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The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca(2+)- and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (∼60 min) Ca(2+) elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca(2+) elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.

The epidermal barrier is thought to protect sensory nerves from overexposure to environmental stimuli, and barrier impairment leads to pathological conditions associated with itch, such as atopic dermatitis (AD). However, it is not known how the epidermal barrier continuously protects nerves for the sensory homeostasis during turnover of the epidermis. Here we show that epidermal nerves are contained underneath keratinocyte tight junctions (TJs) in normal human and mouse skin, but not in human AD samples or mouse models of chronic itch caused by epidermal barrier impairment. By intravital imaging of the mouse skin, we found that epidermal nerve endings were frequently extended and retracted, and occasionally underwent local pruning. Importantly, the epidermal nerve pruning took place rapidly at intersections with newly forming TJs in the normal skin, whereas this process was disturbed during chronic itch development. Furthermore, aberrant Ca2+ increases in epidermal nerves were induced in association with the disturbed pruning. Finally, TRPA1 inhibition suppressed aberrant Ca2+ increases in epidermal nerves and itch. These results suggest that epidermal nerve endings are pruned through interactions with keratinocytes to stay below the TJ barrier, and that disruption of this mechanism may lead to aberrant activation of epidermal nerves and pathological itch.

A novel microdeletion of 14q13.1q13.3 was identified in a patient with developmental delay and intractable epilepsy. The 2.2-Mb deletion included 15 genes, of which TULIP1 (approved gene symbol: RALGAPA1)was the only gene highly expressed in the brain. Western blotting revealed reduced amount of TULIP1 in cell lysates derived from immortalized lymphocytes of the patient, suggesting the association between TULIP1 haploinsufficiency and the patient's phenotype, then 140 patients were screened for TULIP1 mutations and four missense mutations were identified. Although all four missense mutations were common with parents, reduced TULIP1 was observed in the cell lysates with a P297T mutation identified in a conserved region among species. A full-length homolog of human TULIP1 was identified in zebrafish with 72% identity to human. Tulip1 was highly expressed in zebrafish brain, and knockdown of which resulted in brain developmental delay. Therefore, we suggest that TULIP1 is a candidate gene for developmental delay.

Multiphoton imaging has been utilized to analyze in vivo immune cell dynamics over the last 15 years. Particularly, it has deepened the understanding of how immune responses are organized by immune cell migration and interactions. In this review, we first describe the following technical advances in recent imaging studies that contributed to the new findings on the regulation of immune responses and inflammation. Improved multicolor imaging of immune cell behavior has revealed that their interactions are spatiotemporally coordinated to achieve efficient and long-term immunity. The use of photoactivatable and photoconvertible fluorescent proteins has increased duration and volume of cell tracking, even enabling the analysis of inter-organ migration of immune cells. In addition, visualization of immune cell activation using biosensors for intracellular calcium concentration and signaling molecule activities has started to give further mechanistic insights. Then, we also introduce recent imaging analyses of interactions between immune cells and non-immune cells including endothelial, fibroblastic, epithelial, and nerve cells. It is argued that future imaging studies that apply updated technical advances to analyze interactions between immune cells and non-immune cells will be important for thorough physiological understanding of the immune system. Electronic supplementary material The online version of this article (doi:10.1007/s00424-016-1882-x) contains supplementary material, which is available to authorized users.