Faculty of Biology, University of Freiburg, 79104, Freiburg, Germany.
Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg - Bad Krozingen, Medical Center-University of Freiburg, 79110, Freiburg, Germany.
Adv Biol (Weinh). 2022 Jul;6(7):e2000337. doi: 10.1002/adbi.202000337. Epub 2022 Apr 28.
In the rapidly expanding field of molecular optogenetics, the performance of the engineered systems relies on the switching properties of the underlying genetically encoded photoreceptors. In this study, the bacterial phytochromes Cph1 and DrBphP are engineered, recombinantly produced in Escherichia coli, and characterized regarding their switching properties in order to synthesize biohybrid hydrogels with increased light-responsive stiffness modulations. The R472A mutant of the cyanobacterial phytochrome 1 (Cph1) is identified to confer the phytochrome-based hydrogels with an increased dynamic range for the storage modulus but a different light-response for the loss modulus compared to the original Cph1-based hydrogel. Stiffness measurements of human atrial fibroblasts grown on these hydrogels suggest that differences in the loss modulus at comparable changes in the storage modulus affect cell stiffness and thus underline the importance of matrix viscoelasticity on cellular mechanotransduction. The hydrogels presented here are of interest for analyzing how mammalian cells respond to dynamic viscoelastic cues. Moreover, the Cph1-R472A mutant, as well as the benchmarking of the other phytochrome variants, are expected to foster the development and performance of future optogenetic systems.
在快速发展的分子光遗传学领域，工程系统的性能依赖于基础遗传编码光感受器的开关特性。在这项研究中，对细菌的叶绿素 Cph1 和 DrBphP 进行了工程改造，在大肠杆菌中重组表达，并对其开关特性进行了表征，以便合成具有增加的光响应刚度调制的生物杂化水凝胶。鉴定出蓝细菌的光敏色素 1（Cph1）的 R472A 突变体赋予基于光敏色素的水凝胶增加的存储模量的动态范围，但与原始基于 Cph1 的水凝胶相比，损耗模量具有不同的光响应。在这些水凝胶上生长的人心房成纤维细胞的刚度测量表明，在可比的存储模量变化下损耗模量的差异会影响细胞刚度，从而强调了基质粘弹性对细胞机械转导的重要性。本文介绍的水凝胶对于分析哺乳动物细胞如何响应动态粘弹性线索具有重要意义。此外，Cph1-R472A 突变体以及其他光敏色素变体的基准测试有望促进未来光遗传学系统的开发和性能。
