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Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function.

Lee YL, Santé J, Comerci CJ, Cyge B, Menezes LF, Li FQ, Germino GG, Moerner WE, Takemaru K, Stearns T - Mol. Biol. Cell (2014)

Bottom Line: Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies.Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium.This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1(-/-) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford School of Medicine, Stanford University, Stanford, CA 94305.

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Loss of Cby1 results in polycystic kidneys. (A) Kidney cyst formation in Cby1−/− mice. Kidney sections from Cby1+/+ and Cby1−/− adult mice (3 mo old) were stained with hematoxylin and eosin. Cysts are indicated with arrowheads. (B) Renal sections from postnatal day 13 Cby1−/− mice were stained with DBA (red, distal tubules), LTL (green, proximal tubules), and DAPI (blue). Note that some kidney cysts arise from collecting duct/distal tubules (arrowheads). (C) Cby1 localizes to the basal bodies of primary cilia in the renal tubular cells. Kidney sections from 3-mo-old Cby1+/+ mice were double labeled with antibodies against Cby1 (red) and the ciliary/basal body marker acetylated α-tubulin (Ac-tub, green), and the merged image is shown. Inset, high-magnification view with arrowheads pointing to primary cilia. The dotted line encircles a renal tubule. No Cby1 staining was observed in Cby1−/− kidney sections. Scale bars, 200 μm (A), 50 μm (B), 5 μm (C). (D) The number of primary cilia (>2.0 μm) per microscope field at 100× in the renal cortex of Cby1+/+ (WT) and Cby1−/− (KO) mice was counted, with 10 fields of view/mouse. Values are means ± SEM for individual mice.
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Figure 1: Loss of Cby1 results in polycystic kidneys. (A) Kidney cyst formation in Cby1−/− mice. Kidney sections from Cby1+/+ and Cby1−/− adult mice (3 mo old) were stained with hematoxylin and eosin. Cysts are indicated with arrowheads. (B) Renal sections from postnatal day 13 Cby1−/− mice were stained with DBA (red, distal tubules), LTL (green, proximal tubules), and DAPI (blue). Note that some kidney cysts arise from collecting duct/distal tubules (arrowheads). (C) Cby1 localizes to the basal bodies of primary cilia in the renal tubular cells. Kidney sections from 3-mo-old Cby1+/+ mice were double labeled with antibodies against Cby1 (red) and the ciliary/basal body marker acetylated α-tubulin (Ac-tub, green), and the merged image is shown. Inset, high-magnification view with arrowheads pointing to primary cilia. The dotted line encircles a renal tubule. No Cby1 staining was observed in Cby1−/− kidney sections. Scale bars, 200 μm (A), 50 μm (B), 5 μm (C). (D) The number of primary cilia (>2.0 μm) per microscope field at 100× in the renal cortex of Cby1+/+ (WT) and Cby1−/− (KO) mice was counted, with 10 fields of view/mouse. Values are means ± SEM for individual mice.

Mentions: Renal disorders are a major feature of ciliopathies resulting from defects in primary cilia structure or function (Berbari et al., 2009; Gascue et al., 2011). To determine whether loss of Cby1 results in renal defects, we examined the kidneys of mutant Cby1−/− and wild-type Cby1+/+ mice. The kidneys of Cby1−/− mice were similar in size to wild-type kidneys at all ages examined. However, histological analysis revealed the presence of multiple renal cysts in Cby1−/− mice (Figure 1A). Although the extent of the cystic phenotype varied, renal cysts were consistently observed in all adult (>2 mo old) Cby1−/− animals and were detectable in some animals as early as at birth. As shown in Figure 1B, some of the cysts were positive for the collecting duct/distal tubule marker Dolichos biflorus agglutinin (DBA), but the others were negative for both DBA and the proximal tubule marker Lotus tetragonolobus lectin (LTL). LTL-positive cysts were rarely observed. Thus it appears that renal cysts predominantly derive from collecting duct/distal tubules in Cby1−/− mice. Consistent with its function in kidney development, Cby1 was detected in renal tubular cells, localizing to the centrioles at the base of primary cilia (Figure 1C). Next we determined the status of primary cilia in Cby1−/− kidneys. Assessment by immunostaining, as in Figure 1C, showed that primary cilia were present in the renal tubular cells of Cby1−/− mice but that the cilia were less abundant compared with Cby1+/+ controls. The number of primary cilia in the renal cortex was quantified at various ages, blinded to genotype for unbiased counting (Figure 1D). Only cilia that were >2 μm and protruded into the tubular lumen were scored. In Cby1+/+ kidneys from four mice, mean cilium number per microscope field varied from 11.6 to 20.1. In Cby1−/− kidneys from six mice, mean cilium number varied from 2.0 to 17.4, with greater variability than in the wild-type kidneys. Kidneys from three of the mice (KO4, 5, 6) had a significantly lower number of cilia, whereas the other three were not significantly different from wild type. Although we do not yet understand the basis of the variability of the Cby1−/− phenotype, the reduced number of cilia in the kidneys observed in some animals is consistent with Cby1 being important for primary cilium function in renal epithelia.


Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function.

