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SNARE-mediated membrane fusion proceeds via the formation of a fusion pore. This intermediate structure is highly dynamic and can flicker between open and closed states. In cells, cholesterol has been reported to affect SNARE-mediated exocytosis and fusion pore dynamics. Here, we address the question of whether cholesterol directly affects the flickering rate of reconstituted fusion pores in vitro. These experiments were enabled by the recent development of a nanodisc⋅black lipid membrane recording system that monitors dynamic transitions between the open and closed states of nascent recombinant pores with submillisecond time resolution. The fusion pores formed between nanodiscs that bore the vesicular SNARE synaptobrevin 2 and black lipid membranes that harbored the target membrane SNAREs syntaxin 1A and SNAP-25B were markedly affected by cholesterol. These effects include strong reductions in flickering out of the open state, resulting in a significant increase in the open dwell-time. We attributed these effects to the known role of cholesterol in altering the elastic properties of lipid bilayers because manipulation of phospholipids to increase membrane stiffness mirrored the effects of cholesterol. In contrast to the observed effects on pore kinetics, cholesterol had no effect on the current that passed through individual pores and, hence, did not affect pore size. In conclusion, our results show that cholesterol dramatically stabilizes fusion pores in the open state by increasing membrane bending rigidity.

mTORC1 is known to activate sterol regulatory element-binding proteins (SREBPs) including SREBP-2, a master regulator of cholesterol synthesis. Through incompletely understood mechanisms, activated mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi where SREBP-2 is cleaved to translocate to the nucleus and activate gene expression for cholesterol synthesis. Low ER cholesterol is a well-established trigger for SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing cholesterol delivery to the ER. We report here that mTORC1 activation is accompanied by low ER cholesterol and an increase of SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with a rise in ER cholesterol and a decrease in SERBP-2 activity. This rise in ER cholesterol is of lysosomal origin: blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from increasing and, consequently, SREBP-2 is activated without mTORC1 activation. Furthermore, when mTORC1 activity is low, cholesterol is delivered to lysosomes through two membrane trafficking pathways: autophagy and rerouting of endosomes to lysosomes. Indeed, with dual blockade of both pathways by Atg5−/− and dominant-negative rab5, ER cholesterol fails to increase when mTORC1 activity is low, and SREBP-2 is activated. Conversely, overexpressing constitutively active Atg7, which forces autophagy and raises ER cholesterol even when mTORC1 activity is high, suppresses SREBP-2 activation. We conclude that mTORC1 actively suppresses autophagy and maintains endosomal recycling, thereby preventing endosomes and autophagosomes from reaching lysosomes. This results in a reduction of cholesterol in the ER and activation of SREBP-2.

The plasma membrane in mammalian cells is rich in cholesterol, but how the cholesterol is partitioned between the two leaflets of the plasma membrane remains a matter of debate. Recently, Liu et al. used domain 4 (D4) of perfringolysin O as a cholesterol sensor to argue that cholesterol is mostly in the exofacial leaflet (Liu et al., 2017). This conclusion was made by interpreting D4 binding in live cells using in vitro calibrations with liposomes. However, liposomes may be unfaithful in mimicking the plasma membrane, as we demonstrate here. Also, D4 binding is highly sensitive to the presence of cytosolic proteins. In addition, we find that a D4 variant, which requires >35 mol% cholesterol to bind to liposomes in vitro, does in fact bind to the cytoplasmic leaflet of the plasma membrane in a cholesterol-dependent manner. Thus, we believe, based on the current evidence, that it is unlikely that there is a significantly higher proportion of cholesterol in the exofacial leaflet of the plasma membrane compared to the cytosolic leaflet.

