Genetically Encoded Supramolecular Targeting of Fluorescent Membrane Tension Probes within Live Cells: Precisely Localized Controlled Release by External Chemical Stimulation

Abstract

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.

Photocleavable Fluorescent Membrane Tension Probes: Fast Release with Spatiotemporal Control in Inner Leaflets of Plasma Membrane, Nuclear Envelope, and Secretory Pathway

“…Irradiation of the resulting conjugate 5 caused an instantaneous and complete migration of flipper fluorescence to the plasma membrane (PM, Figure 3 B). This change was much too fast for flipper translocation along the secretory pathway [17] . It thus indicated that, as soon as photoreleased from the ER surface within a few minutes (Figures 2 B,C, S2), flipper 1 re‐distributes among all membrane leaflets accessible by free diffusion (Figures 2 D, S4).…”

“…With the alternative genetic engineering approach, probes are attached to ligands for proteins that are expressed at specific sites [15] . With fluorescent flippers, both strategies have been explored, including HaloFlippers 2 to covalently bind to HaloTags [16] and SupraFlippers to non‐covalently bind to streptavidin expressed at the site of interest [17] . This non‐covalent targeting allows the release of SupraFlippers in response to chemical stimulation.…”

Dithiolane quartets: thiol-mediated uptake enables cytosolic delivery in deep tissue

“…6 ). 63 The mechanosensitive flipper probes have been introduced recently to image membrane tension in live cells. SupraFlippers 29 in particular were designed for the controlled release of the mechanophore in the membrane of interest within cells.…”

“…The selective increase of flipper emission was consistent with flipper release from complex 30 by ligand exchange with 31 and the formation of partially planarized, more emissive flippers 32 in the mitochondrial membranes, as previously confirmed by co-localization with GFP in 2D images. 63 …”

“…The selective increase of ipper emission was consistent with ipper release from complex 30 by ligand exchange with 31 and the formation of partially planarized, more emissive ippers 32 in the mitochondrial membranes, as previously conrmed by colocalization with GFP in 2D images. 63 Preliminary results with uorescence lifetime imaging microscopy (FLIM) conrmed detectability quite deep into spheroids (Fig. 8a, s av ¼ 2.83 ns), increasing counts and lifetimes upon ipper release (Fig.…”