Lee YL, Santé J, Comerci CJ, Cyge B, Menezes LF, Li FQ, Germino GG, Moerner WE, Takemaru K, Stearns T - Mol. Biol. Cell (2014)

Loss of Cby1 results in polycystic kidneys. (A) Kidney cyst formation in Cby1−/− mice. Kidney sections from Cby1+/+ and Cby1−/− adult mice (3 mo old) were stained with hematoxylin and eosin. Cysts are indicated with arrowheads. (B) Renal sections from postnatal day 13 Cby1−/− mice were stained with DBA (red, distal tubules), LTL (green, proximal tubules), and DAPI (blue). Note that some kidney cysts arise from collecting duct/distal tubules (arrowheads). (C) Cby1 localizes to the basal bodies of primary cilia in the renal tubular cells. Kidney sections from 3-mo-old Cby1+/+ mice were double labeled with antibodies against Cby1 (red) and the ciliary/basal body marker acetylated α-tubulin (Ac-tub, green), and the merged image is shown. Inset, high-magnification view with arrowheads pointing to primary cilia. The dotted line encircles a renal tubule. No Cby1 staining was observed in Cby1−/− kidney sections. Scale bars, 200 μm (A), 50 μm (B), 5 μm (C). (D) The number of primary cilia (>2.0 μm) per microscope field at 100× in the renal cortex of Cby1+/+ (WT) and Cby1−/− (KO) mice was counted, with 10 fields of view/mouse. Values are means ± SEM for individual mice.
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Figure 1: Loss of Cby1 results in polycystic kidneys. (A) Kidney cyst formation in Cby1−/− mice. Kidney sections from Cby1+/+ and Cby1−/− adult mice (3 mo old) were stained with hematoxylin and eosin. Cysts are indicated with arrowheads. (B) Renal sections from postnatal day 13 Cby1−/− mice were stained with DBA (red, distal tubules), LTL (green, proximal tubules), and DAPI (blue). Note that some kidney cysts arise from collecting duct/distal tubules (arrowheads). (C) Cby1 localizes to the basal bodies of primary cilia in the renal tubular cells. Kidney sections from 3-mo-old Cby1+/+ mice were double labeled with antibodies against Cby1 (red) and the ciliary/basal body marker acetylated α-tubulin (Ac-tub, green), and the merged image is shown. Inset, high-magnification view with arrowheads pointing to primary cilia. The dotted line encircles a renal tubule. No Cby1 staining was observed in Cby1−/− kidney sections. Scale bars, 200 μm (A), 50 μm (B), 5 μm (C). (D) The number of primary cilia (>2.0 μm) per microscope field at 100× in the renal cortex of Cby1+/+ (WT) and Cby1−/− (KO) mice was counted, with 10 fields of view/mouse. Values are means ± SEM for individual mice.
Mentions: Renal disorders are a major feature of ciliopathies resulting from defects in primary cilia structure or function (Berbari et al., 2009; Gascue et al., 2011). To determine whether loss of Cby1 results in renal defects, we examined the kidneys of mutant Cby1−/− and wild-type Cby1+/+ mice. The kidneys of Cby1−/− mice were similar in size to wild-type kidneys at all ages examined. However, histological analysis revealed the presence of multiple renal cysts in Cby1−/− mice (Figure 1A). Although the extent of the cystic phenotype varied, renal cysts were consistently observed in all adult (>2 mo old) Cby1−/− animals and were detectable in some animals as early as at birth. As shown in Figure 1B, some of the cysts were positive for the collecting duct/distal tubule marker Dolichos biflorus agglutinin (DBA), but the others were negative for both DBA and the proximal tubule marker Lotus tetragonolobus lectin (LTL). LTL-positive cysts were rarely observed. Thus it appears that renal cysts predominantly derive from collecting duct/distal tubules in Cby1−/− mice. Consistent with its function in kidney development, Cby1 was detected in renal tubular cells, localizing to the centrioles at the base of primary cilia (Figure 1C). Next we determined the status of primary cilia in Cby1−/− kidneys. Assessment by immunostaining, as in Figure 1C, showed that primary cilia were present in the renal tubular cells of Cby1−/− mice but that the cilia were less abundant compared with Cby1+/+ controls. The number of primary cilia in the renal cortex was quantified at various ages, blinded to genotype for unbiased counting (Figure 1D). Only cilia that were >2 μm and protruded into the tubular lumen were scored. In Cby1+/+ kidneys from four mice, mean cilium number per microscope field varied from 11.6 to 20.1. In Cby1−/− kidneys from six mice, mean cilium number varied from 2.0 to 17.4, with greater variability than in the wild-type kidneys. Kidneys from three of the mice (KO4, 5, 6) had a significantly lower number of cilia, whereas the other three were not significantly different from wild type. Although we do not yet understand the basis of the variability of the Cby1−/− phenotype, the reduced number of cilia in the kidneys observed in some animals is consistent with Cby1 being important for primary cilium function in renal epithelia.

Bottom Line: Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies.Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium.This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1(-/-) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford School of Medicine, Stanford University, Stanford, CA 94305.

Show MeSH
Related in: MedlinePlus