Search

To improve the accuracy of the calculation analysis of crank journal bearings in motorcycle engines and accurately understand lubrication conditions, the oil film conditions of actual crankshafts and journal bearings should be measured. This research study focuses on the oil film pressure generated in the main bearing, and by using an original thin-film pressure sensor with improved durability achieved through the use of DLC (Diamond-like Carbon), it was possible to perform experiments at a maximum of 13,000 rpm and full load, which was not possible before. This established a method for measuring the oil film pressure generated in the main bearing of a high-speed motorcycle engine during operation without changing the surrounding environment. The maximum oil film pressure was 140 MPa, and the oil film pressure generated by each main bearing was successfully measured under different experimental conditions. The timing of pressure onset agreed well between the calculation and experiment stages, but the peak oil film pressure values were different. By varying the temperature of the engine in the calculation model, the calculated values approached the measured values. In the future, we plan to investigate ways to improve the accuracy of the current analytical model.

Abstract Cilia play crucial roles in sensing and transducing extracellular signals. Bidirectional protein trafficking within cilia is mediated by the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes, with the aid of kinesin-2 and dynein-2 motors. The dynein-2 complex drives retrograde trafficking of the IFT machinery after its transportation to the ciliary tip as an IFT cargo. Mutations in genes encoding the dynein-2-specific subunits (DYNC2H1, WDR60, WDR34, DYNC2LI1, and TCTEX1D2) are known to cause skeletal ciliopathies. We here demonstrate that several pathogenic variants of DYNC2LI1 are compromised regarding their ability to interact with DYNC2H1 and WDR60. When expressed in DYNC2LI1-knockout cells, deletion variants of DYNC2LI1 were unable to rescue the ciliary defects of these cells, whereas missense variants, as well as wild-type DYNC2LI1, restored the normal phenotype. DYNC2LI1-knockout cells coexpressing one pathogenic deletion variant together with wild-type DYNC2LI1 demonstrated a normal phenotype. In striking contrast, DYNC2LI1-knockout cells coexpressing the deletion variant in combination with a missense variant, which mimics the situation of cells of compound heterozygous ciliopathy individuals, demonstrated ciliary defects. Thus, DYNC2LI1 deletion variants found in individuals with skeletal ciliopathies cause ciliary defects when combined with a missense variant, which expressed on its own does not cause substantial defects.

INPP5E, a phosphoinositide 5-phosphatase, localizes on the ciliary membrane via its C-terminal prenyl moiety, and maintains the distinct ciliary phosphoinositide composition. The ARL3 GTPase contributes to the ciliary membrane localization of INPP5E by stimulating the release of PDE6D bound to prenylated INPP5E. Another GTPase, ARL13B, which is localized on the ciliary membrane, contributes to the ciliary membrane retention of INPP5E by directly binding to its ciliary targeting sequence. However, as ARL13B was shown to act as a guanine nucleotide exchange factor (GEF) for ARL3, it is also possible that ARL13B indirectly mediates the ciliary INPP5E localization via activating ARL3. We here show that INPP5E is delocalized from cilia in both ARL3-knockout (KO) and ARL13B-KO cells. However, some of the abnormal phenotypes were different between these KO cells, while others were found to be common, indicating the parallel roles of ARL3 and ARL13B, at least concerning some cellular functions. For several variants of ARL13B, their ability to interact with INPP5E, rather than their ability as an ARL3-GEF, was associated with whether they could rescue the ciliary localization of INPP5E in ARL13B-KO cells. These observations together indicate that ARL13B determines the ciliary localization of INPP5E, mainly by its direct binding to INPP5E.

Compositions of proteins and lipids within cilia and on the ciliary membrane are maintained to be distinct from those of the cytoplasm and plasma membrane, respectively, by the presence of the ciliary gate. INPP5E is a phosphoinositide 5-phosphatase that is localized on the ciliary membrane by anchorage via its C-terminal prenyl moiety. In addition, the ciliary membrane localization of INPP5E is determined by the small GTPase ARL13B. However, it remained unclear as to how ARL13B participates in the localization of INPP5E. We here show that wild-type INPP5E, INPP5E(WT), in ARL13B-knockout cells and an INPP5E mutant defective in ARL13B binding, INPP5E(ΔCTS), in control cells were unable to show steady-state localization on the ciliary membrane. However, not only INPP5E(WT) but also INPP5E(ΔCTS) was able to rescue the abnormal localization of ciliary proteins in INPP5E-knockout cells. Analysis using the chemically induced dimerization system demonstrated that INPP5E(WT) in ARL13B-knockout cells and INPP5E(ΔCTS) in control cells were able to enter cilia, but neither was retained on the ciliary membrane due to the lack of the INPP5E–ARL13B interaction. Thus, our data demonstrate that binding of INPP5E to ARL13B is essential for its steady-state localization on the ciliary membrane but is dispensable for its entry into cilia.

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.

