Method Improves 3D Visualization of Lipids

Scientists at Max Planck Institute of Molecular Cell Biology and Genetics have developed a correlative light and electron microscopy workflow to visualize and quantify individual lipids in cellular membranes at ultrastructural resolution. Called Lipid-CLEM, it is reportedly the first reliable way to map the distribution of lipids in 3D within complex cellular structures.

Biological membranes of cells and organelles contain nanodomains composed of lipids and proteins. These regions support key functions such as signaling, sorting, and transport. While protein roles in nanodomains are well understood, lipid distribution poses challenges because lipids move rapidly and existing methods struggle to visualize individual lipid species at high resolution.

To localize lipids now, researchers use "bifunctional lipid probes," which are very small, slightly modified lipids that act like molecular GPS tags. These probes can be added into living cells, then “frozen in place” with light (photo-crosslinking), and later labeled with fluorescence using a chemical reaction (click chemistry). In this way, researchers can track where specific lipids are and not alter and disturb the cell too much.

However, light microscopy alone is not enough to visualize small details in the cell membrane. Higher details can be captured by electron microscopy. Correlative light and electron microscopy (CLEM) combines the strengths of both techniques. Together with the bifunctional lipid probes, Lipid-CLEM shows where labeled lipids are and makes the fine structure of the membranes visible.

To study lipid sorting in early endosomes – a key sorting station inside the cell – cells must be rapidly frozen to stop lipids in their tracks and to preserve the membrane of the cells, explained Mathilda Lennartz, lead author of the study published in Nature Cell Biology. "Later, these lipids can be labeled on very thin slices of the sample, termed 'sections,' of cells using click chemistry. These sections are what we then image using the Lipid-CLEM approach.”

“With Lipid-CLEM, we observed that a specific lipid called sphingomyelin is more common in small vesicles inside the endosome and less common in tubular membrane domains. This separation has already been observed for some proteins,” added Lennartz. “What we concluded from this is that at least some lipids, just like proteins, must also be sorted in the endosome. Interestingly, in our study, sphingomyelin and a protein cargo arrive at the same time in the early endosome but separate into different domains, indicating that lipid and protein trafficking routes can diverge during this sorting.” 

Senior author André Nadler summarized, “Our Lipid-CLEM workflow enables 3D visualization of lipid densities in membrane nanodomains, offering a new way to study lipid organization in complex cellular structures. We finally can look at lipid sorting in membranes with the resolution we need. We believe that our new method Lipid-CLEM will help us to better understand how lipids work in cells, as it allows us to study both lipids and proteins together during membrane organization and function. This may also contribute to a better understanding of membrane dysfunction-related diseases.”