Arl13b regulates ciliogenesis and the dynamic localization of Shh signaling proteins

Department of Human Genetics, School of Medicine, Emory University, Atlanta, GA 30322, USA.
Molecular biology of the cell (Impact Factor: 4.47). 12/2011; 22(23):4694-703. DOI: 10.1091/mbc.E10-12-0994
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Arl13b, a ciliary protein within the ADP-ribosylation factor family and Ras superfamily of GTPases, is required for ciliary structure but has poorly defined ciliary functions. In this paper, we further characterize the role of Arl13b in cilia by examining mutant cilia in vitro and determining the localization and dynamics of Arl13b within the cilium. Previously, we showed that mice lacking Arl13b have abnormal Sonic hedgehog (Shh) signaling; in this study, we show the dynamics of Shh signaling component localization to the cilium are disrupted in the absence of Arl13b. Significantly, we found Smoothened (Smo) is enriched in Arl13b-null cilia regardless of Shh pathway stimulation, indicating Arl13b regulates the ciliary entry of Smo. Furthermore, our analysis defines a role for Arl13b in regulating the distribution of Smo within the cilium. These results suggest that abnormal Shh signaling in Arl13b mutant embryos may result from defects in protein localization and distribution within the cilium.

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Available from: Christine Larkins, Sep 14, 2014

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Article: Arl13b regulates ciliogenesis and the dynamic localization of Shh signaling proteins

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    • "CML cells had large Smo-positive structures that colocalized with glutamylated tubulin, whereas PBMC and AML cells had only diffuse, cytoplasmic Smo staining (Fig. 5C). Two other proteins associated with primary cilium function colocalized with Smo and glutamylated tubulin foci in CML cells (Fig. 5D & Fig. S5): IFT88, a component of intraflagellar transport normally found on the ciliary axoneme [32], and Arl13b, a small GTPase found in the ciliary membrane [33]. Interestingly, these masses in CML cells were associated with protrusions in most cases (Fig. 5C,D & Fig. S5, arrows). "
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    ABSTRACT: Chromosomal translocations observed in myeloproliferative neoplasms (MPNs) frequently fuse genes that encode centrosome proteins and tyrosine kinases. This causes constitutive activation of the kinase resulting in aberrant, proliferative signaling. The function of centrosome proteins in these fusions is not well understood. Among others, kinase centrosome localization and constitutive kinase dimerization are possible consequences of centrosome protein-kinase fusions. To test the relative contributions of localization and dimerization on kinase signaling, we targeted inducibly dimerizable FGFR1 to the centrosome and other subcellular locations and generated a mutant of the FOP-FGFR1 MPN fusion defective in centrosome localization. Expression in mammalian cells followed by western blot analysis revealed a significant decrease in kinase signaling upon loss of FOP-FGFR1 centrosome localization. Kinase dimerization alone resulted in phosphorylation of the FGFR1 signaling target PLCγ, however levels comparable to FOP-FGFR1 required subcellular targeting in addition to kinase dimerization. Expression of MPN fusion proteins also resulted in centrosome disruption in epithelial cells and transformed patient cells. Primary human MPN cells showed masses of modified tubulin that colocalized with centrin, Smoothened (Smo), IFT88, and Arl13b. This is distinct from acute myeloid leukemia (AML) cells, which are not associated with centrosome-kinase fusions and had normal centrosomes. Our results suggest that effective proliferative MPN signaling requires both subcellular localization and dimerization of MPN kinases, both of which may be provided by centrosome protein fusion partners. Furthermore, centrosome disruption may contribute to the MPN transformation phenotype.
    PLoS ONE 03/2014; 9(3):e92641. DOI:10.1371/journal.pone.0092641 · 3.23 Impact Factor
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    • "In this study, we performed immunofluorescence using three different ciliary markers: Arl13b, a small GTPase that is localized on the cilia membrane (Hori et al., 2008; Larkins et al., 2011; Hsiao et al., 2012); acetylated ␣-tubulin, that labels tubulin based axoneme (Piperno and Fuller, 1985); and adenylyl cyclase III, which converts ATP to cyclic AMP and is known to be enriched in neuronal cilia (Defer et al., 2000; Bishop et al., 2007). Through this "
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    ABSTRACT: The primary cilia are considered as "cellular antennae" that sense and interchange information with the extracellular environment. Nearly all mammalian cells have a single primary cilium. In the retina, the outer segment of the photoreceptor is known to be a specialized form of primary cilium, but studies on cilia in other layers of the retina are scarce. In this study, we investigated the expression of primary cilia in the whole thickness of the mouse retina using immunofluorescence with three different ciliary markers: Arl13b, acetylated α-tubulin and adenylyl cyclase III. Our results show positive reactions in the photoreceptor layer, outer plexiform layer and ganglion cell layer, which might suggest the possible presence of primary cilia in these areas, but we could not directly prove the strand-like shape of cilia in those areas. In the outer plexiform layer, all three markers showed intense staining along the neuronal synapses, which suggests that the neuronal processes themselves might share the features of cilia.
    Acta histochemica 04/2013; 115(8). DOI:10.1016/j.acthis.2013.03.005 · 1.71 Impact Factor
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    • "In contrast, Cluap1KO embryos stained positive for Shh ligand, but lacked a defined Shh positive floorplate (Figure 5B,F). Furthermore, staining for Arl13b, a small GTPase that localizes to primary cilia and is necessary for Shh signaling, confirmed an absence of cilia in the neural tubes of Cluap1KO embryos (Figure 5D) [30,31]. To further confirm defects in Hh signaling, whole embryos were analyzed for overall Shh pathway activity by qRT-PCR analysis of Patched-1 and Gli1, two downstream target genes induced by Hh. "
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