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  1. Evaluation Summary:

    Sphingomyelin synthase 2 (SMS2) is a Golgi-localized enzyme that synthesizes sphingomyelin, a critical lipid in the plasma membrane, and mutations in SMS2 underly a rare genetic disorder of bone formation. This study shows that the disease mutations cause retention of SMS2 in the ER, which leads to sphingomyelin being produced in the wrong place and thus to a disrupted sphingomyelin and cholesterol gradient in the membranes of the secretory pathway. Additional experiments would improve the impact of this study in explaining the underlying reasons for some bone development disorders and providing cell biologists with new tools to manipulate lipids in cells.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #3 (Public Review):

    Sokoya, Parolek et al. set out to investigate the pathobiochemistry of osteoporosis with calvarial doughnut lesions (OP-CDL), a bone disorder caused by mutations in the gene encoding for the lipid synthesis enzyme sphingomyelin synthase 2 (SMS2). The authors revealed that two of the most severe mutations lie in a region responsible for the export of the protein from the ER to the Golgi and the plasma membrane (PM). Using cells expressing such catalytically active, but mislocalized SMS2 variants as models and later confirming their findings in fibroblasts derived from patients, the authors characterized the effects of producing sphingomyelin (SM) at the wrong organelle. Both by technically impressive organellar lipidomics and by the use of genetically encoded biosensors for SM, the authors convincingly demonstrated that SMS2 variants accumulate large amounts of SM at the ER. Furthermore, this imbalance in an otherwise tightly controlled subcellular lipid distribution also affected the levels of other lipids (some expected, such as the direct substrates PC and Cer as well as its product DAG, and some unexpected, such as the signaling lipid ceramide-1-phosphate Cer1P) at the ER, but also at the PM. The authors focussed on the subcellular and intrabilayer distributions of SM as well as of cholesterol, two lipids with high affinity for each other, and similar contributions to biophysical properties of the membrane, such as membrane order. SM accumulation in the ER increased ER membrane order as expected, however, this was not accompanied by cholesterol accumulation in the ER. On the other hand, the resulting decrease of SM levels on the outer PM leaflet decreased membrane order and made the cells more susceptible to lipid extracting agents such as methyl-β-cyclodextrin, implying that while cholesterol levels at the ER are well controlled independently of SM, the PM-pool of cholesterol responds to changes in SM levels.

    Overall, this work is an important description of the effects of breaking lipid gradients. It is of high technical quality and makes use of state-of-the-art methods. The first part of the manuscript lays a solid foundation, detailing the mislocalization phenotype and the accompanying lipidome changes, which in itself will be a great resource for the community.
    It is astonishing that changes in the subcellular localization of one lipid can lead to such drastic changes in a collective membrane property in other organelles of the secretory pathway. An increase in ER membrane order, as well as a decrease in PM order, was convincingly shown using environmentally-sensitive reporters. This is important to consider, given that the distinct functions of subcellular organelle rely on different membrane properties (e.g. the barrier function of the PM requires high membrane order whereas the biosynthetic tasks of the ER require loose packing).
    The techniques employed were applied in different cells (engineered HeLa cells, osteosarcoma cells, patient-derived fibroblasts) and show the appropriateness of the used models to investigate processes relevant to the disease.

    The second part of the manuscript currently lacks coherence, especially regarding the connection between SM and Chol gradients. A clear hypothesis of how the respective (mis)localizations arise, would help with a better understanding of the lipid landscape in this disease.
    The use of cytosolic Equinatoxin to investigate SM localization has limitations, mainly that in the SMS2-mutant expressing cells it localizes to yet unidentified punctae in the cell. Given that the nature of these punctae could not be elucidated, artefacts stemming from the probe cannot be excluded. Overall, this hinders interpretation. Most importantly, the central claim - SM production in the ER leads to the breakdown of SM asymmetry in the PM and mislocalization of SM to the inner leaflet - could not be directly addressed.
    The authors focused on the interesting connection between SM and cholesterol. They showed that overall cholesterol levels do not change (neither in whole cells nor in the ER or PM isolates) in the SMS2-mutant cell lines. However, the cholesterol-binding protein D4H is strongly localized to endolysosomes in the disease model. This observation was not discussed, nor independently confirmed. In addition, the use of cholesterol-extracting molecules resulted in higher sensitivity of SMS2-mutant cells. In how far this is due to cholesterol or SM mislocalization or changes in their respective levels was not sufficiently explored and makes an overall assessment difficult.

