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The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

Gerhardt C, Lier JM, Burmühl S, Struchtrup A, Deutschmann K, Vetter M, Leu T, Reeg S, Grune T, Rüther U - J. Cell Biol. (2015)

Bottom Line: Mutations in RPGRIP1L result in severe human diseases called ciliopathies.Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l.We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany Christoph.Gerhardt@hhu.de.

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Rpgrip1l deficiency affects ciliary length. (A and B) MEFs were isolated from E12.5 WT and Rpgrip1l−/− embryos. (A) Comparison of WT and Rpgrip1l−/− ciliary length. Embryonic organs were separated from E12.5 embryos. At least 20 cilia per embryo were analyzed. n values (n refers to the number of embryos) are as follows: MEFs: WT, n = 4; Rpgrip1l−/−, n = 5; limbs: WT, n = 4; Rpgrip1l−/−, n = 5; hearts: WT, n = 4; Rpgrip1l−/−, n = 5; lungs: WT, n = 4; Rpgrip1l−/−, n = 4; livers: WT, n = 4; Rpgrip1l−/−, n = 4. Error bars show standard error of the mean. (B) Elongation of E12.5 Rpgrip1l−/− limb cilia are confirmed in comparison to fluorescence based cilia length measurement (10 cilia; SEM: 2.88 ± 0.14 µm; immunofluorescence: 2.44 ± 0.265 µm). Magnifications (termed as 1 and 2) show two examples of Rpgrip1l−/− limb cilia. Bars: (overview) 10 µm; (magnification) 1 µm. (C) Absolute values of the quantified ciliary length of WT (n = 4 embryos) and Rpgrip1l−/− (n = 5 embryos) limbs at E12.5 based on immunofluorescence. Rpgrip1l-negative cilia (2.44 ± 0.265 µm [standard error of the mean]) are significantly longer than WT cilia (1.25 µm ± 0.05 µm [standard error of the mean]). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig2: Rpgrip1l deficiency affects ciliary length. (A and B) MEFs were isolated from E12.5 WT and Rpgrip1l−/− embryos. (A) Comparison of WT and Rpgrip1l−/− ciliary length. Embryonic organs were separated from E12.5 embryos. At least 20 cilia per embryo were analyzed. n values (n refers to the number of embryos) are as follows: MEFs: WT, n = 4; Rpgrip1l−/−, n = 5; limbs: WT, n = 4; Rpgrip1l−/−, n = 5; hearts: WT, n = 4; Rpgrip1l−/−, n = 5; lungs: WT, n = 4; Rpgrip1l−/−, n = 4; livers: WT, n = 4; Rpgrip1l−/−, n = 4. Error bars show standard error of the mean. (B) Elongation of E12.5 Rpgrip1l−/− limb cilia are confirmed in comparison to fluorescence based cilia length measurement (10 cilia; SEM: 2.88 ± 0.14 µm; immunofluorescence: 2.44 ± 0.265 µm). Magnifications (termed as 1 and 2) show two examples of Rpgrip1l−/− limb cilia. Bars: (overview) 10 µm; (magnification) 1 µm. (C) Absolute values of the quantified ciliary length of WT (n = 4 embryos) and Rpgrip1l−/− (n = 5 embryos) limbs at E12.5 based on immunofluorescence. Rpgrip1l-negative cilia (2.44 ± 0.265 µm [standard error of the mean]) are significantly longer than WT cilia (1.25 µm ± 0.05 µm [standard error of the mean]). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: The absence of particular TZ proteins results in differences in ciliary length (Tammachote et al., 2009; Cui et al., 2011; Dowdle et al., 2011; Garcia-Gonzalo et al., 2011). Such alterations are indicative for ciliary signaling defects (Cui et al., 2011; Dowdle et al., 2011; Garcia-Gonzalo et al., 2011; Ishikawa and Marshall, 2011; Larkins et al., 2011). We measured the length of cilia in Rpgrip1l−/− MEFs and in different organs of Rpgrip1l−/− mouse embryos by using immunofluorescence. Although the cilia in Rpgrip1l−/− MEFs, limbs, livers, and lungs are significantly longer, the length of cardiac cilia is significantly reduced (Fig. 2 A). We evaluated the quality of our fluorescence-based ciliary length measurements by comparing these results with previously published scanning EM (SEM) data. The absolute value we measured for wild-type (WT) limb cilia is 1.25 ± 0.05 µm (standard error of the mean; Fig. 2 C), which correlates with SEM quantifications (Haycraft et al., 2005; Moon et al., 2014). Moreover, we performed SEM analyses to quantify the length of Rpgrip1l−/− limb cilia. The elongation of limb cilia, in the absence of Rpgrip1l, was verified by these SEM experiments (Fig. 2, B and C).


