The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons.

G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation

The subcellular distribution of fluorescently tagged GPR3 in CGNs.

<p>(A) We employed a GPR3-HT expression vector to analyze the localization of GPR3. After transfecting the GPR3-HT vector into the CGNs, the cells were labeled with the TMR HaloTag ligand at forty-eight hours after transfection. A representative image was shown. (B) To evaluate the localization of GPR3 at the plasma membrane, the CGNs were co-transfected with the GPR3-HT and YFP-mem expression vectors, followed by TMR HaloTag ligand labeling. YFP-mem fluorescence was shown in the green pseudo-color to easily visualize the co-localization. (C-F) To evaluate the localization of GPR3 in the endoplasmic reticulum, Golgi body, and endosomes, the GPR3-mAGFL expression vector was transfected into the CGNs. Forty-eight hours after transfection, the cells were immunostained with various organelle markers: anti-KDEL antibody (endoplasmic reticulum), WGA633 (Golgi body), anti-Rab5 antibody (early endosome), and anti-Rab7 antibody (late endosome). Co-localization of GPR3 with the organelle markers was evaluated by confocal microscopy using built-in Z-stack analysis software. Scale bar = 10 μm.</p

The GPR3-mediated modulation of PKA activity was analyzed with a PKA FRET indicator.

<p>(A-C) HEK293 cells were co-transfected with the AKAR3-EV and GPR3 expression plasmids. AKAR3-EV and the mock vector were co-transfected as a control. Forty-eight hours after transfection, the CFP and FRET images were captured using a fluorescent microscope. Some of the AKAR3-EV/mock vector co-transfected cells were treated with 1 mM dbcAMP 15 min before the images were captured. The FRET/CFP ratios in the images were analyzed by the MetaMorph software. (A) Representative images of cells transfected with either the mock vector (top) or the GPR3 vector (bottom) are shown. (B) The YFP/CFP ratio in each group were analyzed identically in each dish (n = 8). The values are expressed as the percentage of the FRET/CFP ratio in the GPR3-transfected or dbcAMP-treated cells compared to the mock-transfected cells, i.e., the percentage of the control vector-transfected cells. The means ± SEM were calculated for each condition. The asterisk (*) represents p < 0.001 compared to the cells transfected with the mock vector. (C) The FRET/CFP ratio in the cytoplasm and plasma membrane were evaluated in a single GPR3-transfected cell. The asterisk (*) represents p < 0.001 compared to the values for the cytoplasm. (D-E) The PKA activity in GPR3-expressing CGNs was evaluated by AKAR3-EV. CGNs were co-transfected with mock/AKAR3-EV or pGPR3-HT/AKAR3-EV expression plasmids. Forty-eight hours after transfection, the CGNs were stained with organelle-specific markers for the plasma membrane, ER, Golgi body, lysosome, and mitochondria. (D) Representative images of CGNs stained with CellMask (plasma membrane), ER tracker (ER), WGA633 (Golgi body), LysoTracker (lysosome), and MitoTracker (mitochondria) were shown (middle raw). The CFP/YFP ratios in the images were visualized and analyzed by the MetaMorph software (right raw). (E) The FRET/CFP ratios were compared in each organelle of the mock/AKAR3-EV- or pGPR3-HT/AKAR3-EV-transfected CGNs. The FRET/CFP ratios were significantly increased in the plasma membrane of the GPR3-HT-expressing CGNs compared to the mock-transfected CGNs. The data represent the means ± SEM for each condition (n = 6). The asterisk (*) represents p < 0.05. (F) The PKA activity in the plasma membrane around soma was analyzed in CGNs transfected with control siRNA+Mock, GPR3 siRNA+Mock, and GPR3 siRNA+ pGPR3-HT, respectively. Forty-eight hours after transfection, the FRET/CFP images were taken. The FRET/CFP ratio in each group were analyzed identically. The data represent the means ± SEM for each condition (n = 10). The asterisk (*) represents p < 0.05 and the double asterisk (**) represents p < 0.005.</p

The PKA activity at the neurite tips was modulated by the local transport of GPR3 in CGNs.

<p>(A-B) Forty-five hours after transfection, GPR3-HT was labeled with the TMR HaloTag ligand for 15 min. After three sequential washes in PBS, the cells were incubated with Blebbistatin or Monastrol for another 2 hours. As a control, a 1:1000 dilution of DMSO was added to the culture medium. After capturing the CFP and FRET images, 1 mM dbcAMP was applied to the bath and additional CFP and FRET images were captured. (A) The representative FRET/CFP images from control, Blebbistatin-treated (5 μM, 50 μM), and Monastrol-treated (0.2 mM, 2 mM) CGNs at the neurite tips were shown. (B) The FRET/CFP ratio in each inhibitor-treated group were analyzed identically. The FRET/CFP ratios are expressed as the percentage of the maximum level stimulated by 1 mM dbcAMP in each cell. The data represent the means ± SEM for each condition (n = 8). The asterisk (*) represents p < 0.005.</p

The local PKA activity was modulated by the expression of GPR3 in the CGNs.

