A functional dual-color indicator is designed for in situ visualization of intracellular glycosylation. Using O-GlcNAcylation as model, the indicator is constructed on a poly-GlcNAc-coated gold nanoparticle (AuNP) by assembling dye labeled lectin (FSWGA) and then another dye-labeled GlcNAc (FGlcNAc) through the two opposite subunits of FSWGA. These dyes possess negligible overlapping emission and can be quenched by AuNP. In the presence of intracellular dissociated GlcNAc residue and O-GlcNAcylated proteins, the assembled FGlcNAc and the conjugate of FSWGA with FGlcNAc are released from AuNP through the dynamic competitive conjugation, which lights up the fluorescence of two dyes, respectively, and provides a simple technique for simultaneously monitoring the level of O-GlcNAcylated proteins and the total amount of GlcNAc groups in living cells. The practicality of the protocol for visually monitoring the biological pathway between intracellular O-GlcNAcylation and cell surface differentiation-related proteins demonstrates a convenient and powerful tool for research of glycosylation equilibrium and related biological processes.
A screening strategy involving designed extractors and collectors was used for the nondestructive quantitation of gangliosides on cell surfaces. The extractors were constructed by functionalizing maleimide silica bubbles with a DNA probe, which contains an endonuclease cleavage site and a boronic acid end to extract cell-surface sialic acid-containing compounds through simple centrifugation. After the extractors containing the extracted compounds were incubated with endonuclease, the released oligonucleotide-gangliosides were selectively collected by silanized collector bubbles through hydrophobic interactions. The in vitro fluorescent signals from the collectors were used for the quantitation of cell-surface gangliosides. By combining with sialidase cleavage, a protocol for the identification of ganglioside subtypes was developed. The successful monitoring of the regeneration of cell-surface gangliosides demonstrates the potential of this strategy in probing related biological processes.
Summary
The plasma membrane repair holds significance for maintaining cell survival and homeostasis. To achieve the sensitive visualization of membrane repair process for revealing its mechanism, this work designs a perforation-induced surface-enhanced Raman spectroscopy (SERS) strategy by conjugating Raman reporter (4-mercaptobenzoic acid) loaded gold nanostars with pore-forming protein streptolysin O (SLO) to induce the SERS signal on living cells. The SERS signal obviously decreases with the initiation of membrane repair and the degradation of SLO pores due to the departure of gold-nanostar-conjugated SLO. Thus, the designed strategy can dynamically visualize the complete cell membrane repair and provide a sensitive method to demonstrate the SLO endocytosis- and exocytosis-mediated repairing mechanism. Using DOX-resistant MCF-7 cells as a model, a timely repair-blocking technology for promoting the highly efficient treatment of drug-resistant cancer cells is also proposed. This work opens an avenue for probing the plasma membrane repairing mechanisms and designing the precision therapeutic schedule.
O-GlcNAcylation is involved in many biological processes including cancerization. Nevertheless, its in-situ quantification in single living cells is still a bottleneck. Here we develop a quantitative SERS imaging strategy for...
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