Xia Shumin, Yim Evelyn K F, Kanchanawong Pakorn
Mechanobiology Institute, Singapore, Republic of Singapore, 117411.
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
ACS Biomater Sci Eng. 2019 Aug 12;5(8):3828-3842. doi: 10.1021/acsbiomaterials.8b01124. Epub 2019 Jan 29.
The mechanical microenvironment serves as an important factor influencing stem cell differentiation. Mechanobiological responses depend strongly on actomyosin contractility and integrin-based cell-extracellular matrix (ECM) interactions mediated by adhesive structures such as focal adhesions (FAs). While the roles of FAs in mechanobiology have been intensively studied in many mesenchymal and migratory cell types, recently it has been recognized that certain pluripotent stem cells (PSCs) exhibited significantly attenuated FA-mediated mechanobiological responses. FAs in such PSCs are sparsely distributed and much less prominent in comparison to "classical" FAs of typical adherent cells. Despite these differences, insights into how FAs in PSCs are structurally organized to perform their functions are still elusive. Using mouse embryonic stem cells (mESCs) to study PSC-ECM interactions, here we surveyed the molecular composition and nanostructural organization of FAs. We found that, despite being small in size, mESC FAs appeared to be compositionally mature, containing markers such as vinculin, zyxin, and α-actinin, and dependent on myosin II contractility. Using super-resolution microscopy, we revealed that mESC FAs were organized into a conserved multilayer nanoscale architecture. However, the nanodomain organization was compressed in mESCs, with the force transduction layer spanning ∼10 nm, significantly more compact than in FAs of other cell types. Furthermore, we found that the position and orientation of vinculin, a key mechanotransduction protein, were modulated in an ECM-dependent manner. Our analysis also revealed that while most core FA genes were expressed, the expression of LIM domain proteins was comparatively lower in PSCs. Altogether our results suggest that while core structural and mechanosensitive elements are operational in mESC FAs, their structural organization and regulatory aspects may diverge significantly from "classical" FAs, which may account for the attenuated mechanobiological responses of these cell types.
机械微环境是影响干细胞分化的重要因素。机械生物学反应强烈依赖于肌动球蛋白收缩性以及由诸如粘着斑（FAs）等粘附结构介导的基于整合素的细胞-细胞外基质（ECM）相互作用。虽然粘着斑在机械生物学中的作用已在许多间充质和迁移细胞类型中得到深入研究，但最近人们认识到某些多能干细胞（PSCs）表现出显著减弱的粘着斑介导的机械生物学反应。与典型贴壁细胞的“经典”粘着斑相比，此类多能干细胞中的粘着斑分布稀疏且不太明显。尽管存在这些差异，但对于多能干细胞中的粘着斑如何进行结构组织以发挥其功能仍知之甚少。利用小鼠胚胎干细胞（mESCs）来研究多能干细胞-细胞外基质相互作用，我们在此调查了粘着斑的分子组成和纳米结构组织。我们发现，尽管mESC粘着斑尺寸较小，但在组成上似乎已经成熟，包含诸如纽蛋白、斑联蛋白和α-辅肌动蛋白等标志物，并且依赖于肌球蛋白II收缩性。利用超分辨率显微镜，我们揭示mESC粘着斑被组织成一种保守的多层纳米级结构。然而，mESCs中的纳米结构域组织被压缩，力转导层跨度约为10纳米，比其他细胞类型的粘着斑明显更紧凑。此外，我们发现关键机械转导蛋白纽蛋白的位置和取向以一种依赖于细胞外基质的方式受到调节。我们的分析还表明，虽然大多数核心粘着斑基因都有表达，但在多能干细胞中LIM结构域蛋白的表达相对较低。我们的结果总体表明，虽然核心结构和机械敏感元件在mESC粘着斑中发挥作用，但其结构组织和调节方面可能与“经典”粘着斑有显著差异，这可能解释了这些细胞类型减弱的机械生物学反应。
