Sharpening a Blurry Image

Lipids have long been known to play key roles in maintaining the plasma membrane’s structure and functionality in mammalian cells. Studying the distribution of these lipids, across the inner and the outer leaflets (sides) of the membrane, is crucial to understanding how cells regulate the biosynthesis of lipids. Cholesterol, one of the major lipids in the mammalian plasma

A simple cartoon representation of a mammalian cell membrane. This image demonstrates the presence of cholesterol on both the inner and the outer leaflets of the membrane. Source: khanacadamy.org

membrane, has been extensively studied for its various roles in cell structure and function. The lack of accurate quantification methods, however, has hindered any further analysis. Shu-Lin Liu et al., nonetheless, in an article published recently in Nature, report their development of promising new methods that allow for in situ quantification of cholesterol in the plasma membrane is mammalian cells with high sensitivity and accuracy. The quantification of the distribution of plasma membrane cholesterol, and being able to modulate its transbilayer distribution can potentially lead to treatment of hypercholesterolemia-linked breast cancer.

The spatiotemporal, asymmetric, distribution of cholesterol in the membrane is maintained by special transporters that respond to stimulants and feedback loops. Due to the complexity of these processes, it is crucial to be able to study the membrane of a living cell, without disrupting its function. To achieve this, Shu-Lin Liu et al. isolated cholesterol-specific bacterial domains (D4) that had been mutated to bind to membrane cholesterol at variable concentrations. These domains were then tagged with solvatochromic fluorophores (dyes) to create tunable sensors that have ranging affinities for cholesterol.

To confirm the accuracy of the sensors, the authors conducted a quantitative mass spectroscopy experiment in which they were able to obtain the concentration of all major lipids found in the membrane of the same HeLa cell, under the same cellular conditions. Upon separation of the lipids, it was found that the total concentration of cholesterol in the membrane agreed with the sum of inner and outer leaflet concentrations obtained via sensor quantification.

Using those sensors to quantify membrane cholesterol, the authors found the concentration in the outer side of the membrane to be significantly higher than that of the inner leaflet. This is consistent with previous findings that high outer-membrane cholesterol concentration determined membrane permeability and assisted in membrane domain formation in the outer leaflet, and the inner membrane cholesterol functions in cell signalling when in low concentrations. This finding further confirms the accuracy of the results obtained from the sensors.

The method that the authors of this paper developed can be a model to later development of sensors that can potentially quantify any membrane lipid, in any cellular membrane. This clear and accurate quantification of a membrane lipid will have an enormous impact on many scientific fields, including cancer research. Being able to regulate and moderate the concentrations of membrane cholesterol can lead to possible detection, and treatment options for hypercholesterolemia-linked breast cancer. As the research of cholesterol-linked cancer grows, it is possible to stumble upon other membrane lipids that participate in the reactions, and it would be interesting to see how the development of new sensors that can detect various lipids will have an affect on the field.

References

1. Liu, S.-L. et al. Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol. Nat Chem Biol advance online publication, (2016).

2. Sheng, R. et al. Cholesterol modulates cell signaling and protein networking by specifically interacting with PDZ domain-containing scaffold proteins. Nat. Commun.3, 1249 (2012).

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6 Replies to “Sharpening a Blurry Image”

  1. Hi Leen. I think you chose a very interesting article, especially since in class we discussed the vast roles of cholesterol, so the clinical implications of the authors’ findings are apparent. I was particularly interested in the determined ratio between outer membrane to inner membrane cholesterol concentration. Did the authors or any other researchers study the effect of changing the the ratio between outer and inner membrane cholesterol? Perhaps there might be some clinical implications associated with changing the cholesterol ratio.

    1. In a short section of the results, the authors report that they tested the significance of maintaining transbilayer asymmetric plasma membrane cholesterol. The authors performed a knock-down of ABCA1 and ABCG1, two crucial cholesterol transporters in HeLa cells.. The knock-down led to the accumulation of inner leaflet cholesterol concentration. When the cells were exposed to growth and proliferation stimuli, the response of the mutants was 3.5-fold greater than that of the WT cells. These results were consistent with the authors’ findings in colorectal cancer cells, which led them to suggest that a similar mutation in the outward transport pathway can be an explanation for the behavior of these cells. The authors also show the extent to which this mutation can be treated, by treating the KD HeLa cells with mevastatin, which inhibits the biosynthesis of cholesterol, and therefore lowers its concentration on the inner membrane leaflet. I hope this answers your question!

  2. It is a big step forward to be able to quantify the amount of cholesterol or other lipids in a certain membrane . One common heart condition is called atherosclerosis, it is caused by accumulation of lipid buildup that hardens and narrows the arteries. My grandfather was a victim of this heart condition, so this actually makes me very glad to know that we are moving forward. I know there are some blood tests out there to find out the levels of cholesterol in your blood, but this paper is saying we can use sensors to quantify it anywhere. If we are able to efficiently and fully quantify the levels of cholesterol, then we can protect others against any possible clot that is caused by the lipid buildup. GREAT!

    1. Using this new technology we will not only be able to quantify membrane lipid concentration, but we will be able to pharmacologically increase or lower the concentrations while monitoring them.

  3. Being able to quantify membrane lipids in situ is certainly of benefit, especially if this method can be further applied to other membrane lipids in any cell. Since cholesterol has been highly studied previously and other, less accurate methods of measuring have been attempted, I am curious about how much of an improvement this method is. How inaccurate were previous quantification methods, and were they also able to be performed in situ? Also, I am interested in the invasiveness or safety of this tagging/quantification process. Beyond being accurate, is this method also safe and reasonable, or does much further work need to be done?

  4. I thought it was really interesting that there might be a possible method for quantifying cholesterol in situ across a membrane with a high degree of accuracy. This can provide for new information for what goes on in abnormal cells and could be used to track what happens to a cell at various time points. Given that the authors write that their method could potentially be used for any membrane lipid in a cellular membrane, this could provide much more individual attention for diseases that involved abnormal cell behavior. It could also be used to see whether there are differences and/or similarities in the membrane lipids in healthy versus diseased subjects.

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