Zemsky Sasha, Ruijgrok Paul V, Bryant Zev
Department of Bioengineering, Stanford University, Stanford, CA, USA.
Graduate Program in Biophysics, Stanford University, Stanford, CA, USA.
bioRxiv. 2025 Aug 23:2025.08.19.666446. doi: 10.1101/2025.08.19.666446.
Understanding the behaviors of contractile actomyosin systems requires precise spatiotemporal control of filamentous myosin activity. Here, we develop a tool for optical control of contractility by extending the MyLOV family of gearshifting motors to create engineered filamentous myosins that change velocity in response to blue light. We characterize these minifilaments using single-molecule tracking assays, contractility assays in reconstituted actin networks, and imaging of contractile phenotypes in S2 cells. The minifilaments change speed and/or direction when illuminated, display speeds that fall within and beyond the relevant physiological range, and display high processivities. Additionally, minifilament-driven contraction rates increase in blue light both and in S2 cells. Finally, we develop an alternative design for minifilaments that only interact processively with actin in blue light. Engineered minifilaments can be used to dissect behaviors such as self-organization and mechanotransduction in contractile systems both and in cells and tissues.
了解收缩性肌动球蛋白系统的行为需要对丝状肌球蛋白活性进行精确的时空控制。在这里，我们通过扩展MyLOV系列换挡马达开发了一种用于光学控制收缩性的工具，以创建能响应蓝光而改变速度的工程化丝状肌球蛋白。我们使用单分子追踪分析、重构肌动蛋白网络中的收缩性分析以及S2细胞中收缩表型成像来表征这些微型丝。微型丝在光照下会改变速度和/或方向，其速度落在相关生理范围之内和之外，并且具有高持续性。此外，微型丝驱动的收缩率在体外重构网络和S2细胞中均在蓝光下增加。最后，我们开发了一种微型丝的替代设计，其仅在蓝光下与肌动蛋白进行持续性相互作用。工程化微型丝可用于剖析收缩系统在体外重构网络以及细胞和组织中的自组织和机械转导等行为。
