The molecular complex of ciliary and golgin protein is crucial for skull development.

Intramembranous ossification, which consists of direct conversion of mesenchymal cells to osteoblasts, is a characteristic process in skull development. One crucial role of these osteoblasts is to secrete collagen-containing bone matrix. However, it remains unclear how the dynamics of collagen trafficking is regulated during skull development. Here, we reveal the regulatory mechanisms of ciliary and golgin proteins required for intramembranous ossification. During normal skull formation, osteoblasts residing on the osteogenic front actively secreted collagen. Mass spectrometry and proteomic analysis determined endogenous binding between ciliary protein IFT20 and golgin protein GMAP210 in these osteoblasts. As seen in Ift20 mutant mice, disruption of neural crest-specific GMAP210 in mice caused osteopenia-like phenotypes due to dysfunctional collagen trafficking. Mice lacking both IFT20 and GMAP210 displayed more severe skull defects compared with either IFT20 or GMAP210 mutants. These results demonstrate that the molecular complex of IFT20 and GMAP210 is essential for the intramembranous ossification during skull development.

Mouse spermatogenesis-associated protein 1 (SPATA1), an IFT20 binding partner, is an acrosomal protein.

BACKGROUND: Intraflagellar transport is a motor-driven trafficking system that is required for the formation of cilia. Intraflagellar transport protein 20 (IFT20) is a master regulator for the control of spermatogenesis and male fertility in mice. However, the mechanism of how IFT20 regulates spermatogenesis is unknown. RESULTS: Spermatogenesis associated 1 (SPATA1) was identified to be a major potential binding partner of IFT20 by a yeast two-hybrid screening. The interaction between SPATA1 and IFT20 was examined by direct yeast two-hybrid, co-localization, and co-immunoprecipitation assays. SPATA1 is highly abundant in the mouse testis, and is also expressed in the heart and kidney. During the first wave of spermatogenesis, SPATA1 is detectable at postnatal day 24 and its expression is increased at day 30 and 35. Immunofluorescence staining of mouse testis sections and epididymal sperm demonstrated that SPATA1 is localized mainly in the acrosome of developing spermatids but not in epididymal sperm. IFT20 is also present in the acrosome area of round spermatids. In conditional Ift20 knockout mice, testicular expression level and acrosomal localization of SPATA1 are not changed. CONCLUSIONS: SPATA1 is an IFT20 binding protein and may provide a docking site for IFT20 complex binding to the acrosome area.

SPEF2 functions in microtubule-mediated transport in elongating spermatids to ensure proper male germ cell differentiation.

Sperm differentiation requires specific protein transport for correct sperm tail formation and head shaping. A transient microtubular structure, the manchette, appears around the differentiating spermatid head and serves as a platform for protein transport to the growing tail. Sperm flagellar 2 (SPEF2) is known to be essential for sperm tail development. In this study we investigated the function of SPEF2 during spermatogenesis using a male germ cell-specific Spef2 knockout mouse model. In addition to defects in sperm tail development, we observed a duplication of the basal body and failure in manchette migration resulting in an abnormal head shape. We identified cytoplasmic dynein 1 and GOLGA3 as novel interaction partners for SPEF2. SPEF2 and dynein 1 colocalize in the manchette and the inhibition of dynein 1 disrupts the localization of SPEF2 to the manchette. Furthermore, the transport of a known SPEF2-binding protein, IFT20, from the Golgi complex to the manchette was delayed in the absence of SPEF2. These data indicate a possible novel role of SPEF2 as a linker protein for dynein 1-mediated cargo transport along microtubules.

Regulation of c-SMAC formation and AKT-mTOR signaling by the TSG101-IFT20 axis in CD4+ T cells.

CD4+ T cells play major roles in the adaptive immune system, which requires antigen recognition, costimulation, and cytokines for its elaborate orchestration. Recent studies have provided new insight into the importance of the supramolecular activation cluster (SMAC), which comprises concentric circles and is involved in the amplification of CD4+ T cell activation. However, the underlying mechanism of SMAC formation remains poorly understood. Here, we performed single-cell RNA sequencing of CD4+ T cells left unstimulated and stimulated with anti-CD3 and anti-CD28 antibodies to identify novel proteins involved in their regulation. We found that intraflagellar transport 20 (IFT20), previously known as cilia-forming protein, was upregulated in antibody-stimulated CD4+ T cells compared to unstimulated CD4+ T cells. We also found that IFT20 interacted with tumor susceptibility gene 101 (TSG101), a protein that endocytoses ubiquitinated T-cell receptors. The interaction between IFT20 and TSG101 promoted SMAC formation, which led to amplification of AKT-mTOR signaling. However, IFT20-deficient CD4+ T cells showed SMAC malformation, resulting in reduced CD4+ T cell proliferation, aerobic glycolysis, and cellular respiration. Finally, mice with T-cell-specific IFT20 deficiency exhibited reduced allergen-induced airway inflammation. Thus, our data suggest that the IFT20-TSG101 axis regulates AKT-mTOR signaling via SMAC formation.

Intraflagellar transport 20: New target for the treatment of ciliopathies.

Cilia are ubiquitous in mammalian cells. The formation and assembly of cilia depend on the normal functioning of the ciliary transport system. In recent years, various proteins involved in the intracellular transport of the cilium have attracted attention, as many diseases are caused by disorders in cilia formation. Intraflagellar transport 20 (IFT20) is a subunit of IFT complex B, which contains approximately 20 protein particles. Studies have shown that defects in IFT20 are associated with numerous system -related diseases, such as those of the urinary system, cardiovascular system, skeletal system, nervous system, immune system, reproductive system, and respiratory system. This review summarizes current research on IFT20.We describe studies related to the role of IFT20 in cilia formation and discuss new targets for treating diseases associated with ciliary dysplasia.