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Results: 8

1.
FIGURE 8:

FIGURE 8:. From: The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content.

Model of Rilp-like protein function. The data presented in this study fit two models for Rilp-like protein control of ciliary membrane content. 1) Protein removal: Rilp-like proteins may promote the removal of membrane and membrane proteins from the primary cilium. 2) Protein exclusion: Rilp-like proteins may prevent entry of membrane proteins into the cilium. Loss of the Rilp-like proteins increases the ciliary membrane protein concentration by either decreasing protein removal or increasing protein entry.

Johanna R. Schaub, et al. Mol Biol Cell. 2013 February 15;24(4):453-464.
2.
FIGURE 1:

FIGURE 1:. From: The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content.

Rilpl2 is up-regulated during ciliogenesis. (A) Schematic diagram comparing Rab-interacting lysosomal protein family members. RH1 and RH2 domains are regions of high sequence similarity. Solid lines represent predicted coiled-coil domains. Dashed lines mark the region specifying the lysosomal function of Rilp, the region of Rilpl2 that interacts with MyoVa, and the region specific to Rilpl1 and Rilpl2 (LD). (B) Immunofluorescence images of the apical surface of MTEC cultures labeled with antibodies for Rilpl2 (green) and the centrosome and cilium markers acTub or pericentrin (red). Images to the right are zoomed in on Rilpl2-expressing cells. Bars, 10 μm. (C) Immunofluorescence image of the apical surface of an MTEC culture (ALI >12 d) labeled with antibodies for Rilpl1 (green) and γ-tubulin (red). Images to the right are zoomed in on Rilpl1-expressing cells. Bar, 10 μm.

Johanna R. Schaub, et al. Mol Biol Cell. 2013 February 15;24(4):453-464.
3.
FIGURE 6:

FIGURE 6:. From: The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content.

Loss of Rilp-like proteins increases signaling proteins in the primary cilium. (A) Representative images of control and Rilpl1/Rilpl2–depleted IMCD3 cells serum starved, fixed, and stained for PKHDCTS-GFP (green), acTub (red), and DNA (DAPI, blue). Bars, 10 μm. (B) Quantification of frequency of PKHDCTS-GFP positive cilia in IMCD3 cells depleted of the indicated protein(s) normalized to control frequency. Results shown are the mean of three independent experiments ± SEM (200 cells/experiment, **p < 0.01, χ2 on pooled data). (C) Representative images of control and Rilpl1/Rilpl2 depleted IMCD3 cells serum starved, fixed, and stained for 5-HT6-GFP (green), acTub (red), and DNA (DAPI, blue). Bars, 10 μm. (D) Quantification of 5-HT6-GFP in cilia (fluorescence intensity/micrometer). Results shown are the mean of cilia from 20 random fields of cells for each of three independent experiments ± SEM (n = 124, 89, 67, 106, respectively; **p < 0.01, t test).

Johanna R. Schaub, et al. Mol Biol Cell. 2013 February 15;24(4):453-464.
4.
FIGURE 5:

FIGURE 5:. From: The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content.

Loss of Rilp-like proteins prevents spheroid formation. (A) Rilpl1 and Rilpl2 depleted individually or together from IMCD3 cells by lentiviral expression of shRNAs. Lysates were probed for Rilpl1, Rilpl2, and p38 as a loading control. Numbers to the left are molecular weights in kilodaltons. (B) Representative images of cell clusters from control and Rilpl1/Rilpl2–depleted IMCD3 cells grown in Matrigel, fixed, and stained for nuclei (DAPI, blue), apical junctions (ZO-1, green), cilia (acTub, red), and basolateral surfaces (β-catenin, purple). Bars, 10 μm. (C) Quantification of the frequency of spheroid formation of IMCD3 cells depleted of the indicated Rilp-like protein(s) normalized to the frequency of spheroid formation of control cells. Results shown are the mean of three independent experiments ± SEM (200 cells/experiment, *p < 0.05, **p < 0.01, t test).

Johanna R. Schaub, et al. Mol Biol Cell. 2013 February 15;24(4):453-464.
5.
FIGURE 2:

FIGURE 2:. From: The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content.

Rilp-like proteins localize to the centrosome and primary cilium. (A) IMCD3 cells were fixed and stained for endogenous Rilpl2 (green), polyglutamylated tubulin (pgTub, red), and DNA (DAPI, blue). Bottom, cells were serum starved. Bars, 10 μm. (B) NIH 3T3 cells were fixed and stained for endogenous Rilpl1 (green), pgTub (red), and DNA (DAPI, blue). Top, cells were serum starved. Bars, 10 μm. Note that Rilpl1 localizes to one of the two γ-tubulin spots. (C) Serum-starved IMCD3 cells stably expressing LAP-Rilpl1 or Rilpl2-LAP were fixed and stained for GFP (Rilpl1/Rilpl2, green), centrioles or cilia (centrin, acTub, pgTub; red), and DNA (DAPI, blue). Bars, 10 μm. (D) IMCD3 FlpIN cells stably expressing Rilpl2-LAP were serum starved, fixed, and stained for GFP (Rilpl2, green), endogenous Rilpl1 (red), and DNA (DAPI, blue). Bar, 10 μm. (E) N2A cells were transfected with LAP-Rilpl1, fixed, and stained for LAP-Rilpl1 (GFP, green), γ-tubulin (blue), and the distal appendage protein Cep164 (red). Images are maximum projections of image stacks obtained by deconvolution microscopy. LAP-Rilpl1 localizes to the distal end of the mother centriole. Bars, 0.5 μm. (F) An asynchronous population of NIH 3T3 cells expressing Rilpl1-LAP was fixed and stained for Rilpl1-LAP (GFP, green), centrin (red), and DNA (DAPI, blue). Rilpl1 is lost at the mother centriole in early mitosis, but asymmetric distribution returns at telophase. Bars, 10 μm. Insets are enlarged images of centrosome/cilium regions.

