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

Abstract

Mechanosensitive flipper probes are attracting interest as fluorescent reporters of membrane order and tension in biological systems. We introduce PhotoFlippers, which contain a photocleavable linker and an ultralong tether between mechanophore and various targeting motifs. Upon irradiation, the original probe is released and labels the most ordered membrane that is accessible by intermembrane transfer. Spatiotemporal control from photocleavable flippers is essential to access open, dynamic or elusive membrane motifs without chemical or physical interference. For instance, fast release with light is shown to place the original small‐molecule probes into the innermost leaflet of the nuclear envelope to image changes in membrane tension, at specific points in time of membrane trafficking along the secretory pathway, or in the inner leaflet of the plasma membrane to explore membrane asymmetry. These results identify PhotoFlippers as useful chemistry tools to enable research in biology.

Flipper Probes for the Community

“…[30] PhotoFlippers release Flipper-TR ® 1 in any MOI. [57] New blinking flipper architectures have been introduced to achieve compatibility with superresolution microscopy. [58] In HydroFlippers, the same architectures are used to simultaneously image changes in membrane compression and hydration, with the former being less dependent of the nature of the membrane and thus preferable to reliably image membrane tension.…”

Experimental Investigations on the Conductance of Lipid Membranes under Differential Hydrostatic Pressure

“…The asymmetrical distribution of membrane components, either between leaflets [ 19 ] or owing to organization into lipid rafts [ 39 , 40 ], may introduce additional limitations for adopting one of the largely utilized membrane bending models [ 41 , 42 , 43 ]. A more feasible approach would be the utilization of membrane tension indicators [ 44 , 45 , 46 , 47 , 48 , 49 ], in combination with an easier-to-model artificial system (i.e., giant vesicles exposed to osmotic pressure [ 45 ]) for calibration and direct measurements on curved membranes. The experiments presented in this work do not provide direct clues for identifying a particular permeation mechanism from the several that are available [ 15 , 16 ]; nonetheless, a comparison between predictions from simulations and experimental results may help with such identification.…”

Alfred Werner Fund, Master's Student Scholarships

“…3A). [8] Different experiments have demonstrated that the photocleavable HaloFlipper is cell permeant, selective to HaloTag, and can be released inside living cells (Fig. 3B).…”

“…3A). [8] This design allows the selective targeting of the photocleavable HaloFlipper 2 to the membrane of interest, and the spatiotemporal controlled quick release of the Flipper-TR 3, which is membrane-impermeant and has high membrane partitioning (Fig. 3A).…”