Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois.
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois.
J Cell Physiol. 2018 Apr;233(4):3672-3683. doi: 10.1002/jcp.26236. Epub 2017 Nov 24.
Cell migration is regulated by several mechanotransduction pathways, which consist of sensing and converting mechanical microenvironmental cues to internal biochemical cellular signals, such as protein phosphorylation and lipid signaling. While there has been significant progress in understanding protein changes in the context of mechanotransduction, lipid signaling is more difficult to investigate. In this study, physical cues of stiffness (10, 100, 400 kPa, and glass), and microrod or micropost topography were manipulated in order to reprogram primary fibroblasts and assess the effects of lipid signaling on the actin cytoskeleton. In an in vitro wound closure assay, primary cardiac fibroblast migration velocity was significantly higher on soft polymeric substrata. Modulation of PIP2 availability through neomycin treatment nearly doubled migration velocity on 10 kPa substrata, with significant increases on all stiffnesses. The distance between focal adhesions and the lamellar membrane (using wortmannin treatment to increase PIP2 via PI3K inhibition) was significantly shortest compared to untreated fibroblasts grown on the same surface. PIP2 localized to the leading edge of migrating fibroblasts more prominently in neomycin-treated cells. The membrane-bound protein, lamellipodin, did not vary under any condition. Additionally, fifteen micron-high micropost topography, which blocks migration, concentrates PIP2 near to the post. Actin dynamics within stress fibers, measured by fluorescence recovery after photobleaching, was not significantly different with stiffness, microtopography, nor with drug treatment. PIP2-modulating drugs delivered from microrod structures also affected migration velocity. Thus, manipulation of the microenvironment and lipid signaling regulatory drugs might be beneficial in improving therapeutics geared toward wound healing.
细胞迁移受几种机械转导途径调控，这些途径包括感知和将机械微观环境线索转换为内部生化细胞信号，如蛋白质磷酸化和脂质信号转导。虽然在理解机械转导背景下的蛋白质变化方面已经取得了重大进展，但脂质信号转导更难研究。在这项研究中，通过操纵刚度（10、100、400 kPa 和玻璃）的物理线索和微柱或微柱形貌，重新编程原代成纤维细胞并评估脂质信号对肌动蛋白细胞骨架的影响。在体外伤口闭合测定中，原代心脏成纤维细胞在柔软的聚合物基质上的迁移速度明显更高。通过新霉素处理调节 PIP2 的可用性几乎使在 10 kPa 基质上的迁移速度增加了一倍，在所有刚度下都有显著增加。与在相同表面上生长的未经处理的成纤维细胞相比，粘着斑和片状膜之间的距离（使用wortmannin 处理通过 PI3K 抑制增加 PIP2）明显最短。与未经处理的成纤维细胞相比，在用新霉素处理的细胞中，PIP2 更明显地定位于迁移的成纤维细胞的前缘。膜结合蛋白，lamellipodin，在任何条件下都没有变化。此外，十五微米高的微柱形貌，阻止了迁移，将 PIP2 集中在柱子附近。通过荧光恢复后漂白测量的应力纤维内的肌动蛋白动力学，与刚度、微形貌或药物处理均无显著差异。从微柱结构递送至 PIP2 调节药物也影响迁移速度。因此，微环境的操纵和脂质信号调节药物可能有助于改善针对伤口愈合的治疗方法。
