The intracellular C-terminal domain of mGluR6 contains ER retention motifs.

Metabotropic glutamate receptor 6 (mGluR6) predominantly localizes to the postsynaptic sites of retinal ON-bipolar cells, at which it recognizes glutamate released from photoreceptors. The C-terminal domain (CTD) of mGluR6 contains a cluster of basic amino acids resembling motifs for endoplasmic reticulum (ER) retention. We herein investigated whether these basic residues are involved in regulating the subcellular localization of mGluR6 in 293T cells expressing mGluR6 CTD mutants using immunocytochemistry, immunoprecipitation, and flow cytometry. We showed that full-length mGluR6 localized to the ER and cell surface, whereas mGluR6 mutants with 15- and 20-amino acid deletions from the C terminus localized to the ER, but were deficient at the cell surface. We also demonstrated that the cell surface deficiency of mGluR6 mutants was rescued by introducing an alanine substitution at basic residues within the CTD. The surface-deficient mGluR6 mutant still did not localize to the cell surface and was retained in the ER when co-expressed with surface-expressible constructs, including full-length mGluR6, even though surface-deficient and surface-expressible constructs formed heteromeric complexes. The co-expression of the surface-deficient mGluR6 mutant reduced the surface levels of surface-expressible constructs. These results indicate that basic residues in the mGluR6 CTD served as ER retention signals. We suggest that exposed ER retention motifs in the aberrant assembly containing truncated or misfolded mGluR6 prevent these protein complexes from being transported to the cell surface.

Spatiotemporal imaging documented the maturation of the cardiomyocytes from human induced pluripotent stem cells.

OBJECTIVES: Cardiomyocytes derived from human induced pluripotent stem cells are a promising source of cells for regenerative medicine. However, contractions in such derived cardiomyocytes are often irregular and asynchronous, especially at early stages of differentiation. This study aimed to determine the differentiation stage of initiation of synchronized and regular contractions, using spatiotemporal imaging and physiological and genetic analyses. METHODS: Knock-in human induced pluripotent stem cell lines were established with clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 to analyze cardiac and pacemaker cell maturation. Time-frequency analysis and Ca2+ imaging were performed, and the expression of related proteins and specific cardiac/pacemaker mRNAs in contracting embryoid bodies was analyzed at various differentiation stages. RESULTS: Time-frequency analysis and Ca2+ imaging revealed irregular, asynchronous contractions at the early stage of differentiation with altered electrophysiological properties upon differentiation. Genes associated with electrophysiological properties were upregulated after 70 days of culturing in differentiation media, whereas pacemaker genes were initially upregulated during the early stage and downregulated at the later stage. CONCLUSIONS: A differentiation period >70 days is required for adequate development of cardiac elements including ion channels and gap junctions and for sarcomere maturation.

Knock-in strategy at 3'-end of Crx gene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation.

Fluorescent reporter gene knock-in induced pluripotent stem cell (iPSC) lines have been used to evaluate the efficiency of differentiation into specific cell lineages. Here, we report a knock-in strategy for the generation of human iPSC reporter lines in which a 2A peptide sequence and a red fluorescent protein (E2-Crimson) gene were inserted at the termination codon of the cone-rod homeobox (Crx) gene, a photoreceptor-specific transcriptional factor gene. The knock-in iPSC lines were differentiated into fluorescence-expressing cells in 3D retinal differentiation culture, and the fluorescent cells also expressed Crx specifically in the nucleus. We found that the fluorescence intensity was positively correlated with the expression levels of Crx mRNA and that fluorescent cells expressed rod photoreceptor-specific genes in the later stage of differentiation. Finally, we treated the fluorescent cells with DAPT, a Notch inhibitor, and found that DAPT-enhanced retinal differentiation was associated with up-regulation of Crx, Otx2 and NeuroD1, and down-regulation of Hes5 and Ngn2. These suggest that this knock-in strategy at the 3'-end of the target gene, combined with the 2A peptide linked to fluorescent proteins, offers a useful tool for labeling specific cell lineages or monitoring expression of any marker genes without affecting the function of the target gene.

Similar numbers of neurons are generated in the male and female rat preoptic area in utero.

The birth date of neurons comprising the sexually dimorphic nucleus of the rat preoptic area (SDN-POA) was determined by bromodeoxyuridine (BrdU) injections at a prescribed time during the embryonic period. Calbindin immunostaining was used as a marker to identity the SDN-POA. The animals were bred from dams injected with BrdU on days 14, 16 or 18 of pregnancy (fertilization defined as day 1). On day 15 after birth (PD), all offspring were euthanized and brain sections were prepared for histology. Neurogenesis in the SDN-POA began around embryonic day (ED) 14 and culminated on ED 18, whereas the preoptic neurons surrounding the SDN-POA generated earlier than did those of the SDN-POA. Although the SDN-POA was significantly larger in males than in females at PD15, the total numbers of neurons comprising the SDN-POA were not significantly different between sexes. Similar aggregates of somatostatin mRNA-positive cells in the central portion of the SDN-POA were observed in both sexes at PD8. On PD15, the aggregates became scattered in males, whereas the aggregates in females remained congested. These data suggest that sexual dimorphism in the SDN-POA results from male-specific postnatal radial spreading of cells rather than cell proliferation during embryonic neurogenesis.

The SK channel blocker apamin inhibits slow afterhyperpolarization currents in rat gonadotropin-releasing hormone neurones.

Gonadotropin-releasing hormone (GnRH) neurones play an essential role in the hypothalamo-pituitary-gonadal axis. As for other neurones, the discharge pattern of action potentials is important for GnRH neurones to properly function. In the case of a luteinizing hormone (LH) surge, for example, GnRH neurones are likely to continuously fire for more than an hour. For this type of firing, GnRH neurones must have a certain intrinsic property. To address this issue, we investigated the voltage-gated Ca(2+) currents and Ca(2+)-activated voltage-independent K(+) currents underlying afterhyperpolarization, because they affect cell excitability. Dispersed GnRH neurones from adult GnRH-EGFP (enhanced green fluorescent protein) transgenic rats were cultured overnight and then used for an electrophysiological experiment involving the perforated patch-clamp configuration. The GnRH neurones showed five subtypes of voltage-gated Ca(2+) currents, i.e. the T-, L-, N-, P/Q- and R-types. The GnRH neurones also showed a slow afterhyperpolarization current (I(sAHP)), but not a medium one. It is reported that the SK channel blocker apamin (10 pm-100 nm) blocks a medium afterhyperpolarization current but not an I(sAHP). In contrast to previous reports, the I(sAHP) observed in rat GnRH neurones was potently blocked by apamin. In addition, the GnRH neurones expressed transcripts for SK1-3 channels. The results indicate that rat GnRH neurones express all five subtypes of voltage-gated Ca(2+) channels and exhibit an apamin-sensitive I(sAHP), which may allow continuous firing in response to a relatively strong depolarizing input.