Molecular Discrimination between Two Conformations of Sphingomyelin in Plasma Membranes

Endapally, Shreya; Frias, Donna; Grzemska, Magdalena; Tomchick, Diana R.; Radhakrishnan, Arun

doi:10.1016/j.cell.2018.12.042

Cited by 116 publications

(205 citation statements)

References 60 publications

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“…Moreover, our findings are crucial for membrane biology and naturally create new questions; how can cholesterol contribute to high order membrane domain formation with sphingomyelin if their diffusion dynamics are so different? Are the fast and slow components of SM and Chol different and related to recently reported different forms of SM 57 ? Do interactions occur between the fast SM and slow cholesterol which have similar diffusion dynamics?…”

Section: Resultsmentioning

confidence: 93%

Nanoscale dynamics of cholesterol in the cell membrane

Pinkwart

Schneider

Lukoseviciute

et al. 2019

Preprint

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Cholesterol constitutes approximately 30-40% of the mammalian plasma membranea larger fraction than any other single component. It is a major player in numerous signalling processes as well as molecular membrane architecture. However, our knowledge on dynamics of cholesterol in the plasma membrane is limited which restricts our understanding of the mechanisms regulating its involvement in cell signalling. Here, advanced fluorescence imaging and spectroscopy approaches were applied on in vitro (model membranes) and in vivo (live cells and embryos) membranes to systematically study the nanoscale dynamics of cholesterol in biological membranes. The results show that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. The data indicate that diffusion of cholesterol and phospholipids is not correlated with membrane domain partitioning. Instead, our data show that the fast diffusion of cholesterol is due to its nanoscale interactions and localization in the membrane.

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Section: Resultsmentioning

confidence: 93%

Nanoscale dynamics of cholesterol in the cell membrane

Pinkwart

Schneider

Lukoseviciute

et al. 2019

Preprint

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“…The existence of cholesterol-sphingomyelin complexes in cell membranes has been demonstrated previously [41][42][43] . It is expected that the organization and dynamics of the molecules forming these nanoscale assemblies would differ from that of PC which is ubiquitously present in the cell membrane.…”

Section: Leaflet Specific Analysis Of Sphingomyelin Fluorescent Analomentioning

confidence: 85%

“…They consist of a ceramide backbone and phosphocholine headgroup exhibiting a narrower cylindrical geometry than phosphatidylcholine and a phase transition above room temperature and thus, exist in the gel phase at room temperature. Sphingomyelins interact with cholesterol to form lipid domains in the membranes, but can also exist freely 41,44,45 . They are predominantly present in the outer leaflet; however, there is some evidence suggesting a pool of SM localized in the inner leaflet 46,47 .…”

Section: Leaflet Specific Analysis Of Sphingomyelin Fluorescent Analomentioning

confidence: 99%

Analysing plasma membrane asymmetry of lipid organisation by fluorescence lifetime and correlation spectroscopy

Gupta

Korte

Herrmann

et al. 2019

Preprint

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Asymmetric organization of plasma membrane lipids 2 ABSTRACT A fundamental feature of a eukaryotic cell membrane is the asymmetric arrangement of lipids in the two leaflets. A cell invests significant energy to maintain this asymmetry and utilizes it to regulate important biological processes such as apoptosis and vesiculation. Here, we employ fluorescence lifetime imaging microscopy (FLIM) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the exofacial and cytoplasmic leaflet of live mammalian cells. We characterize the biophysical properties of fluorescent analogues of phosphatidylcholine (PC), sphingomyelin (SM) and phosphatidylserine (PS) in two mammalian cell membranes. Due to their specific transverse membrane distribution, these probes allow leaflet specific investigation of the plasma membrane. We compare the results with regard to the different temporal and spatial resolution of the methods. Fluorescence lifetimes of fluorescent lipid analogues were found to be in a characteristic range for the liquid ordered phase in the outer leaflet and liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet is supported by free diffusion in the inner leaflet with high average diffusion coefficients. The liquid ordered phase in the outer leaflet is accompanied by slower diffusion and diffusion with intermittent transient trapping.Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogues provides a powerful tool to investigate lateral and trans-bilayer characteristics of plasma membrane in live cells.

