López-Andarias Javier, Straková Karolína, Martinent Rémi, Jiménez-Rojo Noemi, Riezman Howard, Sakai Naomi, Matile Stefan
School of Chemistry and Biochemistry and National Centre of Competence in Research (NCCR) Chemical Biology, University of Geneva, 1211 Geneva, Switzerland.
JACS Au. 2021 Jan 19;1(2):221-232. doi: 10.1021/jacsau.0c00069. eCollection 2021 Feb 22.
To image membrane tension in selected membranes of interest (MOI) inside living systems, the field of mechanobiology requires increasingly elaborated small-molecule chemical tools. We have recently introduced HaloFlipper, i.e., a mechanosensitive flipper probe that can localize in the MOI using HaloTag technology to report local membrane tension changes using fluorescence lifetime imaging microscopy. However, the linker tethering the probe to HaloTag hampers the lateral diffusion of the probe in all the lipid domains of the MOI. For a more global membrane tension measurement in any MOI, we present here a supramolecular chemistry strategy for selective localization and controlled release of flipper into the MOI, using a genetically encoded supramolecular tag. SupraFlippers, functionalized with a desthiobiotin ligand, can selectively accumulate in the organelle having expressed streptavidin. The addition of biotin as a biocompatible external stimulus with a higher affinity for Sav triggers the release of the probe, which spontaneously partitions into the MOI. Freed in the lumen of endoplasmic reticulum (ER), SupraFlippers report the membrane orders along the secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Kinetics of the process are governed by both the probe release and the transport through lipid domains. The concentration of biotin can control the former, while the expression level of a transmembrane protein (Sec12) involved in the stimulation of the vesicular transport from ER to Golgi influences the latter. Finally, the generation of a cell-penetrating and fully functional Sav-flipper complex using cyclic oligochalcogenide (COC) transporters allows us to combine the SupraFlipper strategy and HaloTag technology.
为了对活体内特定感兴趣膜（MOI）的膜张力进行成像，力学生物学领域需要越来越精细的小分子化学工具。我们最近引入了HaloFlipper，即一种机械敏感的翻转探针，它可以利用HaloTag技术定位于MOI，通过荧光寿命成像显微镜报告局部膜张力变化。然而，将探针连接到HaloTag的连接子阻碍了探针在MOI所有脂质结构域中的横向扩散。为了在任何MOI中进行更全面的膜张力测量，我们在此提出一种超分子化学策略，用于将翻转探针选择性定位并可控释放到MOI中，该策略使用了一种基因编码的超分子标签。用脱硫生物素配体功能化的超翻转探针（SupraFlippers）可以选择性地积聚在表达链霉亲和素的细胞器中。添加生物素作为对链霉亲和素具有更高亲和力的生物相容性外部刺激物会触发探针的释放，释放的探针会自发地分配到MOI中。在内质网（ER）腔内释放后，SupraFlippers报告从ER经高尔基体到质膜的分泌途径中的膜有序性。该过程的动力学受探针释放和通过脂质结构域的运输两者控制。生物素的浓度可以控制前者，而参与刺激从ER到高尔基体的囊泡运输的跨膜蛋白（Sec12）的表达水平影响后者。最后，使用环状低聚硫属化物（COC）转运体生成细胞穿透性且功能完全的链霉亲和素 - 翻转探针复合物，使我们能够将SupraFlipper策略与HaloTag技术相结合。
