Genome-edited human stem cells expressing fluorescently labeled endocytic markers allow quantitative analysis of clathrin-mediated endocytosis during differentiation.
Genome-edited human stem cells expressing fluorescently labeled endocytic markers allow quantitative analysis of clathrin-mediated endocytosis during differentiation.
复制标题
DOI：
10.1083/jcb.201710084
复制
发表时间：
2018-09-03
期刊：
影响因子：
--
通讯作者：
Drubin DG
中科院分区：
文献类型：
--
作者：
Dambournet D;Sochacki KA;Cheng AT;Akamatsu M;Taraska JW;Hockemeyer D;Drubin DG
Dambournet等人产生了用内吞标记物标记的基因组编辑的人胚胎干细胞（hESC）。他们通过活细胞成像和铂复制EM在hESC及其同基因后代中比较和定量分析了分化过程中网格蛋白介导的内吞作用的动态。我们开发了一种通用的方法来研究细胞过程如何在分化过程中适应特定的细胞类型。以前的研究报道网格蛋白介导的内吞作用（CME）网站的形态和动力学的实质性差异。然而，将特定的CME特性与不同的分化细胞类型相关联，并确定这些特性在分化过程中是如何发育指定的一直是难以捉摸的。使用基因组编辑的人胚胎干细胞，以及来自它们的同基因成纤维细胞和神经元祖细胞，我们通过活细胞成像和铂复制透射电子显微镜建立了质膜上的CME位点动力学和超微结构在分化过程中精确地重新编程。内吞衔接蛋白AP 2 μ2的表达水平被发现是CME动力学和结构的显著变化的基础。此外，CME对肌动蛋白组装和磷酸肌醇-3激酶活性的依赖性对于每种细胞类型是不同的。总的来说，我们的结果表明，关键的CME属性在分化期间至少部分地通过AP 2 μ2表达调控被重编程。
Dambournet et al. generate genome-edited human embryonic stem cells (hESCs) labeled with endocytic markers. They comparatively and quantitatively analyze the dynamics of clathrin-mediated endocytosis during differentiation through live-cell imaging and platinum replica EM in hESCs and their isogenic progeny. We developed a general approach for investigation of how cellular processes become adapted for specific cell types during differentiation. Previous studies reported substantial differences in the morphology and dynamics of clathrin-mediated endocytosis (CME) sites. However, associating specific CME properties with distinct differentiated cell types and determining how these properties are developmentally specified during differentiation have been elusive. Using genome-edited human embryonic stem cells, and isogenic fibroblasts and neuronal progenitor cells derived from them, we established by live-cell imaging and platinum replica transmission electron microscopy that CME site dynamics and ultrastructure on the plasma membrane are precisely reprogrammed during differentiation. Expression levels for the endocytic adaptor protein AP2μ2 were found to underlie dramatic changes in CME dynamics and structure. Additionally, CME dependency on actin assembly and phosphoinositide-3 kinase activity are distinct for each cell type. Collectively, our results demonstrate that key CME properties are reprogrammed during differentiation at least in part through AP2μ2 expression regulation.
登录
查看更多内容
影响因子：
16.6
作者：
Bucher D;Frey F;Sochacki KA;Kummer S;Bergeest JP;Godinez WJ;Kräusslich HG;Rohr K;Taraska JW;Schwarz US;Boulant S
通讯作者：
Boulant S
DOI：
10.1083/jcb.84.3.560
发表时间：
1980-03
期刊：
The Journal of cell biology
影响因子：
--
作者：
Heuser J
通讯作者：
Heuser J
影响因子：
21.3
作者：
通讯作者：
--
影响因子：
64.5
作者：
Gifford CA;Ziller MJ;Gu H;Trapnell C;Donaghey J;Tsankov A;Shalek AK;Kelley DR;Shishkin AA;Issner R;Zhang X;Coyne M;Fostel JL;Holmes L;Meldrim J;Guttman M;Epstein C;Park H;Kohlbacher O;Rinn J;Gnirke A;Lander ES;Bernstein BE;Meissner A
通讯作者：
Meissner A
影响因子：
9.2
作者：
Collins, Agnieszka;Warrington, Anthony;Taylor, Kenneth A.;Svitkina, Tatyana
通讯作者：
Svitkina, Tatyana