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“…The results of our unbiased screen for lipid-related genes that influence the strength of HH signaling uncovered two pathways--cholesterol and SM synthesis--that both converge on accessible cholesterol as the critical species that regulates the interaction between PTCH1 and SMO. The potentiating effect of SM depletion on HH signaling points to cholesterol itself as the regulatory sterol, since side-chain oxysterols do not form analogous complexes with SM (and the lipid probes used in our studies do not interact with oxysterols) (Endapally et al 2019;Bielska et al 2012;Gale et al 2009 (Infante and Radhakrishnan 2017) . Confining the effects of HH ligands on cholesterol accessibility to the ciliary membrane, rather than the plasma membrane, is required to prevent crosstalk with this important homeostatic pathway in the cell.…”

Section: Discussionmentioning

confidence: 85%

“…Myriocin is a fungal antibiotic that potently inhibits SPTLC2 ( Fig.3A ) and is commonly used to deplete SM in cells (Courtney et al 2018;Tafesse et al 2013Tafesse et al , 2015 . SM depletion by myriocin in NIH/3T3 cells was confirmed using both thin-layer chromatography ( Supplementary Fig.3A ) and flow cytometry of intact cells stained with a fluorescent protein probe (OlyA_E69A) that binds to total SM on the outer leaflet of the plasma membrane ( Supplementary Fig.3B ) (Endapally et al 2019) . Myriocin treatment markedly potentiated the response to SHH in NIH/3T3 cells, as measured by the transcriptional induction of Gli1 ( Fig.3B ).…”

Section: Cellular Sphingomyelin Suppresses Hedgehog Signalingmentioning

confidence: 77%

Sphingomyelin suppresses Hedgehog signaling by restricting cholesterol accessibility at the ciliary membrane

Kinnebrew

Iverson

Patel

et al. 2019

Preprint

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Transmission of the Hedgehog signal across the plasma membrane by Smoothened is proposed to be triggered by its direct interaction with cholesterol. But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find here that Smoothened activation and Hedgehog signaling are driven by a biochemically defined fraction of membrane cholesterol, termed accessible cholesterol. Increasing accessible cholesterol levels by depletion of sphingomyelin, which sequesters cholesterol in complexes, potentiates Hedgehog signaling. By inactivating the transporter-like protein Patched 1, Hedgehog ligands trigger an increase in cholesterol accessibility in the ciliary membrane, the subcellular location for Smoothened signaling. Thus, compartmentalization of Hedgehog signaling in the primary cilium may allow cholesterol accessibility to be used as a second messenger to mediate the communication between Patched 1 and Smoothened, without causing collateral effects on other cellular processes. We thank Kyle Travaglini and Onn Brandman for help with the MATLAB code for automated quantitation of probe fluorescence at primary cilia, Xiaohui Zha and Kevin Courtney for helpful discussions and protocols for sphingomyelin assays, and Suzanne Pfeffer for comments on the manuscript. Author contributionsRR and AR designed the project. BP analyzed the screen data, with the exception of the KEGG analysis which was performed by MK. BP and GP designed and cloned the CRISPR library focused on lipid-related genes. BP, GP and JK generated the mutant cell library and performed the HiSHH-Bottom10% screen. MK performed the LoSHH-Top5% screen. MK performed the experiments related to cholesterol genes and MK and EJI performed experiments related to sphingomyelin genes. MK, GL and KAJ performed the lipid probe staining experiments. MK and BP designed the computational pipeline for probe quantitation at primary cilia. CS and DFC contributed key conceptual and structural insights and experimental suggestions. RR and MK wrote the paper, with input from all authors.

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Molecular Discrimination between Two Conformations of Sphingomyelin in Plasma Membranes

Cited by 116 publications

References 60 publications

Nanoscale dynamics of cholesterol in the cell membrane

Nanoscale dynamics of cholesterol in the cell membrane

Analysing plasma membrane asymmetry of lipid organisation by fluorescence lifetime and correlation spectroscopy

Sphingomyelin suppresses Hedgehog signaling by restricting cholesterol accessibility at the ciliary membrane

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