. blebs protruding from the knob); in many cases the knobs extended far into the lumen. At the ultrastructural level we identified coronally sectioned OSN cilia in the control animals (Figure 5, filled arrowheads top left) but it was difficult to recognize them in the MKS3 mutants (Figure 5, filled arrowheads bottom right). When we turned our attention to individual cilium, ultrastructural differences were pronounced. Cilium from control rats (Figure 6A) showed the typical 9+2 ultrastructural organization of microtubules, characteristic of the proximal segment of OSN cilia. Cilia with two singlet microtubules (Figure 6A insert) were also seen, characteristic of distal segments of OSN cilia (reviewed in [29]). In MKS3 mutant rats, however, cilia with the normal 9+2 microtubule structure were rarely observed (Figure 6B). More commonly, the cilia from MKS3 mutant rats showed a severe perturbation of microtubule organization (Figure 6C–J). In the MKS3 mutants we observed an absence (Figure 6C) or increased number (Figure 6F, G) of singlet microtubules in the center of the cilia; singlets in the periphery of cilia (Figure 6D, E, G, H); doublets in which one of the microtubules is open (Figure 6F, H); or microtubules without any evident organization (Figure 6I, J). However, when we turned our attention to the basal bodies in the dendritic knobs, no significant differences were detected between the two genotypes (Figure 6K, L). All these data point to MKS3 playing an important role in organizing the structure of OSN cilia.


Renal cystic disease proteins play critical roles in the organization of the olfactory epithelium.

Pluznick JL, Rodriguez-Gil DJ, Hull M, Mistry K, Gattone V, Johnson CA, Weatherbee S, Greer CA, Caplan MJ - PLoS ONE (2011)

Swollen knobs and a decreased number of cilia are detected in mutant                                MKS3 mutant rat olfactory epithelium.Representative olfactory epithelium images from normal (A, B) and MKS3                            mutant (C, D) rats. Mutant sections showed enlarged OSN knobs, some of                            which exhibited irregular shapes (e.g., to the left in C), with knobs                            frequently appearing to be “detached” from the OE surface.                            At this magnification, coronally sectioned OSN cilia could be                            distinguished from microvilli as a result of their different sizes.                            Control sections showed multiple easily identifiable cilia (arrowheads,                            only shown in A), while mutant sections showed a decreased number                            (arrowheads in D; none could be recognized in C). Scale bar                             = 1 µm: shown in (D) for A–D.
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pone-0019694-g005: Swollen knobs and a decreased number of cilia are detected in mutant MKS3 mutant rat olfactory epithelium.Representative olfactory epithelium images from normal (A, B) and MKS3 mutant (C, D) rats. Mutant sections showed enlarged OSN knobs, some of which exhibited irregular shapes (e.g., to the left in C), with knobs frequently appearing to be “detached” from the OE surface. At this magnification, coronally sectioned OSN cilia could be distinguished from microvilli as a result of their different sizes. Control sections showed multiple easily identifiable cilia (arrowheads, only shown in A), while mutant sections showed a decreased number (arrowheads in D; none could be recognized in C). Scale bar  = 1 µm: shown in (D) for A–D.
Mentions: To further characterize these anomalies, we used electron microscopy to study the ultrastructure of the OE. Figure 5 shows the general structure of the surface of the OE in control (top) and MKS3 mutant (bottom) animals. In agreement with our confocal observations, cystic animals showed dendritic knobs that were swollen with abnormal shapes (e.g. blebs protruding from the knob); in many cases the knobs extended far into the lumen. At the ultrastructural level we identified coronally sectioned OSN cilia in the control animals (Figure 5, filled arrowheads top left) but it was difficult to recognize them in the MKS3 mutants (Figure 5, filled arrowheads bottom right). When we turned our attention to individual cilium, ultrastructural differences were pronounced. Cilium from control rats (Figure 6A) showed the typical 9+2 ultrastructural organization of microtubules, characteristic of the proximal segment of OSN cilia. Cilia with two singlet microtubules (Figure 6A insert) were also seen, characteristic of distal segments of OSN cilia (reviewed in [29]). In MKS3 mutant rats, however, cilia with the normal 9+2 microtubule structure were rarely observed (Figure 6B). More commonly, the cilia from MKS3 mutant rats showed a severe perturbation of microtubule organization (Figure 6C–J). In the MKS3 mutants we observed an absence (Figure 6C) or increased number (Figure 6F, G) of singlet microtubules in the center of the cilia; singlets in the periphery of cilia (Figure 6D, E, G, H); doublets in which one of the microtubules is open (Figure 6F, H); or microtubules without any evident organization (Figure 6I, J). However, when we turned our attention to the basal bodies in the dendritic knobs, no significant differences were detected between the two genotypes (Figure 6K, L). All these data point to MKS3 playing an important role in organizing the structure of OSN cilia.

Bottom Line: We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs.Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture.Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale School of Medicine New Haven, Connecticut, United States of America.

ABSTRACT
It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases - polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) - localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (G(olf), AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia.

Show MeSH
Related in: MedlinePlus