De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells
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Summary: Zhang et al. describe an interesting method to label newly synthesized lipids with fluorescent fatty acids and track their movement in cells. All reviewers agreed that this could potentially be a useful tool. However, they all raised concerns regarding the rigor of the characterization of this methodology.
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Abstract
Lipids exert dynamic biological functions which are determined both by their fatty acyl compositions and precise spatiotemporal distributions inside the cell. There are more than 1000 lipid species in a typical mammalian cell. However, it remains a daunting task to investigate any of these features in live cells for each of the more than 1000 lipid species. Here we resolved this issue by developing a de novo lipid labeling method for major lipid species, including glycerolipids, glycerophospholipids, and cholesterol esters by using a single fluorescent probe. The method not only allowed us to probe the precise subcellular distribution and trafficking of individual lipid species in live cells, but also uncovered some unexpected biological functions of previously reported lipid metabolic enzymes that were not possible by conventional biochemical methods. We envision that this method will become an indispensable tool for the functional analysis of individual lipid species and numerous lipid metabolic enzymes and transporters in live cells.
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Reviewer #3:
The manuscript by Shi and colleagues delineates an approach for labeling newly synthesized lipids thereby providing a method to examine how lipids move throughout the cell. The premise of this technical approach is that fluorescently labeled fatty acids are fed to a cell in the presence of another lipid which will incorporate the fluorescent acyl tail using the endogenous cellular acyltransferases. Cellular imaging is paired with this approach to show the subcellular accumulation of the lipid. As presented, the data are intriguing, but there are some concerns and questions about the technique that limits the interpretation of the data and could impact the overall utility of this approach. The authors should provide the additional requested data, and resolve the issues raised below to increase confidence that this labeling …
Reviewer #3:
The manuscript by Shi and colleagues delineates an approach for labeling newly synthesized lipids thereby providing a method to examine how lipids move throughout the cell. The premise of this technical approach is that fluorescently labeled fatty acids are fed to a cell in the presence of another lipid which will incorporate the fluorescent acyl tail using the endogenous cellular acyltransferases. Cellular imaging is paired with this approach to show the subcellular accumulation of the lipid. As presented, the data are intriguing, but there are some concerns and questions about the technique that limits the interpretation of the data and could impact the overall utility of this approach. The authors should provide the additional requested data, and resolve the issues raised below to increase confidence that this labeling approach allows for the monitoring of physiologic lipid trafficking pathways.
Specific concerns and questions are delineated below.
The authors initially exploit the remodeling of PLs as described in figure 1a. This involves the addition of lyso-PL and NBD-labeled palmitoyl-CoA. The authors imply from their schematic in Fig 1a that they are using lyso-PLs that are being remodeled at the sn1 position by NBD-labeled palmitoyl-CoA. Unless I am missing something, lyso-PA and other related lyso-PLs are generally remodeled at the sn2 position. Additionally, there is specificity for PUFAs acylation to the lyso-PL. So I am a bit confused about the enzymes that are working in this system. I tried to determine which lyso-PLs that the authors are using, but the methods did not specify if they are using 1- or 2-lyso PLs. This should be clarified so that we can understand the enzymes the authors think are underlying the labeling reaction. On a minor, but related note, the lyso-PL in Figure 1a is missing an -OH group at the sn1 position.
The authors use a cell system where the cells are starved of lipids and other metabolites for 1 hour and then fed a large bolus of lipids as substrates. It appears that the cells can remodel and label some PLs under these conditions, but it is not clear to me that this represents physiologic labeling that can be used to track the de novo labeling and trafficking into subcellular compartments. Nor can it be used to draw strong conclusions about required trafficking or enzymatic pathways under normal conditions. What happens if labeling occurs in complete media or defined media? This might help to resolve this.
