文章摘要

文章信息/Info

Title:: Substrate stiffness-regulated cellular extracellular regulated protein kinases activity visualized by fluorescence resonance energy transfer biosensor

Author(s):: ZHOU Ying; DENG Linhong; OUYANG Mingxing; XU Huadong; Institute of Biomedical Engineering and Health Sciences, School of Pharmacy & Medicine, Changzhou University, Changzhou 213164, China

Keywords:: Substrate stiffness; Polyacrylamide gel; Extracellular regulated protein kinases (ERK); Fluorescence resonance energy transfer; Focal adhesion

摘要:: 体内细胞周围的基质刚度参与调控细胞的一系列反应，如基因表达、干细胞分化、胞内生化信号活动等。本研究利用荧光共振能量转移（fluorescence resonance energy transfer,FRET）技术实时检测细胞中细胞外调节蛋白激酶（extracellular regulated protein kinases，ERK）的活性变化，研究基质刚度的调控作用。当气道平滑肌（ASM）细胞培养在不同刚度的聚丙烯酰胺凝胶基质上，ERK活性与刚度呈正相关性；在较软的基质上活性相对较低，其结果没有显著地受到胞内Src激酶和Myosin收缩力信号的影响。根据细胞通过黏着斑接触基质，实验检测发现黏着斑的形态受到刚度的明显影响，培养在玻璃上时呈典型的斑点状和短直线状，在软的凝胶上呈不规则的形状或弯曲的线条状，且有随凝胶刚度下降而不规则比率增加的趋势。结果显示,基质刚度可能通过黏着斑介导的细胞黏附力调节胞内生化信号。

Abstract:: In vivo cells substrate stiffness regulates various cell responses including gene expression, stem cell differentiation, and biochemical signals. We utilized fluorescence resonance energy transfer（FRET）biosensor to monitor the impact of substrate stiffness onextracellular regulated protein kinases（ERK）activity in airway smooth muscle cells. The results demonstrated a positive correlation between the stiffness of polyacrylamide gel and ERK activity, showed lower ERK activity in cells cultured on softer substrate. This impact was not obviously regulated by Src and cellular contraction signals.According to cells contacting the substrate by focal adhesions (FAs), different FA morphologies were observed related to the substrate stiffness. On hard glass, the morphologies of cellullar FAs showed typical adhesion spots or short straight lines, while on soft gels, it displayed irregular shapes or curved lines with enhanced trend along decreased gel stiffness. The results suggest that substrate stiffness may regulate cellular biochemical activities through adhesion forces.