<p>(A-B) The local PKA activity was compared between the soma and neurite tips in GPR3-transfected CGNs. The CGNs were transfected with Mock+control siRNA, pGPR3-HT+control siRNA, Mock+GPR3 siRNA, and pGPR3-HT+GPR3 siRNA, respectively. Forty-eight hours after transfection, the FRET/CFP images were captured using a fluorescent microscope. After capturing the images, the FRET/CFP intensity ratio was calculated and compared between the soma and neurite tips. (A) Representative FRET/CFP images from CGNs in each condition were shown (left raw). Magnifications of neurite tip were also shown (right raw) (B) The ratios of the PKA activity (tip to soma) in each condition were analyzed identically. The data represent the means ± SEM for each condition (n = 7). The asterisk (*) represents p < 0.005. (C-D) The PKA activity at the neurite tips was analyzed in CGNs transfected with Mock+control siRNA, pGPR3-HT+control siRNA, Mock+GPR3 siRNA, and pGPR3-HT+GPR3 siRNA, respectively. Forty-eight hours after transfection, the FRET/CFP images were captured using a fluorescent microscope. After capturing the images, the cells were treated with 1 mM dbcAMP for 15 min, and FRET/CFP images were captured again in the same cell to evaluated fully activated PKA. (C) Representative FRET/CFP images from the neurite tips of CGNs in each condition were shown. (D) The FRET/CFP ratio in each group were analyzed identically. The FRET/CFP ratios are expressed as the percentage of the maximum level stimulated by 1 mM dbcAMP in each cell. The data represent the means ± SEM for each condition (n = 9). The asterisk (*) represents p < 0.005.</p

Distribution pattern and habitat use of the protandrous shrimp Pandalus latirostris in relation to environmental characteristics in Akkeshi waters on the pacific coast of eastern Hokkaido, Japan

A sampling of the protandric shrimp, Pandalus latirostris, was conducted at fixed sites from July 2015 to March 2020 in Akkeshi Bay and the connecting Lake Akkeshi on the Pacific coast of eastern Hokkaido. Based on the occurrence pattern of P. latirostris, most individuals begin mating as males at Age-1, followed by a sex change to females, spawn eggs as females at Age-2, and hatch their eggs in the spring of Age-3. Pre-hatching ovigerous females were mainly found in eelgrass beds near and in Lake Akkeshi, where the water temperature was relatively high. The females probably migrate there to hatch their eggs, making sure of the high survival and growth of the hatched larvae. Juveniles and small males were also abundant in near and in Lake Akkeshi, indicating that many larvae remained in the eelgrass beds where they hatched. These eelgrass beds play an important role in the maintenance of the shrimp population as a major egg-hatching site and nursery habitat in Akkeshi waters. With ontogenetic development, the occurrence rate of P. latirostris gradually increased on the offshore side of Akkeshi Bay, with its body size being larger. As P. latirostris grow, they will probably disperse to the offshore side of Akkeshi Bay. However, the density of P. latirostris in the eelgrass bed in the bay near the lake was extremely high, with its strong preference at all ontogenetic stages. This eelgrass bed would help maintain the shrimp population as a “key habitat” for the shrimp. The site-specific environmental characteristics of vegetation beds influence the distribution and abundance of individuals within a population of P. latirostris through their growth and the associated change in habitat preference. In this study, the importance of maintaining the diverse habitats of P. latirostris will be discussed as part of the shrimp stock management measures

Fluorescence time-lapse imaging of a single GPR3-HT-expressing CGN.

<p>(A-B) The CGNs were transfected with the pGPR3-HT expression vector and were plated onto 0.03% PEI-coated plates. Forty-five hours after transfection, GPR3-HT was labeled with the TMR HaloTag ligand for 15 min. After three sequential washes in PBS, the cells were incubated with various inhibitors for another two hours. The cells were treated with 1 μM Latrunculin B, 3 μM Nocodazole, 50 μM Blebbistatin, or 2 mM Monastrol in the culture medium, respectively. As a control, a 1:1000 dilution of DMSO was added to the culture medium. The pGPR3-HT fluorescence in the CGNs was captured every 10 minutes using a fluorescent microscope. (A) Representative images of the CGNs from each condition were shown. The arrow indicates the GPR3 fluorescent puncta at each time point. Serial images were shown every 20 min. (B) The directions that the GPR3 puncta moved along the neurite were analyzed in each condition. The directions of puncta movement were divided into three groups: the plus end movement group, the minus end movement group, and the immobile group. The values were then expressed as the percentage of the number of puncta in each direction out of the total numbers of puncta. The numbers of puncta in plus end direction were significantly decreased when Latrunculin B, Nocodazole, Blebbistatin were included in the medium. The data represent the means ± SEM for each condition (n = 16). The asterisk (*) represents p < 0.0001 and the double asterisk (**) represents p < 0.05. (C) The speed of the GPR3 puncta were calculated from the time-lapse images. The mean speed of the GPR3 puncta in plus-end and minus-end directions were separately shown (n = 25). (D-E) The fluorescent intensity of GPR3 in the membranes at the neurite tips were evaluated with or without addition of various actin and tubulin inhibitors. The CGNs were co-transfected with pGPR3-HT and pYFP-mem. Twenty-four hours after transfection, some groups were treated with inhibitors as described above. Forty-six hours after transfection, the cells were labeled with the TMR HaloTag ligand for 2 hours. The cells were then fixed and the fluorescent images of GPR3-HT and YFP-mem were captured using a fluorescent microscope from the cells in each condition. The fluorescent intensity of GPR3 in the membranes at the neurite tips were evaluated by the line profiling method (detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147466#sec002" target="_blank">Materials and methods</a>). (D) Representative images of the CGNs and the line profiling analyses from each condition were shown. (E) The fluorescent intensities of GPR3-HT in the plasma membrane and cytosol at the neurite tips were compared for each condition. The values were then expressed as the ratio of the fluorescent intensity at the plasma membrane to the cytosol at the neurite tips. The data represent the means ± SEM for each condition (n = 7). The asterisk (*) represents p < 0.0001.</p