Molecular Tuning of Styryl Dyes Leads to Versatile and Efficient Plasma Membrane Probes for Cell and Tissue Imaging

Abstract

The plasma membrane (PM) plays a major role in many biological processes; therefore its proper fluorescence staining is required in bioimaging. Among the commercially available PM probes, styryl dye FM1-43 is one of the most widely used. In this work, we demonstrated that fine chemical modifications of FM1-43 can dramatically improve the PM staining. The newly developed probes, SP-468 and SQ-535 were found to display enhanced photophysical properties (reduced crosstalk, higher brightness, improved photostability) and unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian cell lines including neurons (primary culture and tissue imaging). Additionally, we showed that the new probes displayed differences in their internalization pathways compared to their parent FM1-43. Finally, we demonstrated that the modifications made to FM1-43 did not impair the ability of the new probes to stain the PM of plant cells. Overall, this work presents new useful probes for PM imaging in cells and tissues and provides insights on the molecular design of new PM targeting molecules.

Probing Variations of Reduction Activity at the Plasma Membrane Using a Targeted Ratiometric FRET Probe

“…53 In order to specifically target the probe to the PM, the BODIPY unit was connected via a serine moiety to two amphiphilic zwitterionic anchors (Figure 1A) that proved their efficiency and selectivity in our previous works. 18,21,22,27,55 The nonreductive linkage between the PM targeting moieties and the BODIPY donor allows strong anchoring at the PM, which ensures that the donor signal remains on the PM even after cleavage, regardless of the regioselectivity of the TDE reaction and the nature of the thiol nucleophile: thiolated proteins or glutathione (Figure S1).…”

“…PM probes are valuable tools in bioimaging and found interesting applications in structural studies, 18 membrane dynamic in live cells, 19 tracking of exosomes, 20 and in super-resolution microscopy. 21,22,23,23 Unlike those probes that accumulate in the membrane to provide a labeling, functional PM probes are able to report on their environment through a change of fluorescence signal. 24,25 Responsive PM probes were developed to report on lipid order, 26,27 organization of lipid microdomains, 28,29 and loss of transmembrane asymmetry during apoptosis.…”

Current molecular approaches to investigate pre‐synaptic dysfunction

“…SP‐468 (similar absorption and emission spectra to FM1‐43) and SQ‐535 (red‐shifted variant) were engineered from FM1‐43 but in contrast to FM1‐43, these dyes associate with the outer leaflet of the bilayer rather than inserting into the bilayer (Collot et al. 2020). Although proven to stain the plasma membrane in a range of cell types, they remain untested in terms of vesicle recycling, however, the properties of the dyes may make them a more photostable alternative to FM dyes.…”

Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models

“…Nowadays, modified dyes which allow fixation of the stain (FX modifications) are available. Furthermore, fine chemical modifications of FM1-43, which is one of the most widely used FM probes, resulted in new probes, SP-468 (red) and SQ-535 (far red), which have enhanced photophysical properties, e.g., reduced crosstalk, higher brightness, or improved photostability (Collot et al 2019 ). Similar to the long-chain carbocyanine dyes, it cannot be granted that dyes incorporated into the plasma membranes will not cause micro-environmental background by passing on the stain to neighboring cells or due to phagocytosis.…”