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
In this review, the multifunctionality of dithieno[3,2-b:2',3'-d]thiophenes (DTTs) is covered comprehensively. This is of interest because all involved research is very recent, emphasizes timely topics such as mechanochemistry for bioimaging or chalcogen bonds for catalysis and solar cells, and because the newly emerging privileged scaffold is embedded in an inspiring structural space. At the beginning, DTTs are introduced with regard to nomenclature, constitutional isomers and optoelectronic properties. The structural space around DTTs is mapped out next with regard to heteroatom substitution in bridge and core, covering much of the periodic table, eccentric heteroatom doping and bridge expansions. After a brief summary of synthetic approaches to the DTT scaffold, chalcogen bonds are introduced as, together with redox switching and turn-on fluorescence, one of the three conceptual foundations of most multifunctionality. Realized functions cover anion binding, transport (ion carriers, ion channels), catalysis, and the first fluorescent probes to image physical forces in living cells.
Alkyl- and aryl vinyl sulfones were obtained by eosin Y (EY)-mediated visible-light photooxidation of sulfinate salts and the reaction of the resulting S-centered radicals with alkenes. Optimized reaction conditions, the sulfinate and alkene scope, and X-ray structural analyses of several reaction products are provided. A detailed spectroscopic study explains the reaction mechanism, which proceeds through the EY radical cation as key intermediate oxidizing the sulfinate salts.
Tools to image membrane tension in response to mechanical stimuli are badly needed in mechanobiology. We have recently introduced mechanosensitive flipper probes to report quantitatively global membrane tension changes in fluorescence lifetime imaging microscopy (FLIM) images of living cells. However, to address specific questions on physical forces in biology, the probes need to be localized precisely in the membrane of interest (MOI). Herein we present a general strategy to image the tension of the MOI by tagging our newly introduced HaloFlippers to self-labeling HaloTags fused to proteins in this membrane. The critical challenge in the construction of operational HaloFlippers is the tether linking the flipper and the HaloTag: It must be neither too taut nor too loose, be hydrophilic but lipophilic enough to passively diffuse across membranes to reach the HaloTags, and allow partitioning of flippers into the MOI after the reaction. HaloFlippers with the best tether show localized and selective fluorescence after reacting with HaloTags that are close enough to the MOI but remain nonemissive if the MOI cannot be reached. Their fluorescence lifetime in FLIM images varies depending on the nature of the MOI and responds to myriocin-mediated sphingomyelin depletion as well as to osmotic stress. The response to changes in such precisely localized membrane tension follows the validated principles, thus confirming intact mechanosensitivity. Examples covered include HaloTags in the Golgi apparatus, peroxisomes, endolysosomes, and the ER, all thus becoming accessible to the selective fluorescence imaging of membrane tension.
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