ATP-binding cassette transporter A1 (ABCA1) plays an essential role in mediating cholesterol efflux to apolipoprotein A-I (apoA-I), a major housekeeping mechanism for cellular cholesterol homeostasis. After initial engagement with ABCA1, apoA-I directly interacts with the plasma membrane to acquire cholesterol. This apoA-I lipidation process is also known to require cellular signaling processes, presumably to support cholesterol trafficking to the plasma membrane. We report here that one of major signaling pathways in mammalian cells, Akt, is also involved. In several cell models that express ABCA1 including macrophages, pancreatic beta cells and hepatocytes, inhibition of Akt increases cholesterol efflux to apoA-I. Importantly, Akt inhibition has little effect on cells expressing non-functional mutant of ABCA1, implicating a specific role of Akt in ABCA1 function. Furthermore, we provide evidence that mTORC1, a major downstream target of Akt, is also a negative regulator of cholesterol efflux. In cells where mTORC1 is constitutively activated due to tuberous sclerosis complex 2 deletion, cholesterol efflux to apoA-I is no longer sensitive to Akt activity. This suggests that Akt suppresses cholesterol efflux through mTORC1 activation. Indeed, inhibition of mTORC1 by rapamycin or Torin-1 promotes cholesterol efflux. On the other hand, autophagy, one of the major pathways of cholesterol trafficking, is increased upon Akt inhibition. Furthermore, Akt inhibition disrupts lipid rafts, which is known to promote cholesterol efflux to apoA-I. We therefore conclude that Akt, through its downstream targets, mTORC1 and hence autophagy, negatively regulates cholesterol efflux to apoA-I.

Phospholipids and proteins in the plasma membrane (PM) bilayer are well established to be both laterally and transversely asymmetric. However, cholesterol transbilayer distribution within PM remains inconclusive. A fluorescent sterol, dehydroergosterol (DHE), is well-documented to primarily reside in the inner leaflet of PM (∼80%). It remains unclear if native cholesterol shares this surprising asymmetry.The purpose of this study is to first determine cholesterol transbilayer distribution in PM. We developed a protocol that is capable of analyzing cholesterol in a leaflet-specific manner using β-cyclodextrin (βCD). In symmetric large unilamellar vesicles (LUVs), we found that cholesterol flip-flop is rapid at 37°C, leading to 100% extraction/exchange by βCD. However, at 0°C, βCD is only able to remove exactly 50% cholesterol, indicating a complete inhibition of cholesterol flip-flop. We then applied this protocol to erythrocytes and found that only 20-25% cholesterol is accessible by βCD at 0°C, although 100% is accessible at 37°C. Therefore, most cholesterol resides in the inner leaflet of PM in mammalian cells.We then investigated the role of phospholipid transbilayer asymmetry on cholesterol asymmetry. We found that, only in the asymmetric LUVs with long chain (22 carbon) sphingomyelin in the outer leaflet, could we observe cholesterol enrichment in the inner leaflet. Similar experiments with short chain (16 carbon) sphingomyelin and phosphatidylcholine (16:0/18:1) failed to influence cholesterol distribution.We therefore conclude that, like DHE, cholesterol is enriched in the inner leaflet of PM and that this asymmetry is regulated by the phospholipid asymmetry and, more specifically, by long chain sphingolipids. We suggest that the current lipid raft model may need to be revised to reflect this cholesterol asymmetry.

Lateral membrane microdomains, or lipid rafts, are considered fundamental to a wide range of biological processes. The association of cholesterol and sphingolipids promotes transient, nanoscopic protein-protein interactions that enhance membrane signal transduction. In giant unilamellar vesicles (GUVs) or giant plasma membrane vesicles (GPMVs), these microdomains are readily resolvable by optical microscopy. However, similar visible microdomains have not been observed in living cells, which has been a long-standing mystery. A unique feature of the native plasma membrane is the high degree of transbilayer lipid asymmetry and, particularly, with long acyl chain sphingolipids exclusively in the outer leaflet. The impact of asymmetry on optically resolvable microdomains is not known, nor is the role of long acyl chain sphingomyelin (SM). Here, we show that GUVs fail to form visible microdomains if long acyl chain SM is exclusively in the outer leaflet but, if long acyl chain SM is placed in both leaflets, GUVs readily produce visible microdomains. With a novel methodology capable of analyzing cholesterol in the individual leaflets, we obtained evidence that long acyl chain SM, not short acyl chain SM, drives cholesterol into the inner-leaflet of large unilamellar vesicles. This depletes cholesterol from the outer leaflet, thereby providing a mechanism for abolishing visible microdomains. We finally confirm that, in native plasma membrane - human erythrocytes, the majority of cholesterol is indeed in the inner leaflet. We conclude that native plasma membrane cannot form visible microdomains, primarily due to lack of cholesterol in the outer leaflet as the consequence of exofacial long acyl SM. Our findings therefore resolve the long-standing puzzle and uncover a novel fundamental organization principle for the plasma membrane.