  3. Reviewer #2 (Public Review):

    The authors have identified an ER export signal in sphingomyelin synthase (SMS) - this is defective in SMS mutants associated with a certain bone disease. Consequently, disease mutants of the enzyme remain in the ER, producing sphingomyelin locally instead of at the Golgi apparatus. This results in dysregulation of the sphingomyelin gradient through the secretory pathway, loss of the canonical asymmetric distribution of this lipid (localization exclusively to the exoplasmic side of the cell membrane) and causes a pronounced, secondary outcome: a change in cholesterol organization at the plasma membrane. These changes may explain the bone disease phenotype. More generally, the authors reveal new cell biology and introduce new tools to perturb lipid gradients in cells.

    This is a very interesting, technically excellent paper that offers new insights but stretches some of its conclusions.

    The key results are:
    1. Bone disease-linked SMS2 variants under study are ER-localized whereas their wild-type counterparts are located in the Golgi apparatus;
    2. SM levels in the ER are high (but surprisingly unaffected at the PM) in SMS-knockout cells expressing disease mutants, and, as a consequence of unspecific lipid scrambling activity in this organelle, SM is distributed "symmetrically" across the bilayer (in the wild-type situation, SM is locked into the exoplasmic/lumenal leaflet in the Golgi apparatus and plasma membrane);
    3. Cholesterol levels at the PM are the same in SMS-knockout cells expressing disease mutants versus wild-type cells, but the cholesterol is organized differently in the former as evinced by detection with a biosensor and susceptibility of the cells to cyclodextrin treatment. This is consistent with data obtained with membrane fluidity probes.

  4. Reviewer #1 (Public Review):

    Sphingomyelin (SM) is an abundant lipid of the plasma membrane and is synthesized from ceramide and PC by two enzymes, SMS1 and SMS2, with the former being in the Golgi and the latter in the plasma membrane. Mutations in SMS2 have been found to underly a rare genetic disorder of bone formation, and previous work from the authors has shown that in two cases this arises from autosomal dominant missense mutations in the N-terminal cytoplasmic tail of SMS2 (I62S and M64R). Their previous study reported that these mutations cause SMS2 to accumulate in the ER instead of the plasma membrane, with cells from the patients containing elevated levels of SM, suggesting that the lipid is being synthesized by the SMS2 in the ER.

    This new study extends this work by using both organelle fractionation and in vivo imaging to confirm that these mutations in SMS2 do indeed result in substantially elevated levels of SM in the ER. They also find changes in phospholipid desaturation in the ER which suggests a compensatory adjustment, but interestingly, levels of cholesterol in the ER do not appear to increase despite the known affinity of SM for cholesterol. SM is normally only found on the outer leaflet of membranes and so is not exposed to the cytoplasm. The authors use an in vivo probe for SM to argue that in the mutant cells SM is being present on the inner leaflet of both the ER and plasma membrane, and with a concomitant reduction in cholesterol levels, and lipid order, in the outer leaflet of the plasma membrane. These findings lead them to comment on the robustness of the intramembrane system to a major perturbation of lipid distribution and to speculate that these changes in lipid distribution may contribute to the bone deposition defects in the patients.

    Overall the data is clearly presented, well-controlled, and quantified. The cell biology of lipids in general, and SM in particular, is an interesting and important topic. The first part of the paper demonstrating the elevated levels of SM in the ER is very convincing with both fractionation and in vivo imaging showing clear changes in the mutants. The second part, using in vivo imaging with an SM reporter to show the appearance of SM in the cytosolic face of membranes in the mutant cells, is less convincing, and although there are clear differences, the authors admit that the nature of the difference is perplexing. Indeed there seems little direct evidence at present to support the conclusion that SM is being translocated to the cytoplasmic face of the ER. Thus, significant further work is required to support the conclusions on exposure of SM to the cytosolic face of membranes.