Limits...
Bardet-Biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling.

Wiens CJ, Tong Y, Esmail MA, Oh E, Gerdes JM, Wang J, Tempel W, Rattner JB, Katsanis N, Park HW, Leroux MR - J. Biol. Chem. (2010)

Bottom Line: The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions.Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane.Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

ABSTRACT
The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions. Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane. Overproduction of GDP- or GTP-locked variants of ARL6/BBS3 in vivo influences primary cilium length and abundance. ARL6/BBS3 also modulates Wnt signaling, a signal transduction pathway whose association with cilia in vertebrates is just emerging. Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations. By determining the structure of GTP-bound ARL6/BBS3, coupled with functional assays, we provide a mechanistic explanation for such pathogenic alterations, namely altered nucleotide binding. Our findings therefore establish a previously unknown role for ARL6/BBS3 in mammalian ciliary (dis)assembly and Wnt signaling and provide the first structural information for a BBS protein.

Show MeSH

Related in: MedlinePlus

ARL6 localizes at the distal end of the basal body in a ring-shaped pattern. A, immunocytochemistry using the ARL6C antibody shows ARL6 localizing to the distal end of mother centrioles and basal bodies in hTERT-RPE cells. Inset, magnified image of the selected area. B, confocal microscopy images using the ARL6N antibody in hTERT-RPE cells shows that ARL6 localizes in a ring-like pattern, just distal to the mother centrioles, in nonciliated and ciliated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2871489&req=5

Figure 5: ARL6 localizes at the distal end of the basal body in a ring-shaped pattern. A, immunocytochemistry using the ARL6C antibody shows ARL6 localizing to the distal end of mother centrioles and basal bodies in hTERT-RPE cells. Inset, magnified image of the selected area. B, confocal microscopy images using the ARL6N antibody in hTERT-RPE cells shows that ARL6 localizes in a ring-like pattern, just distal to the mother centrioles, in nonciliated and ciliated cells.

Mentions: To perform our immunocytochemistry experiments, we employed polyclonal antibodies directed against N- and C-terminal epitopes of human ARL6 (named ARL6N and ARL6C, respectively); by Western blot analysis, both recognize ARL6 but not the closely related ARF6 protein, used as a control for specificity (supplemental Fig. 2). In mouse medullary collecting duct epithelial cells (IMCD3), the two anti-ARL6 antibodies specifically stain two peri-nuclear puncta that co-localize with the centrosomal marker γ-tubulin (Fig. 4, A and B). The ARL6 signal at centrosomes could be observed throughout the cell cycle, indicating a specific association with the dynamic microtubule organizing center; in interphase cells, ARL6 was associated with the basal body, which sports a cilium that is detected using an antibody against acetylated tubulin (Fig. 4B). We confirmed the centrosomal/basal body localization of ARL6 in a second ciliated cell line, namely retinal pigmented epithelial cells (hTERT-RPE) (Fig. 5). Importantly, we also observed co-localization of ARL6 with basal bodies in several ciliated mammalian tissue sections, including testis, brain, and kidney (supplemental Fig. 3, A–C). These data demonstrate that ARL6/BBS3 associates with centriolar structures/basal bodies in a variety of ciliated cells in vivo.


Bardet-Biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling.

Wiens CJ, Tong Y, Esmail MA, Oh E, Gerdes JM, Wang J, Tempel W, Rattner JB, Katsanis N, Park HW, Leroux MR - J. Biol. Chem. (2010)

ARL6 localizes at the distal end of the basal body in a ring-shaped pattern. A, immunocytochemistry using the ARL6C antibody shows ARL6 localizing to the distal end of mother centrioles and basal bodies in hTERT-RPE cells. Inset, magnified image of the selected area. B, confocal microscopy images using the ARL6N antibody in hTERT-RPE cells shows that ARL6 localizes in a ring-like pattern, just distal to the mother centrioles, in nonciliated and ciliated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871489&req=5

Figure 5: ARL6 localizes at the distal end of the basal body in a ring-shaped pattern. A, immunocytochemistry using the ARL6C antibody shows ARL6 localizing to the distal end of mother centrioles and basal bodies in hTERT-RPE cells. Inset, magnified image of the selected area. B, confocal microscopy images using the ARL6N antibody in hTERT-RPE cells shows that ARL6 localizes in a ring-like pattern, just distal to the mother centrioles, in nonciliated and ciliated cells.
Mentions: To perform our immunocytochemistry experiments, we employed polyclonal antibodies directed against N- and C-terminal epitopes of human ARL6 (named ARL6N and ARL6C, respectively); by Western blot analysis, both recognize ARL6 but not the closely related ARF6 protein, used as a control for specificity (supplemental Fig. 2). In mouse medullary collecting duct epithelial cells (IMCD3), the two anti-ARL6 antibodies specifically stain two peri-nuclear puncta that co-localize with the centrosomal marker γ-tubulin (Fig. 4, A and B). The ARL6 signal at centrosomes could be observed throughout the cell cycle, indicating a specific association with the dynamic microtubule organizing center; in interphase cells, ARL6 was associated with the basal body, which sports a cilium that is detected using an antibody against acetylated tubulin (Fig. 4B). We confirmed the centrosomal/basal body localization of ARL6 in a second ciliated cell line, namely retinal pigmented epithelial cells (hTERT-RPE) (Fig. 5). Importantly, we also observed co-localization of ARL6 with basal bodies in several ciliated mammalian tissue sections, including testis, brain, and kidney (supplemental Fig. 3, A–C). These data demonstrate that ARL6/BBS3 associates with centriolar structures/basal bodies in a variety of ciliated cells in vivo.

Bottom Line: The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions.Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane.Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

ABSTRACT
The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions. Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane. Overproduction of GDP- or GTP-locked variants of ARL6/BBS3 in vivo influences primary cilium length and abundance. ARL6/BBS3 also modulates Wnt signaling, a signal transduction pathway whose association with cilia in vertebrates is just emerging. Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations. By determining the structure of GTP-bound ARL6/BBS3, coupled with functional assays, we provide a mechanistic explanation for such pathogenic alterations, namely altered nucleotide binding. Our findings therefore establish a previously unknown role for ARL6/BBS3 in mammalian ciliary (dis)assembly and Wnt signaling and provide the first structural information for a BBS protein.

Show MeSH
Related in: MedlinePlus