Sphingomyelinase‐Mediated Multitimescale Clustering of Ganglioside GM1 in Heterogeneous Lipid Membranes

Lee, Hyun‐Ro; Choi, Siyoung Q.

doi:10.1002/advs.202101766

Cited by 5 publications

(4 citation statements)

References 89 publications

(143 reference statements)

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“…The RGG domain of LAF-1 forms liquid-like condensates on freestanding planar membranes Freestanding planar lipid membranes were created on the surface of the hexagonal holes of a transmission electron microscopy (TEM) grid, as described previously 19,20 (Supplementary Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…However, neither of these systems provides simultaneous access to both surfaces of the membrane. Therefore, we adapted a recently developed technique in which suspended planar lipid membranes are formed over the hexagonal holes of an electron microscopy grid 19,20 . Using this approach, we observed liquid-liquid phase separation (LLPS) simultaneously on both surfaces of a multiplexed planar membrane array.…”

Section: Introductionmentioning

confidence: 99%

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Lateral compression of lipids drives transbilayer coupling of liquid-like protein condensates

Lee

Park

Feng

et al. 2022

Preprint

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Liquid-liquid phase separation of proteins has recently been observed on the surfaces of biological membranes, where it plays a role in diverse cellular processes, from assembly of focal adhesions and the immunological synapse, to biogenesis of trafficking vesicles. Interestingly in each of these cases, proteins on both surfaces of the membrane are thought to participate, suggesting that protein phase separation could be coupled across the membrane. To explore this possibility, we used an array of freestanding planar lipid membranes to observe protein phase separation simultaneously on both surfaces of lipid bilayers. When proteins known to engage in phase separation bound to the surfaces of these membranes, two-dimensional, protein-rich phases rapidly emerged. These phases displayed the hallmarks of a liquid, coarsening over time by fusing and re-rounding. Interestingly, we observed that protein-rich domains on one side of the membrane colocalized with those on the other side, resulting in transbilayer coupling. How do liquid-like protein phases communicate across the lipid bilayer? Our results, based on lipid probe partitioning and the differential mobility of proteins and lipids, collectively suggest an entropic coupling mechanism, which relies on the ability of protein phase separation to locally reduce the entropy of the underlying lipid membrane, most likely by increasing lipid packing. Regions of reduced entropy then colocalize across the bilayer to minimize the overall free energy of the membrane. These findings suggest a previously unknown mechanism by which cellular signals originating from one side of the membrane, triggered by protein phase separation, can be transferred to the opposite side.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Lateral compression of lipids drives transbilayer coupling of liquid-like protein condensates

Lee

Park

Feng

et al. 2022

Preprint

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“…1 D-H). Others have found that SMase activity reduces the area of lipid rafts, and this reduction in area increases the density of smaller GM1 gangliosides [ 50 ]. Using the inverse pharmacologic approach of inhibiting nSMase activity, our data corroborated this finding.…”

Section: Discussionmentioning

confidence: 99%

Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo

Risner,

Ribeiro,

McGrady

et al. 2023

Cell Commun Signal

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Background Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC). Methods Here, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport. Results Following twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus. Conclusion Overall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs.

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“…Cholesterol is thereby displaced-at least partially-by ceramide and thus, the association of cholesterol with CAV1 is decreased. Lipid membranes were shown to generate two distinct types of condensed GM1 gangliosides (sialic acid-containing oligosaccharides covalently attached to ceramide lipids)-containing microdomains: rapidly formed but short-lived GM1 clusters that are formed in ceramide-rich domains and are predominantly involved in apoptosis, and long-lasting high-density GM1 condensations in the liquidordered phase, namely raft-like regions accounting for proliferation-promoting signaling (50,51). As a more permanent plasma membrane modification, the latter one might account for signalosome-amplified signaling events upon CAV1 being present within the plasma membrane.…”

Section: The Impact Of a Proper Cav Localization And Function For The Rt Response Of Malignant Prostate Epithelial Cellsmentioning

confidence: 99%

Stromal Fibroblasts Counteract the Caveolin-1-Dependent Radiation Response of LNCaP Prostate Carcinoma Cells

et al. 2022

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In prostate cancer (PCa), a characteristic stromal–epithelial redistribution of the membrane protein caveolin 1 (CAV1) occurs upon tumor progression, where a gain of CAV1 in the malignant epithelial cells is accompanied by a loss of CAV1 in the tumor stroma, both facts that were correlated with higher Gleason scores, poor prognosis, and pronounced resistance to therapy particularly to radiotherapy (RT). However, it needs to be clarified whether inhibiting the CAV1 gain in the malignant prostate epithelium or limiting the loss of stromal CAV1 would be the better choice for improving PCa therapy, particularly for improving the response to RT; or whether ideally both processes need to be targeted. Concerning the first assumption, we investigated the RT response of LNCaP PCa cells following overexpression of different CAV1 mutants. While CAV1 overexpression generally caused an increased epithelial-to-mesenchymal phenotype in respective LNCaP cells, effects that were accompanied by increasing levels of the 5′-AMP-activated protein kinase (AMPK), a master regulator of cellular homeostasis, only wildtype CAV1 was able to increase the three-dimensional growth of LNCaP spheroids, particularly following RT. Both effects could be limited by an additional treatment with the SRC inhibitor dasatinib, finally resulting in radiosensitization. Using co-cultured (CAV1-expressing) fibroblasts as an approximation to the in vivo situation of early PCa it could be revealed that RT itself caused an activated, more tumor-promoting phenotype of stromal fibroblats with an increased an increased metabolic potential, that could not be limited by combined dasatinib treatment. Thus, targeting fibroblasts and/or limiting fibroblast activation, potentially by limiting the loss of stromal CAV1 seems to be absolute for inhibiting the resistance-promoting CAV1-dependent signals of the tumor stroma.

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Sphingomyelinase‐Mediated Multitimescale Clustering of Ganglioside GM1 in Heterogeneous Lipid Membranes

Cited by 5 publications

References 89 publications

Lateral compression of lipids drives transbilayer coupling of liquid-like protein condensates

Lateral compression of lipids drives transbilayer coupling of liquid-like protein condensates

Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo

Stromal Fibroblasts Counteract the Caveolin-1-Dependent Radiation Response of LNCaP Prostate Carcinoma Cells

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