The labeling looks non-uniform in mitochondria as evidenced by the images in figure 2a. Why is the labeling only at the outer edge of the mito in these cells in this figure? What happens if labeling goes longer? Similarly, the authors quantify "30 cell images" or the like in the figures for Pearson correlations. How were the 30 cells selected, and since labeling across the mitochondria is not uniform, how were images selected? A much larger number of images scanned in an unbiased manner would increase confidence.
Likewise, what happens if the labeling is allowed to proceed beyond 15 min. Can the authors provide a 30 min and 1 hr image?
There are a number of conclusions drawn about specific pathways required for the trafficking of accumulation of labeled lipids. I realize that some of these studies are used as a specific proof-of-concept for the approach. However, there are many studies that go beyond proof-of-concept and draw conclusions about biology. Many of the studies are somewhat superficial and the conclusions reached by the authors should be tempered given that they have not deeply investigated this new biology.
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Reviewer #2:
In this study, Zhang et al. use a semi-novel method to track acyltransferase activity using fluorescently labeled palmitic acid (NBC-16:0) to track where specific lipids are incorporated with subcellular specificity. They show that NBD-16:0 can be incorporated into different lipid classes that segregate based previously known on subcellular specificity. While this is an interesting technique, it is difficult to determine how much fidelity this method has in recapitulating biological function without additional experimentation, orthogonal measurements, and a more descriptive methods section.
Comments:
The authors do not specify which lysophospholipids were used in their study. In the method section they specify that they came from Avanti, but there are >100 different lPLs in their catalog. Also, the authors give a range of …
Reviewer #2:
In this study, Zhang et al. use a semi-novel method to track acyltransferase activity using fluorescently labeled palmitic acid (NBC-16:0) to track where specific lipids are incorporated with subcellular specificity. They show that NBD-16:0 can be incorporated into different lipid classes that segregate based previously known on subcellular specificity. While this is an interesting technique, it is difficult to determine how much fidelity this method has in recapitulating biological function without additional experimentation, orthogonal measurements, and a more descriptive methods section.
Comments:
The authors do not specify which lysophospholipids were used in their study. In the method section they specify that they came from Avanti, but there are >100 different lPLs in their catalog. Also, the authors give a range of lPL concentrations in the methods, but do not specify which concentration was used for which experiment. Without this information and other unspecified aspect of their studies, interpreting subsequent experiments is difficult.
One potential advantage of this method is that it is a method to track endogenous lipids in live cells, however the authors show the NBD-16:0 transporting to lipid species where palmitate is almost never measured. For example, the use of the transport of NBD-16:0 to CL as evidence this is working. However natural cardiolipins are almost completely devoid of 16:0. In mammalian cells >80% of the fatty acids in CL is 18:2 with most of the remaining being 18:1 and 18:3. Similarly (assuming you are using sn-1 lPL-16:0), phospholipids with two 16:0 are extremely rare in mammalian cells with the exception of lung surfactant. Further, 16:0 composes <5% of cholesteryl esters in typical cells. The authors should be clearer about how this discrepancy between natural sorting of palmitate and the sorting of NBD-16:0 supports this as an accurate model of acyltransferase activity and intracellular transport.
The authors state that PA is primarily remodeled in the ER and transported to the mitochondria as a precursor to CLs (lines 108-111). This statement needs a source. In most studies I am aware of, the vast majority of both PA and 16:0 are primarily converted to TGs or PC/PE with only a small fraction going towards the CDP-DAG pathway required for CL biosynthesis. Are C2C12 cells unique in this regard? Does lPA stimulation specificity induce CL production? Does any of the NBD get into the TG or phospholipid fractions?
This study would be much stronger if another fluorescently labeled fatty acid was added. A comparison of the sorting of 20:4 and 16:0 would be very informative. This is especially true if the studies were done in the context of a known acyltransferase, for example LPCAT3.
This study would also be strengthened by an orthogonal technique showing similar sorting. For example, separation of the organelles and measurement of labeled fatty acids by MS or nano-SIM analysis would greatly strengthen these studies.