The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

Gerhardt C, Lier JM, Burmühl S, Struchtrup A, Deutschmann K, Vetter M, Leu T, Reeg S, Grune T, Rüther U - J. Cell Biol. (2015)

Rpgrip1l deficiency affects ciliary length. (A and B) MEFs were isolated from E12.5 WT and Rpgrip1l−/− embryos. (A) Comparison of WT and Rpgrip1l−/− ciliary length. Embryonic organs were separated from E12.5 embryos. At least 20 cilia per embryo were analyzed. n values (n refers to the number of embryos) are as follows: MEFs: WT, n = 4; Rpgrip1l−/−, n = 5; limbs: WT, n = 4; Rpgrip1l−/−, n = 5; hearts: WT, n = 4; Rpgrip1l−/−, n = 5; lungs: WT, n = 4; Rpgrip1l−/−, n = 4; livers: WT, n = 4; Rpgrip1l−/−, n = 4. Error bars show standard error of the mean. (B) Elongation of E12.5 Rpgrip1l−/− limb cilia are confirmed in comparison to fluorescence based cilia length measurement (10 cilia; SEM: 2.88 ± 0.14 µm; immunofluorescence: 2.44 ± 0.265 µm). Magnifications (termed as 1 and 2) show two examples of Rpgrip1l−/− limb cilia. Bars: (overview) 10 µm; (magnification) 1 µm. (C) Absolute values of the quantified ciliary length of WT (n = 4 embryos) and Rpgrip1l−/− (n = 5 embryos) limbs at E12.5 based on immunofluorescence. Rpgrip1l-negative cilia (2.44 ± 0.265 µm [standard error of the mean]) are significantly longer than WT cilia (1.25 µm ± 0.05 µm [standard error of the mean]). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig2: Rpgrip1l deficiency affects ciliary length. (A and B) MEFs were isolated from E12.5 WT and Rpgrip1l−/− embryos. (A) Comparison of WT and Rpgrip1l−/− ciliary length. Embryonic organs were separated from E12.5 embryos. At least 20 cilia per embryo were analyzed. n values (n refers to the number of embryos) are as follows: MEFs: WT, n = 4; Rpgrip1l−/−, n = 5; limbs: WT, n = 4; Rpgrip1l−/−, n = 5; hearts: WT, n = 4; Rpgrip1l−/−, n = 5; lungs: WT, n = 4; Rpgrip1l−/−, n = 4; livers: WT, n = 4; Rpgrip1l−/−, n = 4. Error bars show standard error of the mean. (B) Elongation of E12.5 Rpgrip1l−/− limb cilia are confirmed in comparison to fluorescence based cilia length measurement (10 cilia; SEM: 2.88 ± 0.14 µm; immunofluorescence: 2.44 ± 0.265 µm). Magnifications (termed as 1 and 2) show two examples of Rpgrip1l−/− limb cilia. Bars: (overview) 10 µm; (magnification) 1 µm. (C) Absolute values of the quantified ciliary length of WT (n = 4 embryos) and Rpgrip1l−/− (n = 5 embryos) limbs at E12.5 based on immunofluorescence. Rpgrip1l-negative cilia (2.44 ± 0.265 µm [standard error of the mean]) are significantly longer than WT cilia (1.25 µm ± 0.05 µm [standard error of the mean]). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: The absence of particular TZ proteins results in differences in ciliary length (Tammachote et al., 2009; Cui et al., 2011; Dowdle et al., 2011; Garcia-Gonzalo et al., 2011). Such alterations are indicative for ciliary signaling defects (Cui et al., 2011; Dowdle et al., 2011; Garcia-Gonzalo et al., 2011; Ishikawa and Marshall, 2011; Larkins et al., 2011). We measured the length of cilia in Rpgrip1l−/− MEFs and in different organs of Rpgrip1l−/− mouse embryos by using immunofluorescence. Although the cilia in Rpgrip1l−/− MEFs, limbs, livers, and lungs are significantly longer, the length of cardiac cilia is significantly reduced (Fig. 2 A). We evaluated the quality of our fluorescence-based ciliary length measurements by comparing these results with previously published scanning EM (SEM) data. The absolute value we measured for wild-type (WT) limb cilia is 1.25 ± 0.05 µm (standard error of the mean; Fig. 2 C), which correlates with SEM quantifications (Haycraft et al., 2005; Moon et al., 2014). Moreover, we performed SEM analyses to quantify the length of Rpgrip1l−/− limb cilia. The elongation of limb cilia, in the absence of Rpgrip1l, was verified by these SEM experiments (Fig. 2, B and C).

Bottom Line: Mutations in RPGRIP1L result in severe human diseases called ciliopathies.Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l.We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany Christoph.Gerhardt@hhu.de.

Show MeSH
Related in: MedlinePlus