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Expression and reconstitution of the bioluminescent Ca2+ reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes (2016)

Chan, Harvey Y. S. ; Cheung, Man Chun ; Gao, Yi ; [et al.]

Springer

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Publication Date: 2022-05-25

Description: © The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Science China Life Sciences 59 (2016): 811-824, doi:10.1007/s11427-016-5094-6.

Description: In order to develop a novel method of visualizing possible Ca2+ signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca2+ reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca2+ signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca2+ transients (generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca2+ transients were generated from day 1 onward. That ATP was inducing Ca2+ release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca2+ response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

Description: This work was supported by the Hong Kong Theme-based Research Scheme award (T13-706/11-1), the Hong Kong Research Grants Council (RGC) General Research Fund awards (662113, 16101714, 16100115), the ANR/RGC joint research scheme award (A-HKUST601/13), and the Innovation and Technology Commission (ITCPD/17-9). HYSC was supported by a Hong Kong University Grants Council post-graduate studentship (T13-706/11- 11PG).

Keywords: Ca2+ signaling ; Apo-aequorin expression ; Bioluminescence ; HES2 human embryonic stem cells ; hESC-derived cardiospheres ; IP3 and ryanodine receptors

Repository Name: Woods Hole Open Access Server

Type: Article

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Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains (2015)

Barnes, Mark ; van Rensburg, Gerrit ; Li, Wai-Ming ; [et al.]

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Publication Date: 2022-05-26

Description: Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of American Society for Biochemistry and Molecular Biology for personal use, not for redistribution. The definitive version was published in Journal of Biological Chemistry 290 (2015): 625-639, doi:10.1074/jbc.M114.614735.

Description: The ability of its four heterogeneous nuclear ribonucleoprotein-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of Coding Region Determinant-Binding Protein (CRD-BP). However, the particular RNA binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved Glycine-X-X-Glycine (GXXG) motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA-binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP a well as in the future design of inhibitors against CRDBP function.

Description: This research was supported in part by a Discovery Grant (# 227158) from Natural Sciences & Engineering Research Council (NSERC) (to CHL), University of Northern British Columbia Research Project Awards (to MB, GVR, KM and SM), and the French National Research Agency/Hong Kong Research Grants Council Joint Research Scheme (# A-HKUST601/13) (to ALM). DTK was a recipient of NSERC Undergraduate Student Research Awards and a BC Cancer Agency Summer Studentship.

Keywords: CRD-BP ; RNA binding proteins ; Mutagenesis ; KH domain ; Zebrafish ; Granule formation ; Ribonucleoprotein ; RNA-protein interaction ; mRNA ; Molecular biology