In figure 1A, the authors draw a schematic with an sn-2 lyso-PL in the figure. Sn-2 lyso-PLs as labile and will acyl migrate to the sn-1 position without careful handling of the PL in a basic solution. The authors make no mention of this type of handling in the method section. This figure should either be corrected or more details of how they handled their lysophospholipids should be provided.
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Reviewer #1:
My general assessment of this work is that it is full of good ideas and presents a novel and general approach to examine lipid remodeling in cells and perhaps subsequent transport of lipids, mainly to mitochondria, but it lacks the scientific rigor necessary to be fully confident that their conclusions firmly support their claims. Often, insufficient information about the methods are provided and the manuscript is hard to follow critically.
More specific comments:
I am surprised that acyl-CoAs are transported into cells. I don't know of any precedent for this. Usually fatty acids are imported into cells and then converted to acyl-CoAs as part of the mechanism of import. Could it be that the acyl--CoAs are hydrolysed before uptake only to be reformed inside the cells? I would suggest feeding the NBD-palmitate plus the …
Reviewer #1:
My general assessment of this work is that it is full of good ideas and presents a novel and general approach to examine lipid remodeling in cells and perhaps subsequent transport of lipids, mainly to mitochondria, but it lacks the scientific rigor necessary to be fully confident that their conclusions firmly support their claims. Often, insufficient information about the methods are provided and the manuscript is hard to follow critically.
More specific comments:
I am surprised that acyl-CoAs are transported into cells. I don't know of any precedent for this. Usually fatty acids are imported into cells and then converted to acyl-CoAs as part of the mechanism of import. Could it be that the acyl--CoAs are hydrolysed before uptake only to be reformed inside the cells? I would suggest feeding the NBD-palmitate plus the lysolipids to the cells as a control to see whether this is the case.
In fig 1 as an example they choose a region to blow up. As one can see there is a large variation, even in the blowups of mitochondrial labeling and if one looks at the originals the variation is confirmed. How have they chosen these areas? Furthermore, in figure 1 there is quite a bit of label with MLCL outside of the mitochondria, in particular in regions that they did not choose to blow up. What are these structures? Remodeling of MLCL is thought to take place in mitochondria.
They speak of transport of lipids from ER to mitochondria, but in fact the demonstration of this is very weak from what they show in the time course in supp fig 1. I am also disturbed by the difference in patterns of the NBD-PA patterns in a and b. They should be the same, but there are problems, maybe focus? I would say anyway that there is no clear evidence that the NBD PA first appears in the ER then goes to mitos. It could be synthesized in both compartments from their data.
The product characterization by TLC is insufficient. There are no standards, no characterization. Would they have seen the free NBD-palm by their methods?
When they use mutants and find less "transport" the mitochondrial signal as seen by mitotracker is always more diffuse. This indicates to me that there is another problem.
In fig 3 the fluorescent pictures do not correspond to what is seen in the quantification. There is more yellow in e than in h.
How did they add cholesterol at 50 or 100 micromolar? It is soluble at less than 1 micromolar in aqueous solution. The cholesterol experiments are puzzling. From what we know about StAR protein it recognizes cholesterol not esters. There is no precedent for cholesterol ester transport into mitochondria. Can they rule out that the esters are transported to the surface of the mitochondria and the NBD-Palm cleaved off and transported into the mitochondria?
The MAG and DAG experiments are overinterpreted. It could just be a kinetic problem since the MAG gets converted to DAG before TAG
They compare to externally added NBD lipids, but we don't know which ones they used. Are they using short chain NBD phospholipids. I could not find this in their manuscript. If they do not have the same NBD-palm in the sn-2 position then the comparison is meaningless.
The excitation and emission spectra of their probes are sometimes overlapping. How did they deal with this? Are they sure that they are not seeing FRET?
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Summary: Zhang et al. describe an interesting method to label newly synthesized lipids with fluorescent fatty acids and track their movement in cells. All reviewers agreed that this could potentially be a useful tool. However, they all raised concerns regarding the rigor of the characterization of this methodology.
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