Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, direct visualization of Pcdh trans interactions across cells remains challenging. Here, we present Förster resonance energy transfer (FRET)-based indicators for directly visualizing Pcdh trans interactions. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. They enabled successful visualization of specific trans interactions of Pcdh and revealed that the Pcdh trans interaction is highly sensitive to changes in extracellular Ca2+ levels. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes.

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in CNS. However, its interactions have yet not been visualized in neurons. We previously reported PcdhγB2-FRET probes to be applicable only for cell lines. Herein, we newly designed PcdhγB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single PcdhγB2 molecule and succeeded in visualizing PcdhγB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals were observed at contact sites between neurites and eliminated by EGTA addition. Live imaging revealed that the FRET-negative γB2 signals were rapidly moving along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe would allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.

SUMMARYThis paper describes an along-wind vibration of frame-type pylons of Hakucho Suspension Bridge found from longterm vibration monitoring data. It is shown that the vibration is very harmonic although its amplitude is small and it occurs under certain ranges of wind direction (10~30 degree from the bridge perpendicular axis) and of wind velocity (13~25 m/s). The dominant frequency of the vibration is either 0.6 Hz or 0.8 Hz depending on the wind velocity and these correspond to the natural frequencies of the in-plane pylon dominant modes of the suspension bridge. A series of the wind tunnel experiments using a spring-supported scaled model are conducted under uniform flow and the alongwind pylon vibration was observed and its vibration characteristics is found to be consistent to those observed in Hakucho Bridge. It is also found that the vibration is very sensitive to the incident angle of wind and to the distance between the two columns.key words: suspension bridge, in-plane oscillation, vortex induced vibration, wind tunnel experiment --

CCCTC-binding factor (CTCF) has a key role in higher-order chromatin architecture that is important for establishing and maintaining cell identity by controlling gene expression. In the mature cerebellum, CTCF is highly expressed in Purkinje cells (PCs) as compared with other cerebellar neurons. The cerebellum plays an important role in motor function by regulating PCs, which are the sole output neurons, and defects in PCs cause motor dysfunction. However, the role of CTCF in PCs has not yet been explored. Here we found that the absence of CTCF in mouse PCs led to progressive motor dysfunction and abnormal dendritic morphology in those cells, which included dendritic self-avoidance defects and a proximal shift in the climbing fibre innervation territory on PC dendrites. Furthermore, we found the peculiar lamellar structures known as “giant lamellar bodies” (GLBs), which have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe disease. GLBs are localized to PC dendrites and are assumed to be associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model in which CTCF was deleted. GLBs were not noted in PC dendrites at infancy but instead developed over time. In conjunction with GLB development in PC dendrites, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Our results revealed the important role of CTCF during normal development and in maintaining PCs and provide new insights into the molecular mechanism of GLB formation during neurodegenerative disease.

Loss of Purkinje cells (PCs) in the cerebellum causes severe motor deficits. The peculiar lamellar structures known as “giant lamellar bodies” (GLBs) have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe’s disease. GLBs are localized to PC dendrites and are associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model, in which the CCCTC-binding factor (CTCF) was deleted. CTCF orchestrates gene expression by organizing the three-dimensional chromatin structure. The mouse model showed progressive motor dysfunction and abnormal dendritic morphology in PCs, including dendritic self-avoidance defects and proximal shift in the climbing fibre innervation territory on PC dendrites. GLBs were not clearly found in PC dendrites at infancy but instead developed with age. In conjunction with GLB development, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Thus, our results are the first experimental demonstration that GLBs represent a pathological alteration of PCs and suggest different genetic backgrounds involved in the induction of GLBs.