Researchers have, for the first time, visualized individual sugars within the glycocalyx – the sugar layer surrounding each cell in the human body – at ångström resolution, using a combination of microscopy and bioorthogonal chemistry.
By allowing scientists to study glycocalyx components in their native environment with spatial resolution down to a single sugar unit, the technique, developed by researchers at the Max Planck Institute for the Science of Light and at the Max Planck Institute for Biochemistry, could open the door to breakthroughs in our understanding of the role of glycosylation in cancer biology and lead to new diagnostic markers and therapeutic targets.
The glycocalyx has a fundamental role in a range of cellular processes in health and disease, including immune system regulation, cell signaling, and cancer development. But our understanding has been limited by an inability to structurally characterize cell-surface glycans. This is due to the glycocalyx’s thickness, which can range from several dozens to hundreds of nanometers, its structural complexity, being composed of thousands of densely packed components; as well as its sensitivity. As a result, methods used must be high-resolution, minimally invasive, and have high labeling specificity to obtain species information.
“All these requirements are met by the latest optical super-resolution microscopy methods, which are the methodological basis for our results,” says Leonhard Möckl, co-corresponding author of the study and Physical Glycosciences research group leader at MPL.
The researchers combined resolution enhancement by sequential imaging (RESI) – a DNA-PAINT-based microscopy method pioneered by the Jungmann Lab – with bioorthogonal chemistry in which the cell’s metabolism is used to attach specific markers to target structures. The latter makes use of copper-free click chemistry, pioneered by the Bertozzi Lab, which was awarded with a Nobel Prize in 2022.
“It took us two years to optimize the labeling protocols to get everything right for imaging. We faced the classic cycle of failing, understanding why, redesigning the approach, and optimizing,” says Möckl. “The ‘eureka’ moment was certainly when we got the first molecular resolution map of individual sugars. This was a dream of mine during my PhD, and I was chasing it for almost 15 years. It was mesmerizing when it finally came true.”
The method overcomes the limitations of mass spectrometry, which has been used to study total amounts of sugar structures in the glycocalyx, but can’t retain spatial information. “Recent
findings from our group indicate that the relative special arrangement of glycocalyx components at the nanoscale communicates critical information to the exterior,” says Möckl. “Having established a method that can reveal these arrangements allows us, for the first time, to understand this so far uncharacterized axis. And obtaining insights into the interplay between cell state and organization of cell-surface glycosylation could have far-reaching consequences for the clinic.”
This research was, essentially, a proof-of-concept, and the researchers plan to look at other sample types, such as primary cells or different cell states. “We’re just at the beginning,” says Möckl. “The tool in our hands is immensely useful to chart a completely unknown territory of cell biology: the molecular organization of cell-surface glycosylation.”
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