No abstract

We describe the development of click-expansion microscopy (Click-ExM), which integrates click-labeling into ExM to enable a"one-stop-shop" method for nanoscale imaging of various types of biomolecules.Using 18 clickable labels for click-ExM imaging of DNA, RNA, proteins, lipids, glycans and small molecules, we demonstrate its universality, compatibility with signal-ampli cation techniques, and broad applications in cellular and tissue imaging. The click-labeling and ExM steps could be nished within two days. This step-by-step protocol is related to the publication "Click-ExM enables expansion microscopy for all biomolecules" in Nature Methods.

Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per- O -acetylated, which, however, can induce a long-overlooked side reaction, non-enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N -azidoacetylgalactosamine (GalNAz) as next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di- O -acetylated GalNAz (1,3-Ac 2 GalNAz) and 1,3-di- O -propionylated GalNAz (1,3-Pr 2 GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr 2 GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG.

Metabolic labeling of glycans with bioorthogonal reporters has been widely used for glycan imaging and glycoproteomic profiling. One of the intrinsic limitations of metabolic glycan labeling is the lack of cell-type selectivity. The recently developed liposome-assisted bioorthogonal reporter (LABOR) strategy provides a promising means to overcome this limitation, but the mechanism of LABOR has not been investigated in detail. In this work, we performed a mechanistic study on LABOR and explored its multiplexing capability. Our studies support an endocytosis-salvage mechanism. The ligand-targeted liposomes encapsulating azidosugars are internalized into the endosome via the receptor-mediated endocytosis. Unlike the conventional drug delivery, LABOR does not rely on the endosomal escape pathways. Rather, the liposomes are allowed to enter the lysosome, inside which the azidosugars are released from the liposomes. The released azidosugars then intercept the salvage pathways of monosaccharides and get transported into the cytosol by lysosomal sugar transporters. Based on this mechanism, we expanded the scope of LABOR by evaluating a series of ligand-receptor pairs for targeting sialoglycans in various cell types. Different ligand types including small molecules, antibodies, aptamers, and peptides could be easily implemented into LABOR. Finally, we demonstrated that the sialoglycans in two distinct cell populations in a co-cultured system could be selectively labeled with two distinct chemical reporters by performing a multiplexed LABOR labeling.