Tracking Individual Membrane Translocation Accelerates Precision Microscopic Drug Screening Efforts

A new single-protein analysis technique gives researchers an unprecedented ability to study proteins called scramblases, which have critical roles in biology. The development of the new technique, in a study led by investigators at Weill Cornell Medicine and Ruhr University Bochum in Germany, expands the toolkit available to cell biologists and biophysicists and could someday be useful in devising new strategies against multiple diseases.

Scramblases operate within cell membranes to rearrange the fat-related molecules, known as lipids, that make up those membranes. Their disruption of the usual layered organization of the membrane is essential for many important biological processes. In the study, published June 15 in Nature Structural & Molecular Biology, the researchers developed a fluorescence imaging-based technique-the first of its kind-for measuring the activity rates of individual scramblase proteins. Their demonstrations of the technique uncovered new findings on key scramblases, and showcased the technique's broad applicability.

I'm excited about this new platform as it is versatile and provides unprecedented information on exactly how fast a single scramblase works."

Anant Menon, Professor of Biochemistry and Biophysics, Weill Cornell Medicine

The study's co-senior author is Dr. Thomas Günther-Pomorski, professor of biochemistry at Ruhr University Bochum. The first author is Dr. Sarina Veit, a postdoctoral associate in the Pomorski Lab.

Scramblases are ubiquitous in biology, with roles in the assembly of cell membranes, modification of proteins with sugars, cell survival, muscle development and molecular trafficking. In principle, being able to modulate the activity of specific scramblases, for example with a drug, could be useful across a wide range of clinical applications.

Strategies for studying how scramblases work have been limited, however. Typically, scientists have purified scramblase proteins, setting them in cell-membrane-like tiny lipid spheres called vesicles, then recording, and averaging, what multiple scramblases are doing. This "ensemble" or "bulk analysis" approach pioneered by the Menon lab and now widely used, is unable to precisely measure the transport rate of a single scramblase and misses what could be large differences between individual scramblase proteins-differences that could inform a better understanding of how these proteins work, and how they become dysfunctional.

In the study, the researchers incorporated fluorescently-tagged scramblases within the vesicles, then imaged the vesicles after depositing them onto a glass slide. Using sophisticated microscopy and analytical methods, they identified vesicles containing just one scramblase and measured the rate of scrambling in these.

They used this approach firstly to evaluate a scramblase protein called VDAC1. Best known as a channel protein in the membranes surrounding mitochondria-the oxygen-burning reactors that help produce chemical fuel molecules within cells-VDAC1 was found recently by the Menon Lab to be a scramblase as well. Two copies of VDAC must pair up to provide a pathway for lipid movement, and the researchers' analysis showed that these pairs (dimers) have a wide range of scrambling rates, from fewer than 100 to more than 1,000 lipids per second.

"These findings indicate that only certain dimer conformations are capable of rapid scrambling, directly validating predictions from computer simulations," Dr. Menon said.

The team demonstrated the versatility of their approach by using it to measure lipid-scrambling by opsin, a cell-membrane receptor that is involved in light-detection in the eye, but-as Dr. Menon and colleagues have shown - "moonlights" as a scramblase. The findings revealed that individual opsin scramble lipids faster than VDAC dimers, achieving rates in excess of 10,000 lipids per second.

The researchers envision the use of this new platform to study, for example, how scramblase function is affected by changes in the composition of surrounding lipids in a cell membrane, or by different drug molecules. Additionally, they hope to combine their functional studies of scramblases with high-resolution imaging, to reveal how scramblases' shapes relate to their activity rates. The team also plans to use the new platform to study other lipid-moving proteins called flippases and floppases, Dr. Menon said.