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Destabilization of the IFT-B cilia core complex due to mutations in IFT81 causes a Spectrum of Short-Rib Polydactyly Syndrome

View Article: PubMed Central - PubMed

ABSTRACT

Short-rib polydactyly syndromes (SRPS) and Asphyxiating thoracic dystrophy (ATD) or Jeune Syndrome are recessively inherited skeletal ciliopathies characterized by profound skeletal abnormalities and are frequently associated with polydactyly and multiorgan system involvement. SRPS are produced by mutations in genes that participate in the formation and function of primary cilia and usually result from disruption of retrograde intraflagellar (IFT) transport of the cilium. Herein we describe a new spectrum of SRPS caused by mutations in the gene IFT81, a key component of the IFT-B complex essential for anterograde transport. In mutant chondrocytes, the mutations led to low levels of IFT81 and mutant cells produced elongated cilia, had altered hedgehog signaling, had increased post-translation modification of tubulin, and showed evidence of destabilization of additional anterograde transport complex components. These findings demonstrate the importance of IFT81 in the skeleton, its role in the anterograde transport complex, and expand the number of loci associated with SRPS.

No MeSH data available.


IFT81 mutations induce cilia defects and abnormal Hh signaling.(A,B) ARL13B and Pericentrin staining of the centrosome and cilia in green in control and R98-443 chondrocytes. (C) Cilia length of control, R98-443 and rescued R98-443 chondrocytes with IFT81 vector showing that rescued cells partially corrected cilia length phenotype. (D) Percentage of cells with cilia in control and patient chondrocytes showing no difference in number. (E,F) Cilia staining with ARL13B and Pericentrin (both green) in rescued R98-443 chondrocytes showing average length cilia. (F,F’) demonstrate that the IFT81-GFP fusion protein co-localized with Acetyl-Tubulin in the cilia. (G,H) GLI3 levels in control and R98-443 chondrocytes with and without SAG stimulation showing altered GLI3FL/R ratios. I-J GLI3FL levels were restored to control levels with R98-443 chondrocytes rescued with vectors of IFT81 fusion protein (GFP and DDK flag).
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f3: IFT81 mutations induce cilia defects and abnormal Hh signaling.(A,B) ARL13B and Pericentrin staining of the centrosome and cilia in green in control and R98-443 chondrocytes. (C) Cilia length of control, R98-443 and rescued R98-443 chondrocytes with IFT81 vector showing that rescued cells partially corrected cilia length phenotype. (D) Percentage of cells with cilia in control and patient chondrocytes showing no difference in number. (E,F) Cilia staining with ARL13B and Pericentrin (both green) in rescued R98-443 chondrocytes showing average length cilia. (F,F’) demonstrate that the IFT81-GFP fusion protein co-localized with Acetyl-Tubulin in the cilia. (G,H) GLI3 levels in control and R98-443 chondrocytes with and without SAG stimulation showing altered GLI3FL/R ratios. I-J GLI3FL levels were restored to control levels with R98-443 chondrocytes rescued with vectors of IFT81 fusion protein (GFP and DDK flag).

Mentions: Although many of the mutations causing SRPS affect retrograde transport proteins, at a molecular level they disrupt the normal trafficking of molecules inside the cilia5154748. These alterations frequently lead to defects in ciliogenesis, cilia architecture and contribute to alter Hedgehog signaling activity. To investigate whether ciliogenesis was affected in mutant cells, we quantified both the abundance and length of cilia in mutant chondrocytes (Fig. 3A–D). The percentages of ciliated cells were similar between case and control cells (no difference detected by t-test p = 0.05) (Fig. 3D), but there was significant variability in cilia length among affected chondrocytes, with a significant increase in average cilia length in mutant cells (t-test P < 0.0001, Fig. 3C). To determine if expression of IFT81 could rescue the cilia phenotype, we expressed wild-type IFT81 (Origene) and again measured cilia length. As shown in Fig. 3, mutant cells transfected with wild-type IFT81 had reduced average cilia length compared with non-transfected cells, with cilia lengths similar to wild type cells.


