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Calcium transients drive cells to discharge prostaglandin E2 (PGE2). We visualized PGE2-induced protein kinase A (PKA) activation and quantitated PGE2 secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE2-producer cells that express either an optogenetic or a chemogenetic calcium channel stimulator: OptoSTIM1 or Gq-DREADD, respectively. Second, we prepared reporter cells expressing the Gs-coupled PGE2 reporter EP2 and the PKA biosensor Booster-PKA, which is based on the principle of Förster resonance energy transfer (FRET). Upon the stimulation-induced triggering of calcium transients, a single producer cell discharges PGE2 to stimulate PKA in the surrounding reporter cells. Due to the flow of the medium, the PKA-activated area exhibited a comet-like smear when HeLa cells were used. In contrast, radial PKA activation was observed when confluent MDCK cells were used, indicating that PGE2 diffusion was restricted to the basolateral space. By fitting the radius of the PKA-activated area to a simulation model based on simple diffusion, we estimated that a single HeLa cell secretes 0.25 fmol PGE2 upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE2 discharge rate is comparable to the diffusion rate. Thus, our method quantitatively envisions that a single calcium transient affects more than 1000 neighboring cells via PGE2.Key words: prostaglandin E2, imaging, intercellular communication, biosensor, quantification

Cell adhesion is essential for proper tissue architecture and function in multicellular organisms. Cell adhesion molecules not only maintain tissue integrity but also possess signaling properties that contribute to diverse cellular events such as cell growth, survival, differentiation, polarity, and migration; however, the underlying molecular basis remains poorly defined. Here we identify that the cell adhesion signal initiated by the tight-junction protein claudin-6 (CLDN6) regulates nuclear receptor activity. We show that CLDN6 recruits and activates Src-family kinases (SFKs) in second extracellular domain-dependent and Y196/200-dependent manners, and SFKs in turn phosphorylate CLDN6 at Y196/200. We demonstrate that the CLDN6/SFK/PI3K/AKT axis targets the AKT phosphorylation sites in the retinoic acid receptor γ (RARγ) and the estrogen receptor α (ERα) and stimulates their activities. Interestingly, these phosphorylation motifs are conserved in 14 of 48 members of human nuclear receptors. We propose that a similar link between diverse cell adhesion and nuclear receptor signalings coordinates a wide variety of physiological and pathological processes.

Prostaglandin E 2 (PGE 2 ) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE 2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin–Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors, we demonstrate that calcium transients in a single cell cause PGE 2 -mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE 2 discharge in response to the increasing cytoplasmic Ca 2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE 2 discharge is regulated temporally by calcium transients and ERK activity.

Rab-dependent membrane trafficking is critical for changing the structure and function of dendritic spines during synaptic plasticity. Here, we developed highly sensitive sensors to monitor Rab protein activity in single dendritic spines undergoing structural long-term potentiation (sLTP) in rodent organotypic hippocampal slices. During sLTP, Rab10 was persistently inactivated (>30 min) in the stimulated spines, whereas Rab4 was transiently activated over ~5 min. Inhibiting or deleting Rab10 enhanced sLTP, electrophysiological LTP, and AMPA receptor (AMPAR) trafficking during sLTP. In contrast, disrupting Rab4 impaired sLTP only in the first few minutes and decreased AMPAR trafficking during sLTP. Thus, our results suggest that Rab10 and Rab4 oppositely regulate AMPAR trafficking during sLTP, and inactivation of Rab10 signaling facilitates the induction of LTP and associated spine structural plasticity.

Behavioural time scale plasticity (BTSP) is non-Hebbian plasticity induced by integrating presynaptic and postsynaptic components separated by a behaviourally relevant time scale (seconds) 1 . BTSP in hippocampal CA1 neurons underlies place cell formation. However, the molecular mechanisms that enable synapse-specific plasticity on a behavioural time scale are unknown. Here we show that BTSP can be induced in a single dendritic spine using two-photon glutamate uncaging paired with postsynaptic current injection temporally separated by a behavioural time scale. Using an improved Ca 2+ /calmodulin-dependent kinase II (CaMKII) sensor, we did not detect CaMKII activation during this BTSP induction. Instead, we observed dendritic, delayed and stochastic CaMKII activation (DDSC) associated with Ca 2+ influx and plateau potentials 10–100 s after BTSP induction. DDSC required both presynaptic and postsynaptic activity, which suggests that CaMKII can integrate these two signals. Also, optogenetically blocking CaMKII 15–30 s after the BTSP protocol inhibited synaptic potentiation, which indicated that DDSC is an essential mechanism of BTSP. IP 3 -dependent intracellular Ca 2+ release facilitated both DDSC and BTSP. Thus, our study suggests that non-synapse-specific CaMKII activation provides an instructive signal with an extensive time window over tens of seconds during BTSP. Induction of behavioural time scale plasticity leads to dendritic, delayed and stochastic Ca 2+ /calmodulin-dependent kinase II activation, findings that clarify the mechanisms that underlie place cell formation.

