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Viewed and Photographed on a Laser Scanning Confocal Type (WT) and Tubby Mouse Retina and Brain Extracts

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Viewed and Photographed on a Laser Scanning Confocal Type (WT) and Tubby Mouse Retina and Brain Extracts Sun et al. Cilia 2012, 1:21 http://www.ciliajournal.com/content/1/1/21 RESEARCH Open Access Tubby is required for trafficking G protein-coupled receptors to neuronal cilia Xun Sun1, James Haley1, Oleg V Bulgakov1, Xue Cai2, James McGinnis2 and Tiansen Li1* Please see related Commentary article by Mukhopadhyay and Jackson http://www.ciliajournal.com/content/2/1/1 Background Abstract The tubby-like proteins are defined by a highly con- Background: Tubby is the founding member of the served carboxyl terminal half of their primary sequence tubby-like family of proteins. The naturally occurring known as the tubby signature domain [1,2]. This family tubby mutation in mice causes retinitis pigmentosa, of proteins includes the prototype tubby, and TULP1, 2 hearing loss and obesity. Tubby has been proposed to and 3, for tubby-like proteins 1, 2 and 3 [3-5]. Other function as an accessory factor in ciliary trafficking. We than members of the tubby family, search of sequence directly examined a role for tubby in ciliary trafficking databases reveals no significant homology with known Tub in vivo. proteins or functional motifs. The tubby gene ( )was originally discovered by way of a spontaneously arisen Methods: We used immunofluoresence labeling to obesity model in mice, and other members of the family examine the subcellular localization of rhodopsin, were subsequently identified by homology cloning [3]. somatostatin receptor 3 (SSTR3) and melanin Mutations in human TULP1 are a cause of retinitis pig- concentrating hormone receptor 1 (MCHR1), all of mentosa [6]. Loss of TULP1 function in mice replicates which are G protein-coupled receptors (GPCR), in the this rapid photoreceptor degeneration phenotype [7,8]. retina and brain of wild type (WT) and tubby mutant Prior to photoreceptor degeneration in the mouse retina, mice. pronounced ectopic distribution of rhodopsin is apparent tubby Results: In mouse retina, rhodopsin is not fully indicating a defect in trafficking across the connecting cilia transported across the connecting cilia to the outer to reach their normal destination, the outer segments segments with ensuing photoreceptor degeneration. In [9]. Loss of TULP3 function in mice leads to neural tubby the mouse brain, SSTR3 and MCHR1 fail to tube patterning defects and embryonic lethality [10], and localize at the neuronal primary cilia in regions where the cellular basis can be traced to a failure of Hedgehog these receptors play critical roles in neural signaling. The signaling due to defective ciliary trafficking [11]. Little is tubby mutant does not manifest a generalized defect in known about Tulp2, but its Chlamydomonas ortholog ciliogenesis or protein trafficking. was identified as one of strongly induced genes during Conclusions: Tubby plays a critical role in trafficking flagellar regeneration [12] and it was also reported as a can- select GPCRs to the cilia. This role is reminiscent of didate gene for human obesity in linkage analysis [13]. The tubby-like proteins 1 and 3, which have been proposed tubby signature domain binds polyphosphorylated phos- to facilitate trafficking of rhodopsin and select GPCRs in phatidylinositol [14], but their N-terminal domain is much photoreceptors and the developing neural tube, more diverse. In the best characterized example, the TULP3 respectively. Thus tubby-like proteins may be generally N-terminal domain binds to the IFT-A complex, which is involved in transciliary trafficking of GPCRs. part of the essential cellular machinery for ciliary transport, through a short conserved motif. In cultured cells, TULP3 Keywords: Cilia, Neuronal cilia, Connecting cilia facilitates membrane receptor trafficking to primary cilia. Thus it serves as bipartite bridges through their phosphoinositide-binding tubby domain and N-terminal * Correspondence: [email protected] 1Neurobiology Neurodegeneration and Repair Laboratory (N-NRL), National IFT-binding motif, coordinating multiple signaling path- Eye Institute, MSC0610, 6 Center Drive, Bethesda, MD 20892, USA ways including membrane receptor trafficking [15]. Full list of author information is available at the end of the article © 2012 Sun et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sun et al. Cilia 2012, 1:21 Page 2 of 12 http://www.ciliajournal.com/content/1/1/21 Originally designated rd5 [16], the spontaneously arisen target genes of tubby have remained unknown. In an- tubby mouse mutant manifests retinal degeneration, hear- other series of studies, tubby was proposed to be a ing loss and obesity, a tripartite phenotype that resembles MerTK ligand that mediates phagocytosis of the photo- mouse models of Bardet-Biedl syndrome (BBS) [17,18]. The receptor outer segments by retinal pigment epithelia [26]. tubby mutation is a G-to-T transversion that abolishes the These findings represented