Molecular phenotyping of single pancreatic islet leader beta cells by "Flash-Seq"
Molecular phenotyping of single pancreatic islet leader beta cells by "Flash-Seq"
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DOI：
10.1016/j.lfs.2023.121436
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发表时间：
2023-01-28
期刊：
影响因子：
6.1
通讯作者：
Rutter, Guy A.
中科院分区：
文献类型：
--
作者：
Chabosseau, Pauline;Yong, Fiona;Rutter, Guy A.
目的：胰岛中胰腺β细胞内胞质Ca 2+的空间组织性增加是高葡萄糖刺激胰岛素分泌的基础。最近的数据已经揭示了β细胞亚群的存在，包括启动Ca 2+波的“领导者”。领导细胞是否具有独特的分子特征，或定位，是未知的。主要方法：高速共聚焦Ca 2+成像用于识别领导细胞和连接分析，运行在MATLAB和Python下，以识别高度连接的“枢纽”细胞。为了探索β细胞亚群之间的转录组学差异，通过隐蔽荧光蛋白PA-mCherry的光活化来标记单个前导细胞或跟随细胞，并进行单细胞RNA测序（“Flash-Seq”）。关键发现：在单个胰岛中鉴定了不同的Ca 2+波类型，其中前导细胞存在于73%中（38个胰岛中的28个被成像）。在29%的胰岛中也观察到无标度的幂律粘附行为，尽管这些胰岛中的“枢纽”细胞与领导细胞不重叠。在前导细胞和跟随细胞之间差异表达的转录物（295; padj < 0.05）包括参与纤毛生物发生和转录调控的基因。为这些发现提供了一些支持，ADCY 6免疫反应性在领导者中往往高于跟随者细胞，而纤毛数量和长度在前者中往往较低。最后，领导细胞位于显着更接近三角洲，但不是阿尔法，细胞在欧几里德空间比followercells.Significance：一个离散的转录组和独特的本地化的存在意味着这些功能在定义专门的功能的领导人的作用。这些数据还提出了δ细胞和前导细胞之间的局部信号传导有助于胰岛Ca 2+波的启动和传播的可能性。
Aims: Spatially-organized increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including "leaders" which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown.Main methods: High speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected "hub" cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing ("Flash-Seq").Key findings: Distinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29 % of islets, though "hub" cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj < 0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Providing some support for these findings, ADCY6 immunoreactivity tended to be higher in leader than follower cells, whereas cilia number and length tended to be lower in the former. Finally, leader cells were located significantly closer to delta, but not alpha, cells in Euclidian space than were follower cells.Significance: The existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. These data also raise the possibility that localised signalling between delta and leader cells contributes to the initiation and propagation of islet Ca2+ waves.