Chemically-activatable alkyne-tagged probe for imaging microdomains in lipid bilayer membranes

Abstract

A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the lipid raft membrane domains by Raman microscopy. The Raman probe, in which ring A of its steroid backbone is replaced with an alkynyl group, was designed to enable activation of the alkyne signal through the Eschenmoser-Tanabe fragmentation reaction of the oxidized cholesterol precursor in lipid bilayer membranes. The alkynyl steroid analogue was observed to form liquid-ordered raft-like domains on a model giant-liposome system in a similar manner as cholesterol, and the large alkyne signal of the accumulated probe at 2120 cm−1 was mapped on the microdomains with a Raman microscope. The alkyne moiety of the probe was confirmed to be converted from the α,β-epoxy ketone group of its precursor by reaction with p-toluensulfonyl hydrazine under a mild condition. Through the reaction, the alkyne signal of the probe was activated on the lipid bilayer membrane of liposomes. Furthermore, the signal activation of the probe was also detected on living cells by stimulated Raman scattering microscopy. The ring-A-opened alkyne steroid analogue, thus, provides a first chemically-activatable Raman probe as a promising tool for potentially unravelling the intracellular formation and trafficking of cholesterol-rich microdomains.

Noncanonical Function of a Small-Molecular Virulence Factor Coronatine against Plant Immunity: An In Vivo Raman Imaging Approach

“…While the results presented here were obtained by spontaneous Raman microscopy, the use of newly emerged ATRI coupled with coherent Raman scattering microscopies would offer faster image acquisition, greater detection sensitivity, and less photodamage. 32−37 In contrast, the nuclear localization of COI1 and JAZ was clearly observed in P 35S -COI1:YFP and P 35S -JAZ9:YFP guard cells (Figure 5c). Thus, a distinct difference in localization was confirmed between 5 and the COI1-JAZ coreceptor in Arabidopsis guard cells (Figures 5a and 5c).…”

A decade of alkyne-tag Raman imaging (ATRI): applications in biological systems

“…They used chemical activation to generate and visualize alkyne-tagged cholesterol in HeLa cells. 102 Our group has reported sphingomyelin mimics with monoalkyne and diyne modification. 103 Using diyne, we have visualized and quantitatively analyzed sphingomyelin distribution in artificial monolayer membranes.…”

“…They used chemical activation to generate and visualize alkyne-tagged cholesterol in HeLa cells. 102 …”

Mammalian cell and tissue imaging using Raman and coherent Raman microscopy

“…Raman probes also vary greatly in size depending on their intended use. Small molecules such as alkynyl steroids have been referred to as probes in their application to the study of liposomal membrane microdomains (Yamaguchi et al, 2017) despite their similar size as compared to Raman tags. Large NPs, Raman-active themselves or complexed with compounds on their surface, are also considered probes.…”