ECM dimensionality tunes actin tension to regulate endoplasmic reticulum function and spheroid phenotype

Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane (rBM) in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with rBM in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging rBM in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with low cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized BM membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function, and underscore the importance of tissue mechanics in organoid homeostasis. RNAseq data from MCF10A cultured in 2D or 3D at three timepoints患者来源类器官（patient-derived organoids）与细胞球状体（cellular spheroids）能够以极高的保真度重现组织生理学特性。本研究探讨了三维（3D）环境中与重构基底膜（reconstituted basement membrane, rBM）的相互作用，如何维持乳腺上皮球状体的极化抗应激组织表型。在三维环境中与rBM相互作用的细胞，其总细丝蛋白及肌动蛋白结合型细丝蛋白的表达水平均有所降低，同时皮层肌动蛋白张力减弱，这一变化可增加质膜突起，进而促进质膜负曲率形成以及与蛋白质分泌相关的质膜蛋白结合。与之相反，在二维（2D）环境中与rBM相互作用的细胞，其皮层肌动蛋白张力较高，可诱导细丝蛋白解折叠并增强其与内质网（endoplasmic reticulum, ER）的结合。增强的细丝蛋白-内质网相互作用会提升PKR样内质网激酶效应因子的表达水平，并增加内质网-质膜接触位点，进而破坏钙稳态并降低细胞活力。因此，皮层肌动蛋白张力较低的细胞内质网应激水平更低，存活能力更强。一致性的实验结果显示，细胞球状体中的皮层肌动蛋白张力可调控极化基底膜沉积以及对外源性应激的敏感性。本研究结果表明，皮层肌动蛋白张力介导的细丝蛋白解折叠参与调控内质网功能，并凸显了组织力学在类器官稳态维持中的重要意义。本研究包含来自于在二维或三维培养体系中培养三个时间点的MCF10A细胞的RNA测序（RNAseq）数据。