Destabilization of the IFT-B cilia core complex due to mutations in IFT81 causes a Spectrum of Short-Rib Polydactyly Syndrome
IFT81 mutations induce cilia defects and abnormal Hh signaling.(A,B) ARL13B and Pericentrin staining of the centrosome and cilia in green in control and R98-443 chondrocytes. (C) Cilia length of control, R98-443 and rescued R98-443 chondrocytes with IFT81 vector showing that rescued cells partially corrected cilia length phenotype. (D) Percentage of cells with cilia in control and patient chondrocytes showing no difference in number. (E,F) Cilia staining with ARL13B and Pericentrin (both green) in rescued R98-443 chondrocytes showing average length cilia. (F,F’) demonstrate that the IFT81-GFP fusion protein co-localized with Acetyl-Tubulin in the cilia. (G,H) GLI3 levels in control and R98-443 chondrocytes with and without SAG stimulation showing altered GLI3FL/R ratios. I-J GLI3FL levels were restored to control levels with R98-443 chondrocytes rescued with vectors of IFT81 fusion protein (GFP and DDK flag).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5035930&req=5

f3: IFT81 mutations induce cilia defects and abnormal Hh signaling.(A,B) ARL13B and Pericentrin staining of the centrosome and cilia in green in control and R98-443 chondrocytes. (C) Cilia length of control, R98-443 and rescued R98-443 chondrocytes with IFT81 vector showing that rescued cells partially corrected cilia length phenotype. (D) Percentage of cells with cilia in control and patient chondrocytes showing no difference in number. (E,F) Cilia staining with ARL13B and Pericentrin (both green) in rescued R98-443 chondrocytes showing average length cilia. (F,F’) demonstrate that the IFT81-GFP fusion protein co-localized with Acetyl-Tubulin in the cilia. (G,H) GLI3 levels in control and R98-443 chondrocytes with and without SAG stimulation showing altered GLI3FL/R ratios. I-J GLI3FL levels were restored to control levels with R98-443 chondrocytes rescued with vectors of IFT81 fusion protein (GFP and DDK flag).
Mentions: Although many of the mutations causing SRPS affect retrograde transport proteins, at a molecular level they disrupt the normal trafficking of molecules inside the cilia5154748. These alterations frequently lead to defects in ciliogenesis, cilia architecture and contribute to alter Hedgehog signaling activity. To investigate whether ciliogenesis was affected in mutant cells, we quantified both the abundance and length of cilia in mutant chondrocytes (Fig. 3A–D). The percentages of ciliated cells were similar between case and control cells (no difference detected by t-test p = 0.05) (Fig. 3D), but there was significant variability in cilia length among affected chondrocytes, with a significant increase in average cilia length in mutant cells (t-test P < 0.0001, Fig. 3C). To determine if expression of IFT81 could rescue the cilia phenotype, we expressed wild-type IFT81 (Origene) and again measured cilia length. As shown in Fig. 3, mutant cells transfected with wild-type IFT81 had reduced average cilia length compared with non-transfected cells, with cilia lengths similar to wild type cells.

View Article: PubMed Central - PubMed

ABSTRACT

Short-rib polydactyly syndromes (SRPS) and Asphyxiating thoracic dystrophy (ATD) or Jeune Syndrome are recessively inherited skeletal ciliopathies characterized by profound skeletal abnormalities and are frequently associated with polydactyly and multiorgan system involvement. SRPS are produced by mutations in genes that participate in the formation and function of primary cilia and usually result from disruption of retrograde intraflagellar (IFT) transport of the cilium. Herein we describe a new spectrum of SRPS caused by mutations in the gene IFT81, a key component of the IFT-B complex essential for anterograde transport. In mutant chondrocytes, the mutations led to low levels of IFT81 and mutant cells produced elongated cilia, had altered hedgehog signaling, had increased post-translation modification of tubulin, and showed evidence of destabilization of additional anterograde transport complex components. These findings demonstrate the importance of IFT81 in the skeleton, its role in the anterograde transport complex, and expand the number of loci associated with SRPS.

No MeSH data available.