Calcium transients drive cells to discharge prostaglandin E (PGE ). We visualized PGE -induced protein kinase A (PKA) activation and quantitated PGE secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE -producer cells that express either an optogenetic or a chemogenetic calcium channel stimulator: OptoSTIM1 or Gq-DREADD, respectively. Second, we prepared reporter cells expressing the Gs-coupled PGE reporter EP2 and the PKA biosensor Booster-PKA, which is based on the principle of Förster resonance energy transfer (FRET). Upon the stimulation-induced triggering of calcium transients, a single producer cell discharges PGE to stimulate PKA in the surrounding reporter cells. Due to the flow of the medium, the PKA-activated area exhibited a comet-like smear when HeLa cells were used. In contrast, radial PKA activation was observed when confluent MDCK cells were used, indicating that PGE diffusion was restricted to the basolateral space. By fitting the radius of the PKA-activated area to a simulation model based on simple diffusion, we estimated that a single HeLa cell secretes 0.25 fmol PGE upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE discharge rate is comparable to the diffusion rate. Thus, our method quantitatively envisions that a single calcium transient affects more than 1000 neighboring cells via PGE .Key words: prostaglandin E , imaging, intercellular communication, biosensor, quantification.

Calcium transients drive cells to discharge prostaglandin E2 (PGE2). We visualized PGE2-induced protein kinase A (PKA) activation and quantitated PGE2 secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE2-producer cells that express either an optogenetic or a chemogenetic calcium channel stimulator: OptoSTIM1 or Gq-DREADD, respectively. Second, we prepared reporter cells expressing the Gs-coupled PGE2 reporter EP2 and the PKA biosensor Booster-PKA, which is based on the principle of Förster resonance energy transfer. Upon the stimulation-induced triggering of calcium transients, a single producer cell discharges PGE2 to stimulate PKA in the surrounding reporter cells. Due to the flow of the medium, the PKA-activated area exhibited a comet-like smear when HeLa cells were used. In contrast, radial PKA activation was observed when confluent MDCK cells were used, indicating that PGE2 diffusion was restricted to the basolateral space. By fitting the radius of the PKA-activated area to a simulation model based on simple diffusion, we estimated that a single HeLa cell secretes 0.25 fmol PGE2 upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE2 discharge rate is comparable to the diffusion rate. Thus, our method quantitatively envisions that a single calcium transient affects more than 1000 neighboring cells via PGE2.Keywords: prostaglandin E2, imaging, intercellular communication, biosensor, quantification

Prostaglandin E.sub.2 (PGE.sub.2) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE.sub.2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin--Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors, we demonstrate that calcium transients in a single cell cause PGE.sub.2-mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE.sub.2 discharge in response to the increasing cytoplasmic Ca.sup.2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE.sub.2 discharge is regulated temporally by calcium transients and ERK activity.

Prostaglandin E 2 (PGE 2 ) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE 2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin–Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors, we demonstrate that calcium transients in a single cell cause PGE 2 -mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE 2 discharge in response to the increasing cytoplasmic Ca 2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE 2 discharge is regulated temporally by calcium transients and ERK activity.

Rab-dependent membrane trafficking is critical for changing the structure and function of dendritic spines during synaptic plasticity. Here, we developed highly sensitive sensors to monitor Rab protein activity in single dendritic spines undergoing structural long-term potentiation (sLTP) in rodent organotypic hippocampal slices. During sLTP, Rab10 was persistently inactivated (>30 min) in the stimulated spines, whereas Rab4 was transiently activated over ~5 min. Inhibiting or deleting Rab10 enhanced sLTP, electrophysiological LTP, and AMPA receptor (AMPAR) trafficking during sLTP. In contrast, disrupting Rab4 impaired sLTP only in the first few minutes and decreased AMPAR trafficking during sLTP. Thus, our results suggest that Rab10 and Rab4 oppositely regulate AMPAR trafficking during sLTP, and inactivation of Rab10 signaling facilitates the induction of LTP and associated spine structural plasticity.