The intraflagellar transport (IFT) machinery, which includes the IFT-A and IFT-B complexes, mediates bidirectional trafficking of ciliary proteins. In addition to these complexes, the BBSome, which is composed of eight subunits that are encoded by the causative genes of Bardet-Biedl syndrome (BBS), has been proposed to connect the IFT machinery to ciliary membrane proteins, such as G protein-coupled receptors, to mediate their export from cilia. However, little is known about the connection between the IFT machinery and the BBSome. Using the visible immunoprecipitation assay, we here identified the interaction between IFT38 from the IFT-B complex and BBS1, BBS2 and BBS9 from the BBSome. Furthermore, by analyzing phenotypes of IFT38-knockout cells exogenously expressing wild-type IFT38 or its mutant lacking the ability to interact with BBS1+BBS2+BBS9, we showed that knockout cells expressing the IFT38 mutant have restored ciliogenesis; however, similar to BBS1-knockout cells, they demonstrated significant accumulation of GPR161 within cilia upon stimulation of Hedgehog signaling. These results indicate that the IFT-B–BBSome interaction is required for the export of GPR161 across the ciliary gate.

In the intraflagellar transport (IFT) machinery, the IFT-B and IFT-A complexes mediate anterograde and retrograde ciliary protein trafficking, respectively. Among the 16 subunits of the IFT-B complex, several subunits are essential for ciliogenesis, whereas others, which are associated peripherally with the complex, are dispensable for ciliogenesis but play a role in protein trafficking. IFT22-knockout (KO) cells established in this study demonstrated no defects in ciliogenesis or ciliary protein trafficking. In stark contrast, IFT70A and IFT70B double-knockout cells did not form cilia, even though IFT70 is associated peripherally with the IFT-B complex via the IFT52–IFT88 dimer, and other IFT-B subunits assembled at the ciliary base in the absence of IFT70. Exogenous expression of either IFT70A or IFT70B restored the ciliogenesis defect of IFT70-KO cells, indicating their redundant roles. IFT70 has 15 consecutive tetratricopeptide repeats (TPRs) followed by a short helix (α36). Deletion of the first TPR or α36 of IFT70A greatly reduced its ability to interact with the IFT52–IFT88 dimer. Exogenous expression of any of the IFT70A deletion mutants in IFT70-KO cells could not restore ciliogenesis. These results show that IFT70 plays an essential role in ciliogenesis, although it is dispensable for assembly of the residual IFT-B subunits.

The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can be visualized by directly observing the beads bearing immunoprecipitates under a fluorescence microscope. This assay can examine a large number of protein combinations at one time, without requiring time-consuming procedures, including SDS-PAGE and immunoblotting. Furthermore, the VIP assay can examine complicated one-to-many and many-to-many protein interactions. Another important point of the VIP assay is the use of nanobodies for immunoprecipitation. A Nanobody is a single-domain antibody derived from Camelidae (camels and relatives). Because of its small size, high-affinity, high-specificity, and stability, anti-GFP Nanobody expressed in E. coli can be purified on a large scale, and used virtually inexhaustibly for immunoprecipitation experiments. Here we describe protocols for preparation of GST-tagged anti-GFP Nanobody and the VIP assay.

Protein trafficking within cilia is mediated by the intraflagellar transport (IFT) machinery composed of large protein complexes. The BBSome consists of eight BBS proteins encoded by causative genes of Bardet-Biedl syndrome (BBS), and has been implicated in the trafficking of ciliary membrane proteins, including G protein-coupled receptors (GPCRs), by connecting the IFT machinery to cargo GPCRs. Membrane recruitment of the BBSome to promote cargo trafficking has been proposed to be regulated by the Arf-like small GTPase ARL6/BBS3, through its interaction with the BBS1 subunit of the BBSome. We here investigated how the BBSome core subcomplex composed of BBS1, BBS2, BBS7, and BBS9 assembles and interacts with ARL6, and found that the ARL6-BBS1 interaction is reinforced by BBS9. BBS1-knockout (KO) cells showed defects in the ciliary entry of other BBSome subunits and ARL6, and in ciliary retrograde trafficking and the export of the GPCRs, Smoothened and GPR161. The trafficking defect of these GPCRs was rescued by the exogenous expression of wild-type BBS1, but not by its mutant lacking BBS9-binding ability. Our data thus indicate that the intact BBSome is required for retrograde trafficking of GPCRs out of cilia.

We recently showed that Rab11 is involved not only in formation of recycling vesicles containing the transferrin (Tfn)–transferrin receptor (TfnR) complex at perinuclear recycling endosomes but also in tethering of recycling vesicles to the plasma membrane (PM) in concert with the exocyst tethering complex. We here aimed at identifying SNARE proteins responsible for fusion of Tfn–TfnR-containing recycling vesicles with the PM, downstream of the exocyst. We showed that exocyst subunits, Sec6 and Sec8, can interact with SNAP23 and SNAP25, both of which are PM-localizing Qbc-SNAREs, and that depletion of SNAP23 and/or SNAP25 in HeLa cells suppresses fusion of Tfn–TfnR-containing vesicles with the PM, leading to accumulation of the vesicles at the cell periphery. We also found that VAMP2, an R-SNARE, is colocalized with endocytosed Tfn on punctate endosomal structures, and that its depletion in HeLa cells suppresses recycling vesicle exocytosis. These observations indicate that fusion of recycling vesicles with the PM downstream of the exocyst is mediated by SNAP23/25 and VAMP2, and provide novel insight into non-neuronal roles of VAMP2 and SNAP25.