PDF(Pubmed)
Abstract：
Intracellular cargo delivery via distinct transport routes relies on vesicle carriers. A key trafficking route distributes cargo taken up by clathrin-mediated endocytosis (CME) via early endosomes. The highly dynamic nature of the endosome network presents a challenge for its quantitative analysis, and theoretical modelling approaches can assist in elucidating the organization of the endosome trafficking system. Here, we introduce a new computational modelling approach for assessment of endosome distributions. We employed a model of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with inherited mutations causing dilated cardiomyopathy (DCM). In this model, vesicle distribution is defective due to impaired CME-dependent signaling, resulting in plasma membrane-localized early endosomes. We recapitulated this in iPSC-CMs carrying two different mutations, TPM1-L185F and TnT-R141W (MUT), using 3D confocal imaging as well as super-resolution STED microscopy. We computed scaled distance distributions of EEA1-positive vesicles based on a spherical approximation of the cell. Employing this approach, 3D spherical modelling identified a bi-modal segregation of early endosome populations in MUT iPSC-CMs, compared to WT controls. Moreover, spherical modelling confirmed reversion of the bi-modal vesicle localization in RhoA II-treated MUT iPSC-CMs. This reflects restored, homogeneous distribution of early endosomes within MUT iPSC-CMs following rescue of CME-dependent signaling via RhoA II-dependent RhoA activation. Overall, our approach enables assessment of early endosome distribution in cell-based disease models. This new method may provide further insight into the dynamics of endosome networks in different physiological scenarios.
摘要：
通过不同运输路线的细胞内货物递送依赖于囊泡载体。关键的贩运途径通过早期内体分配由网格蛋白介导的内吞作用（CME）吸收的货物。内体网络的高度动态性对其定量分析提出了挑战，和理论建模方法可以帮助阐明内体贩运系统的组织。这里,我们引入了一种新的计算建模方法来评估内体分布。我们采用了诱导多能干细胞衍生的心肌细胞（iPSC-CM）模型，其遗传突变导致扩张型心肌病（DCM）。在这个模型中,由于CME依赖性信号传导受损，囊泡分布有缺陷，导致质膜定位的早期内体。我们在携带两种不同突变的iPSC-CM中概述了这一点，TPM1-L185F和TnT-R141W(MUT),使用3D共聚焦成像以及超分辨率STED显微镜。我们根据细胞的球形近似计算了EEA1阳性囊泡的缩放距离分布。采用这种方法，3D球形建模确定了MUTiPSC-CM中早期内体种群的双峰分离，与WT对照相比。此外，球形建模证实了RhoAII处理的MUTiPSC-CM中双峰囊泡定位的回归。这反映了恢复，在通过RhoAII依赖性RhoA激活挽救CME依赖性信号后，MUTiPSC-CM内早期内体的均匀分布。总的来说,我们的方法能够评估基于细胞的疾病模型中的早期内体分布.这种新方法可以进一步深入了解不同生理场景中的内体网络的动力学。
