Spatial organization and crosstalk of vimentin and actin stress fibers regulate the osteogenic differentiation of human adipose-derived stem cells
Spatial organization and crosstalk of vimentin and actin stress fibers regulate the osteogenic differentiation of human adipose-derived stem cells
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DOI：
10.1096/fj.202000378rr
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发表时间：
2020-11-17
期刊：
影响因子：
4.8
通讯作者：
Dai, Jingxing
中科院分区：
文献类型：
--
作者：
Fan, Tingyu;Qu, Rongmei;Dai, Jingxing
人脂肪干细胞(HASCs)具有多向分化潜能，是组织工程的理想种子细胞。微丝、微管和中间丝负责支持细胞内的空间。波形蛋白是一种在间充质来源的细胞中特异表达的III型中间丝蛋白，它可以作为支架发挥作用，赋予细胞抗拉伸和抗剪应力的能力。肌动蛋白应力纤维(ASF)在应力信号转导中起着重要的物理作用，为细胞提供硬度，促进成骨。通过直接的物理接触、交联剂和空间相互作用，波形蛋白和肌动蛋白网络以交叉实体的形式存在。空间相互作用发生在细胞骨架子系统的重叠区域，这可能影响细胞形态、细胞力学和细胞命运。然而，在成骨过程中细胞骨架子系统之间的空间组织是如何变化的，尤其是波形蛋白和ASF之间的空间组织是如何变化的，其对细胞命运的影响机制仍不清楚。本研究通过WB实验检测Vimentin、ASF等蛋白的表达变化。用荧光显微镜三维扫描重建细胞，分别计算Vimentin和ASF细胞骨架的空间厚度以及它们之间重叠区域的厚度，观察Vimentin和ASF在细胞内的空间重组。用细胞松弛素D(肌动蛋白聚合的抑制物)和波形蛋白上调/下调的细胞来验证波形蛋白和ASF之间的空间组织的变化及其对成骨的影响。然后，通过下调热休克蛋白27(HSP27)的表达来阐明波形蛋白和ASF在成骨过程中空间组织的调控机制。在成骨过程中，波形蛋白和肌动蛋白应力纤维的数量和空间位置呈现相反的趋势。通过控制细胞核上的锚点，中间丝波形蛋白可以减少肌动蛋白应激纤维的空间比例，这可以通过HSP27来调节。此外，肌动蛋白应激纤维的解聚导致成骨分化能力降低，导致成骨和脂肪生成同时存在，这可被波形蛋白所拮抗。我们的数据表明，波形蛋白和肌动蛋白应激纤维的空间重组是调节hASCs分化状态的关键因素。它们的空间重叠区域不利于hASCs的成骨，为进一步探讨hASCs成骨机制提供了新的视角。
Human adipose-derived stem cells (hASCs) are ideal seed cells for tissue engineering due to their multidirectional differentiation potential. Microfilaments, microtubules, and intermediate filaments are responsible for supporting the intracellular space. Vimentin, a type III intermediate filament protein that is specifically expressed in cells of mesenchymal origin, can function as a scaffold and endow cells with tension and shear stress resistance. Actin stress fibers (ASF) act as an important physical device in stress signal transduction, providing stiffness for cells, and promoting osteogenesis. Through direct physical contact, cross-linkers, and spatial interactions, vimentin and actin networks exist as intersecting entities. Spatial interactions occur in the overlapping area of cytoskeleton subsystems, which could affect cell morphology, cell mechanics, and cell fate. However, how does the spatial organization between the cytoskeletal subsystems changed during osteogenesis, especially between vimentin and ASF, is still not understood, and its mechanism effect on cell fate remains unclear. In our study, WB experiment was used to detect the expression changes in Vimentin, ASF, and other proteins. Cells were reconstructed by three-dimensional scanning with fluorescence microscope, and the spatial thickness of vimentin and ASF cytoskeletons and the thickness of the overlapping area between them were calculated, respectively, so as to observe the spatial reorganization of vimentin and ASF in cells. Cytochalasin D (an inhibitor of actin polymerization) and vimentin upregulated/downregulated cells were used to verify the change in the spatial organization between vimentin and ASF and its influence on osteogenesis. Then, heat shock protein 27 (HSP27) was downregulated to illuminate the regulatory mechanisms of spatial organization between vimentin and ASF during osteogenesis. The amounts and the spatial positions of vimentin and actin stress fiber exhibited opposite trends during osteogenesis. Through controlling the anchor sites on the nucleus, intermediate filaments vimentin can reduce the spatial proportion of actin stress fibers, which can be regulated by HSP27. In addition, depolymerization of actin stress fibers lead to lower osteogenic differentiation ability, resulting in osteogenesis and lipogenesis existed simultaneously, that can be resisted by vimentin. Our data indicate that the spatial reorganization of vimentin and actin stress fibers is a key factor in the regulation of the differentiation state of hASCs. And their spatial overlapping area is detrimental to hASCs osteogenesis, providing a new perspective for further exploring the mechanism underlying hASCs osteogenesis.