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Summary: cIPAD is a fluorescent indicator that allows the visualization of trans-interactions of clustered protocadherin (Pcdh), a cell adhesion molecule that mediates neuronal self-recognition. We describe steps for using HEK293T cells to visualize Pcdh trans-interactions across cells as a preliminary experiment before using dissociated mouse neurons. We then detail procedures for visualizing Pcdh trans-interactions between processes originating from the same neurons, which are considered as Pcdh-mediated neuronal self-recognition.For complete details on the use and execution of this protocol, please refer to Kanadome et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract 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.

Summary: Clustered protocadherin (Pcdh), a cell adhesion protein, is involved in the self-recognition and non-self-discrimination of neurons by conferring diversity on the cell surface. Although the roles of Pcdh in neurons have been elucidated, it has been challenging to visualize its adhesion activity in neurons, which is a molecular function of Pcdh. Here, we present fluorescent indicators, named IPADs, which visualize the interaction of protocadherin-α4 isoform (α4). IPADs successfully visualize not only homophilic α4 trans-interactions, but also combinatorial homophilic interactions between cells. The reversible nature of IPADs overcomes a drawback of the split-GFP technique and allows for monitoring the dissociation of α4 trans-interactions. Specially designed IPADs for self-recognition are able to monitor the formation and disruption of α4 trans-interactions between processes originating from the same neurons. We expect that IPADs will be useful tools for obtaining spatiotemporal information on Pcdh interactions in neuronal self-recognition and non-self-discrimination processes.

Abstract 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 the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported Pcdh-B2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we -B2 designed-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved 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 might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism. [ABSTRACT FROM AUTHOR]