Johanna R. Schaub, et al. Mol Biol Cell. 2013 February 15;24(4):453-464.
6.
FIGURE 4:

FIGURE 4:. From: The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content.

Rilpl2 ciliary localization is dynamic. (A) IMCD3 cells expressing Rilpl2-LAP (green) and tdTom-Inv (proximal cilium, red) were imaged for 90 min under serum starvation conditions. Images shown were taken at the marked time points after the start of imaging, keeping the primary cilium in focus. Note the absence of Rilpl2-LAP from the cilium at t = 0, its appearance at t = 9 min, and its presence from t = 44 min until the end of imaging (t = 90 min). Still frames from Supplemental Movie S1. Bar, 2 μm (B) Rilpl2-LAP tubulovesicular structure in a live IMCD3 cell. Still frame from Supplemental Movie S2. Bar, 10 μm (C) IMCD3 cells expressing Rilpl2-LAP were serum starved, fixed, and stained for Rilpl2-LAP (GFP, green), pgTub (red), and DNA (DAPI, blue). Image is a maximum projection of a z-stack obtained by deconvolution microscopy. Bar, 5 μm (D) Serum-starved IMCD3 cell expressing Rilpl2-LAP (green) and tdTom-Inv (proximal cilium, red) was imaged for 90 min. Images shown are from the marked time points. Note the tubulovesicular structure at the base of the cilium. Still frames from Supplemental Movie S3. Bar, 1 μm.

Johanna R. Schaub, et al. Mol Biol Cell. 2013 February 15;24(4):453-464.
7.
FIGURE 7:

FIGURE 7:. From: The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content.

Loss of Rilp-like proteins increases basal level of Smo in cilia. (A) Rilpl1 and Rilpl2 were depleted individually or together from MEFs by lentiviral expression of shRNAs. Lysates were probed for Rilpl1, Rilpl2, and p38 as a loading control. Numbers to the left are molecular weights in kilodaltons. Protein loaded: 150 μg (top) or 500 μg (bottom). Note that Rilpl2 expression is below detectable levels in all samples. (B) Representative immunofluorescence images of control and Rilpl1/Rilpl2–depleted MEFs serum starved, treated with 100 nM SAG for 24 h, fixed, and stained for Smo (green), pgTub (red), and DNA (DAPI, blue). Bars, 10 μm. (C) Quantification of the frequency of Smo-positive cilia in SAG-treated MEFs depleted of the indicated proteins relative to control cells. Results shown are the mean of three independent experiments ± SEM (200 cells/experiment). (D) Representative immunofluorescence images of control and Rilpl1/Rilpl2 MEFs serum starved, treated with vehicle, fixed, and stained for Smo (green), pgTub (red), and DNA (DAPI, blue). Individual cilia from each image are highlighted to the right. Bars, 10 μm. (E) Quantification of the frequency of Smo-positive cilia in MEFs depleted of the indicated proteins relative to control cells. Results shown are the mean of three independent experiments ± SEM (200 cells/experiment, **p < 0.01, t test).

Johanna R. Schaub, et al. Mol Biol Cell. 2013 February 15;24(4):453-464.
8.
FIGURE 3:

FIGURE 3:. From: The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content.

The C-terminal regions of Rilpl1 and Rilpl2 are necessary and sufficient for centrosomal localization. (A) NIH 3T3 cells were transfected with GFP-ΔC-Rilpl2 or GFP-ΔN-Rilpl2, fixed, and stained for GFP (green), acTub or centrin (red), and DNA (DAPI, blue). Top and bottom, cells were serum starved. ΔN-Rilpl2 is sufficient for centrosomal and ciliary localization. Bars, 10 μm. (B) NIH 3T3 (top and middle) or IMCD3 (bottom) cells were transfected with ΔC-Rilpl1-GFP or ΔN-Rilpl1-GFP, serum starved, fixed, and stained for GFP (green), centrin/pgTub (red), and DNA (DAPI, blue). ΔN-Rilpl1 is sufficient centrosomal and ciliary localization. Bars, 10 μm. (C) Cilium formation in IMCD3 cells transfected with GFP, Rilpl2-GFP (full-length), GFP-ΔC-Rilpl2, or GFP-ΔN-Rilpl2. Results shown are the mean of three independent experiments ± SEM (100 cells/experiment; **p < 0.01). (D) Cilium formation in IMCD3 cells transfected with GFP, Rilpl1-GFP (full-length), ΔC-Rilpl1-GFP, or ΔN-Rilpl1-GFP. Results shown are the mean of three independent experiments ± SEM (100 cells/experiment; **p < 0.01). (E) Amino acid sequence alignment of the 13–amino acid Rilp-like domain (LD) of Rilpl1 and Rilpl2 from several vertebrates and the Rilp homologue of invertebrates. Amino acid changes are noted in red. (F) IMCD3 cells were transfected with GFP-tagged Rilpl1, Rilpl1-deltaLD, Rilp, or Rilp-LD, fixed, and stained for GFP (green), γ-tubulin (red), and DNA (DAPI, blue). Bars, 10 μm. Insets are enlarged images of the centrosomal/ciliary regions. The average percentage of transfected cells with centrosomal localization of GFP-tagged protein from three independent experiments is noted to the right (100 cells/experiment; **p < 0.01, t test).

Johanna R. Schaub, et al. Mol Biol Cell. 2013 February 15;24(4):453-464.

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