Fluorescent dyes and probes for super-resolution microscopy of microtubules and tracheoles in living cells and tissues

Abstract

Nanoscopy compatible fluorescent tubulin probes can be used to stain microtubules and chitin-rich taenidia in the insect tracheoles.

Enhancing biocompatibility of rhodamine fluorescent probes by a neighbouring group effect

“…We have previously demonstrated that the performance of the tubulin probes depends on the chosen taxane ligand, and that the optimal ligand is different for each fluorophore 8 . Based on thisknowledge, we synthesized a new series of tubulin probes by coupling the 4’-regioisomers of TMR-COOH and 580CP-COOH fluorophores to a Larotaxel derivative ( LTX-C8-NH 2 , ( SI-4 )) and 610CP-COOH and SiR-COOH to a Cabazitaxel derivative ( CTX-C8-NH 2 , ( SI-3 )) (Fig.…”

“…Rhodamine probes are prone to aggregation, which significantly contributes to their fluorogenicity and might impair interaction with the target 8 . We observed a high correlation between fluorescence increase after binding to tubulin and after SDS addition, which indicates that affinity towards tubulin is sufficient to dissociate the probe from the aggregates (Supplementary Fig.…”

“…Cabazitaxel or Larotaxel are anticancer drugs displaying superior resistance to efflux pumps and brain blood permeability 26, 27 . The cytotoxicity of tubulin probes is implied by the design, which exploits the attachment of fluorophores to the 3’-position of taxanes 8 . We hypothesized that NGE enhanced spirolactonization should increase the cell membrane permeability, resulting in stronger staining of tubulin and higher cytotoxicity.…”

“…This property is exploited to generate fluorogenic probes, which are sensitive to a number of environmental factors: pH, ion concentration, enzymatic activity and its local microenvironment polarity or light 6, 7 . Since the spirolactone is a more hydrophobic molecule compared to the zwitterion, its presence makes rhodamine-based fluorescent probes more cell-permeable 8, 9 . Regular rhodamines and carbon-substituted analogues (carbopyronines) have the equilibrium shifted towards zwitterion form, which results in relatively poor membrane permeability 8, 9 .…”

“…Since the spirolactone is a more hydrophobic molecule compared to the zwitterion, its presence makes rhodamine-based fluorescent probes more cell-permeable 8, 9 . Regular rhodamines and carbon-substituted analogues (carbopyronines) have the equilibrium shifted towards zwitterion form, which results in relatively poor membrane permeability 8, 9 . To overcome this issue, several studies attempted to induce spirolactone form preference by introducing electron-withdrawing groups into the xanthene core 10, 11, 12 or in the benzoic acid substituent 13 .…”