Abstract
Here we report a small molecule tubulin probe for single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy and MINFLUX nanoscopy, which can be used in living and fixed cells. We explored a series of taxane derivatives containing spontaneously blinking far-red dye hydroxymethyl silicon–rhodamine (HMSiR) and found that the linker length profoundly affects the probe permeability and off-targeting in living cells. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and the 6′-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameter of ≤50 nm was routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin allows a complementary use of different nanoscopy techniques for investigating microtubule functions and developing imaging methods. For the first time, we resolved the inner microtubule diameter of 16 ± 5 nm by optical nanoscopy and thereby demonstrated the utility of a self-blinking dye for MINFLUX imaging.
Highlights
As one of the major components of the cytoskeleton, tubulin is involved in trafficking of biomolecules, cell movement and division
Paclitaxel is one of the most effective anticancer drugs used for the treatment of solid tumors such as ovarian, breast, and lung cancers.[1]. It acts by stabilizing microtubules and thereby blocking cell progression through mitosis. It was originally isolated from the bark of the yew tree Taxus brevifolia in 1971,2 and a tremendous repertoire of analogues has been reported by research groups and pharmaceutical companies ever since.[1,3]
The development of tubulin probes for single-molecule localization microscopy (SMLM) in living cells is lagging behind, with only one published example composed of a spiropyran derivative and colchicine, which requires UV illumination.[11]
Summary
As one of the major components of the cytoskeleton, tubulin is involved in trafficking of biomolecules, cell movement and division. Paclitaxel is one of the most effective anticancer drugs used for the treatment of solid tumors such as ovarian, breast, and lung cancers.[1] It acts by stabilizing microtubules and thereby blocking cell progression through mitosis It was originally isolated from the bark of the yew tree Taxus brevifolia in 1971,2 and a tremendous repertoire of analogues has been reported by research groups and pharmaceutical companies ever since.[1,3]. The development of tubulin probes for single-molecule localization microscopy (SMLM) in living cells is lagging behind, with only one published example composed of a spiropyran derivative and colchicine, which requires UV illumination.[11] We are bridging this gap by synthesizing taxane analogs coupled to the spontaneously blinking fluorophore hydroxymethyl silicon−rhodamine (HMSiR).[12]
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