Mechanosensitive Fluorescent Probes to Image Membrane Tension in Mitochondria, Endoplasmic Reticulum, and Lysosomes

Abstract

land § These two authors contributed equally ABSTRACT. Measuring forces inside cells is particularly challenging. With the development of quantitative microscopy, fluorophores which allow the measurement of forces became highly desirable. We have previously introduced a mechanosensitive flipper probe, which responds to the change of plasma membrane tension by changing fluorescence lifetime and thus allows tension imaging by FLIM. Herein, we describe the design, synthesis, and evaluation of flipper probes that selectively label intracellular organelles, i.e., lysosomes, mitochondria, and the endoplasmic reticulum. The probes respond uniformly to osmotic shocks applied extracellularly, thus confirming sensitivity toward changes in membrane tension.At rest, different lifetimes found for different organelles relate to known differences in membrane organization rather than membrane tension and allow co-labeling in the same cells. At the organelle scale, lifetime heterogeneity provides unprecedented insights on ER tubules and sheets, and nuclear membranes.Examples on endosomal trafficking or increase of tension at mitochondrial constriction sites outline the potential of intracellularly targeted fluorescent tension probes to address essential questions that were previously beyond reach.The importance of mechanical forces in biological processes is only starting to emerge. 1-3 Plasma membrane tension is a topic of particular current interest because mounting evidence suggests its involvement in regulating various biochemical processes in cells. 2 Although membrane tension should also regulate membranous organelles' functions, standard techniques of force measurements, such as optical tweezers or force microscopes are difficult to apply inside of cells. 3 Therefore, the role of membrane tension in

A Chalcogen‐Bonding Cascade Switch for Planarizable Push–Pull Probes

“…This implied that the effective fluorescence quantum yield of the planarized cascade switch 4 should be F So > 60 %, higher than the F So > 38 %oforiginal 1 (Table 1). According to FLIM images of L d and L o GUVs, [5,6] fluorescence lifetimes of cascade switch 4 were shorter than the original 1 (Figure 4c,F igure S17 in the Supporting Information, Table 1). However, like intensity ratios,l ifetime increases upon planarization from L d to L o membranes were slightly higher for 4 (t Lo /t Ld = 2.07) than for 1 (t Lo /t Ld = 1.90;T able 1).…”

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Planarizable push-pull probes have been introduced to demonstrate physical forces in biology.However,the donors and acceptors needed to polarizem echanically planarized probes are incompatible with their twisted resting state.T he objective of this study was to overcome this "flipper dilemma" with chalcogen-bonding cascade switches that turn on donors and acceptors only in response to mechanical planarization of the probe.T his concept is explored by molecular dynamics simulations as well as chemical double-mutant cycle analysis. [5,6] The current best is constructed around twisted dithienothiophene (DTT) dithienothiophene S,S-dioxide (DTTO2) conjugates ( Figure 1a,D ' = S, A' = SO 2 ). [5,6] The current best is constructed around twisted dithienothiophene (DTT) dithienothiophene S,S-dioxide (DTTO2) conjugates ( Figure 1a,D ' = S, A' = SO 2 ).

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A Coumarin Triflate Reagent Enables One‐Step Synthesis of Photo‐Caged Lipid Metabolites for Studying Cell Signaling

“…[4][5][6][7] In particular,u nderstanding fast processes such as signal transduction requires the abilityt om easure reaction kinetics in living cells. [8][9][10] Current methods for rapidlym odulating cellular small molecules are based on optogenetic proteins, [11,12] chemical dimerizers [13,14] and photo-caged [15][16][17][18][19][20][21] or photo-switchable [19,[22][23][24] chemicalp robes. [8][9][10] Current methods for rapidlym odulating cellular small molecules are based on optogenetic proteins, [11,12] chemical dimerizers [13,14] and photo-caged [15][16][17][18][19][20][21] or photo-switchable [19,[22][23][24] chemicalp robes.…”

“…We next assessed the GPCR ligandsa mong the new photocaged compounds (6,(8)(9)(10)(11)(12)(13)i nl ive cell fluorescencem icroscopy assays.W ef irst determined cellular uptake in HeLa Kyoto cells by fluorescencem icroscopy using the intrinsic fluorescence of the diethylamino-coumarin group as readout.A ll compounds proved to be membrane permeable, despite the fact that some molecules( 6, 10, 11, 12)a re intrinsicallyp ositively or negatively charged ( Figure 2B,F igure S4). While the compounds mostly exhibited the unspecific localization in cellular membranes typical for diethylamino-coumarin-caged molecules, overall fluorescencei ntensities varied significantly among compounds ( Figure S5).…”

“…In Ta ble S1 for details) in HeLa Kyoto cells. We found that the caged fatty acids (7,8) can be used at all tested concentrations, whereas caged LPA (6), caged PAF( 10)a nd caged edelfosin (12)h ave narrower suitable concentration windows, while caged PGE 2 (13)w as found to be not suitable for leave-on experiments( Figure S8, see supplementary information for details). As ac ontrol we added the free ligandst oc ells not expressing the GPCRs and observed no PKCe recruitment to the plasma membrane or Ca 2 + transients ( Figure S7).…”