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Lipids are difficult to detect with light microscopy. Using a new chemical labeling strategy, a Dresden-based team led by André Nadler at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) and Alf Honigmann at the Biotechnology Center (BIOTEC) of TU Dresden has overcome this limitation, enabling new insights into lipids.

The researchers were able to answer a long-standing question: how do cells transport specific lipids to the membranes of their target organelles? The new lipid-imaging technique will help understand the role of lipid transport in health and disease. The findings were in the journal Nature.

Lipid molecules, or fats, are crucial to all forms of life. Cells need lipids to build membranes, separate and organize biochemical reactions, store energy, and transmit information.

Every cell can create thousands of different lipids, and when they are out of balance, metabolic and neurodegenerative diseases can arise. It is still not well understood how cells sort different types of lipids between cell organelles to maintain the composition of each membrane.

A major reason is that lipids are difficult to study, since microscopy techniques to precisely trace their location inside cells have so far been missing.

In a long-standing collaboration, Nadler, a chemical biologist at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany, teamed up with Honigmann, a bioimaging specialist at Biotechnology Center (BIOTEC) at the TU-Dresden University of Technology, to develop a method that enables visualizing lipids in cells using standard fluorescence microscopy.

After the first successful proof of concept, the duo brought mass-spectrometry expert Andrej Shevchenko (MPI-CBG), Björn Drobot at the Helmholtz-Zentrum Dresden-Rossendorf (HZDR), and the group of Martin Hof from the J. Heyrovsky Institute of Â鶹ÒùÔºical Chemistry in Prague on board to study how lipids are transported between cellular organelles.

Artificial lipids under the sunbed

"We started our project with synthesizing a set of minimally modified lipids that represent the main lipids present in organelle membranes. These modified lipids are essentially the same as their native counterparts, with just a few different atoms that allowed us to track them under the microscope," explains Kristin Böhlig, a Ph.D. student in the Nadler group and chemist who was in charge of creating the modified lipids.

The modified lipids mimic natural lipids and are "bifunctional," which means they can be activated by UV light, causing the lipid to bind or crosslink with nearby proteins. The modified lipids were loaded in the membrane of living cells and, over time, transported into the membranes of organelles. The researchers worked with human cells in cell culture, such as bone or intestinal cells, as they are ideal for imaging.

"After the treatment with UV light, we were able to monitor the lipids with fluorescence microscopy and capture their location over time. This gave us a comprehensive picture of lipid exchange between cell membrane and organelle membranes," concludes Kristin.

In order to understand the microscopy data, the team needed a custom image analysis pipeline. "To address our specific needs, I developed an image analysis pipeline with automated image segmentation assisted by artificial intelligence to quantify the lipid flow through the cellular organelle system," says Juan Iglesias-Artola, who did the image analysis.

Speedy lipid transport by proteins

By combining the image analysis with mathematical modeling done by Drobot at the HZDR, the research team discovered that between 85% and 95% of the lipid transport between the membranes of cell organelles is organized by carrier proteins that move the lipids, rather than by vesicles.

This non-vesicular transport is much more specific with regard to individual lipid species and their sorting to the different organelles in the cell. The researchers also found that the lipid transport by proteins is ten times faster than by vesicles. These results imply that the lipid compositions of organelle membranes are primarily maintained through fast, species-specific, non-vesicular lipid transport.

In a parallel set of experiments, the group of Shevchenko at the MPI-CBG used ultra-high-resolution mass spectrometry to see how the different lipids change their structure during the transport from the cell membrane to the organelle membrane.

A boost for lipids in cell biology and disease

This new approach provides the first-ever quantitative map of how lipids move through the cell to different organelles. The results suggest that non-vesicular lipid transport has a key role in the maintenance of each organelle membrane composition.

Honigmann, research group leader at the BIOTEC, says, "Our lipid-imaging technique enables the mechanistic analysis of lipid transport and function directly in cells, which has been impossible before. We think that our work opens the door to a new era of studying the role of lipids within the cell."

Imaging of lipids will allow further discoveries and help to reveal the underlying mechanisms in diseases caused by lipid imbalances. The new technique could potentially help to develop new druggable targets and therapeutic approaches for lipid-associated diseases, such as nonalcoholic fatty liver disease.

'We knew that we were onto something big'

Nadler, research group leader at MPI-CBG, looks back at the start of the study: "Imaging lipids in cells has always been one of the most challenging aspects of microscopy. Our project was no different. Alf Honigmann and I started discussing about solving the lipid imaging problem as soon as we got hired in close succession at MPI-CBG in 2014/15 and we quickly decided to go for it.

"It still took us almost five years from the start of the project to the point in autumn 2019 when the two of us finally produced a sample with a beautiful plasma membrane stain. That's when we knew that we were onto something big. As a reward, certain well-known global events meant we were required to shut down our laboratories a few months later.

"In the end, the delay was for the best. Before the revolution in the use of artificial intelligence in image segmentation, we would not have been able to properly quantify the imaging data, so our conclusions would have been much more limited."

Researchers still need to determine which lipid-transfer proteins drive the selective transport of different lipid species. They also need to identify the energy sources that power lipid transport and ensure that each organelle keeps its own unique membrane composition.