Loss-of-function mutations in the gene encoding FAM161A were recently discovered as the cause for RP28, an autosomal recessive form of retinitis pigmentosa. To initiate the characterization of the cellular role of FAM161A in the retina, we focused on its subcellular localization and conducted in vitro studies to identify FAM161A-interacting proteins and associated cellular structures. Immunohistochemistry revealed the presence of mouse FAM161A in the photoreceptor inner segments, the synaptic regions of the outer and inner plexiform layers and the ganglion cells. In mouse and human retinal sections from unfixed eyes, FAM161A localized to the ciliary region linking photoreceptor outer and inner segments. High-resolution immunofluorescence and immunoelectron microscopy mapped FAM161A to the connecting cilium, the basal body region and the adjacent centriole. Ectopic FAM161A was found in the centrosome and concentrated at the base of primary cilia in cultured cells. In addition, overexpressed FAM161A was clearly associated with microtubules during interphase and mitosis. The presence of FAM161A increased microtubule acetylation and stabilization. We further show that the evolutionarily conserved UPF0564 domain of FAM161A is crucial for its binding to microtubules and mediates homo- and heterotypic FAM161A and FAM161B interaction. In conclusion, our study shows that FAM161A is a microtubule-associated ciliary protein presumably involved in microtubule stabilization to maintain the microtubule tracks and/or in transport processes along microtubules in photoreceptors and other retinal cell types.

The sequestosome 1 gene encodes the p62 protein and is the major genetic risk factor associated with Paget's disease of bone. In 2004, p62 was reported to up-regulate osteoclast differentiation by activating the transcription factors Nfatc1 and NF-κB. Here, we characterized the osteoclastogenic potential of murine p62-derived cells compared with WT cells. Our data confirmed previous findings indicating that p62 is induced during murine osteoclast differentiation. Surprisingly, an indispensable role for p62 in osteoclast differentiation was not reproducible because p62-deficient osteoclasts exhibited robust activation of Nfatc1, NF-κB, and osteoclast marker enzymes. Thus, we concluded that osteoclast differentiation is not negatively influenced by knocking out p62. On the contrary, our results revealed that p62 deficiency accelerates osteoclastogenesis. Differentiation potential, multinucleation status, and soluble receptor activator of NF-κB ligand (sRANKL) sensitivity were significantly elevated in p62-deficient, murine bone marrow-derived stem cells. Moreover, femur ultrastructures visualized by micro-computed tomography revealed pronounced accumulation of adipocytes and trabecular bone material in distal femora of obese p62 mice. Increased tartrate-resistant acid phosphatase activity, along with increased trabecular bone and accumulation of adipocytes, was confirmed in both paraffin-embedded decalcified and methyl methacrylate-embedded nondecalcified bones from p62 mice. Of note, Paget's disease-like osteolytic lesions and increased levels of the bone turnover markers CTX-I and PINP were also observed in the p62 mice. Our results indicate that p62 predominantly suppresses murine osteoclast differentiation and highlight previously undetected Paget's disease-like phenotypes in p62 mice .

Retinitis pigmentosa (RP) is an inherited disease of the retina leading to vision impairment due to progressive photoreceptor cell death. Homozygous and compound heterozygous null mutations in the CRX-regulated FAM161A gene of unknown function were identified as a cause for autosomal recessive RP (RP28) in patients from India, Germany, Israel, the Palestinian territories, and the USA. The FAM161A protein has been found to be localized to the connecting cilium, the basal body, and the adjacent centriole in mammalian photoreceptors and was also present in synaptic layers and ganglion cells of the retina. In addition, FAM161A was shown to be part of microtubule-organizing centers in cultured cells and associates with the intracellular microtubule network. Moreover, FAM161A directly binds to microtubules and increases the acetylation of α-tubulin. An evolutionary highly conserved, C-terminal protein domain (UPF0564) of FAM161A was shown to mediate microtubule association, homo- and heterotypic interaction among UPF0564-containing proteins and binding to several ciliopathy-associated proteins. In summary, FAM161A is a novel centrosomal-ciliary protein that likely is implicated in the regulation of microtubule-based cellular processes in the retina.

In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from wild type or genetically manipulated mouse lines. These cells represent a standardized and theoretically unlimited source for osteoclast-associated research projects.

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