advances in the molecular dis- donor splice site in the penultimate exon (exon 11), result- section of tubby function, but how they relate to the ing in an aberrant transcript [19]. This leads to the substitu- in vivo role of tubby and the tubby mutant phenotype has tion of the tubby C-terminal 44 amino acids with 24 been less clear. In this study, we examined the subcellular different residues encoded by the intron. The spontaneous distribution of a number GPCRs and show that tubby is tub/tub mutation in the tubby mouse (Tub ) appears to cause essential for GPCR trafficking in the neuronal and sensory a loss of function, as targeted disruption of the tub gene cilia. gives a similar phenotype [20]. The original study on tubby mice found moderate and progressive hearing loss, and a Methods moderate retinal degeneration [16]. A modifier gene Animals Mtap1a modulates the severity of hearing loss and retinal All animal care and procedures were approved by Animal degeneration in the tubby mutant mice [21,22]. One inter- Care and Use Committees at the Dean A. McGee Eye In- esting feature of the tubby mutant that is shared with the stitute and the National Eye Institute. Mice were main- Tulp1 knockout mouse is the extracellular accumulation of tained in an animal facility under a 12-h light/12-h dark rhodopsin-laden vesicles in the interphotoreceptor space lighting cycle. Genotyping for tubby mutation was based surrounding the photoreceptor inner segments, which on a published protocol [27]. WT and tubby mutant mice peaks at around 17 to 21 days of age when rhodopsin is at 1 month of age were used for analysis of the brain tissue, rapidly synthesized to build up the outer segments [16]. and mice at 1 month and at 12 days were used to analyze The vesicles are relatively uniform in size averaging 0.1 to the retinal tissues. 0.2 μm in diameter and bounded by a single membrane. This distinct phenotype is also seen in transgenic mice car- Generation of tubby antibody rying a C-terminal rhodopsin mutation known to affect A His-tagged fusion protein encompassing the N-terminal specifically the trafficking of rhodopsin to the outer seg- 200 amino acid residues of mouse tubby protein was ments [23]. It was, therefore, hypothesized that the extra- expressed in E. coli, purified and used to generate a poly- cellular vesicle accumulation might be a hallmark of defect clonal antibody in rabbit. The antibody was affinity- in the directional transport of nascent rhodopsin to the purified. outer segments, thus implying a role for tubby and TULP1 in rhodopsin trafficking in photoreceptors [9]. In further Immunoblotting analysis support of this hypothesis, mice doubly mutant for tubby Mice were euthanized and their retinas and brain were dis- and Tulp1 haveamuchmoresevereretinalphenotypethan sected out. Tissues were homogenized in RIPA buffer, either mutant alone, manifesting a complete failure of rho- boiledinLaemmlibufferandseparatedon10%SDS-PAGE dopsin trafficking and outer segment formation, and rapid gels. Proteins were blotted to polyvinylidene difluoride cell death. These data would appear to suggest that tubby (PVDF) membrane by electrotransfer. After blocking with may function synergistically with TULP1 in a pathway that 5% non-fat milk, the membranes were incubated with pri- facilitates rhodopsin trafficking to the outer segments [9]. mary antibodies overnight at room temperature. After Differing from TULP1, which is photoreceptor-specific, washing, membranes were incubated with peroxidase- W tubby has a wider range of expression but appears enriched conjugated secondary antibodies. SuperSignal West Pico in neuronal tissues. Chemiluminescent Substrate (Thermo Fisher Scientific, Based on a structure-directed approach, it has been Rockford, IL, USA) was used for detection. For proposed that tubby-like proteins are a unique family of normalization, protein samples were separated on stand- bipartite transcription factors [14,24]. The molecular ard SDS-PAGE and probed with an anti-actin antibody. architecture of tubby-like proteins is seen as well suited for a function in transcriptional modulation. There is Immunofluorescence the nuclear localization signal at the N terminus of For immunofluorescence, eyes were enucleated, placed in tubby, an ability of the N-terminal domain to activate fixative and their anterior segments and lens were transcription when fused to a DNA binding motif and removed. Brains were placed directly in a fixative contain- the ability of the conserved C-terminal tubby domain to ing 2% paraformaldehyde in phosphate buffered saline bind DNA and phosphatidylinositol 4, 5-bisphosphate (PBS) for a total duration of 1.5 to 2 hours. Tissues were (PIP2). That the tubby domain binds specifically to PIP2 embedded in 3% agarose and sectioned at 75-μmthickness has been well established [25] but the transcriptional using a vibratome. Sections were collected into PBS buffer Sun et al. Cilia 2012, 1:21 Page 3 of 12 http://www.ciliajournal.com/content/1/1/21 and remained free floating for the duration of the immu- Results nostaining process.
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