Evaluation of the available cholesterol concentration in the inner leaflet of the plasma membrane of mammalian cells

Abstract

Cholesterol is an essential component of the mammalian plasma membrane involved in diverse cellular processes. Our recent quantitative imaging analysis using ratiometric cholesterol sensors showed that the available cholesterol concentration in the inner leaflet of the plasma membrane (IPM) is low in unstimulated cells and increased in a stimulus-specific manner to trigger cell signaling events. However, the transbilayer distribution of cholesterol in the plasma membrane of mammalian cells remains controversial. Here we report a systematic and rigorous evaluation of basal IPM cholesterol levels in a wide range of mammalian cells with different properties employing cholesterol sensors derived from the D4 domain of the Perfringolysin O toxin and a sterol-transfer protein, Osh4. Results consistently showed that, although basal IPM cholesterol levels vary significantly among cells, they remain significantly lower than cholesterol levels in the outer leaflets. We found that IPM cholesterol levels were particularly low in all tested primary cells. These results support the universality of the low basal IPM cholesterol concentration under physiological conditions. We also report here the presence of sequestered IPM cholesterol pools, which may become available to cytosolic proteins under certain physiological conditions. We hypothesize that these pools may partly account for the low basal level of available IPM cholesterol. In conclusion, we provide new experimental data that confirm the asymmetric transbilayer distribution of the plasma membrane cholesterol, which may contribute to regulation of various cellular signaling processes at the plasma membrane.

Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes

“…Given these considerations, we propose that the outer leaflet pool of cholesterol, especially in the SM-rich ciliary membrane ( Kinnebrew et al, 2019 ), is a more plausible target of regulation by PTCH1 to control SMO activity. We emphasize that our model for PTCH1 function does not depend on (and does not inform) the outcome of current debates around the distribution of cholesterol between the inner and outer leaflets of the plasma membrane ( Buwaneka et al, 2021 ; Courtney et al, 2018 ; Liu et al, 2017 ; Steck and Lange, 2018 ).…”

“…These modified PFOD4 probes do not differentiate between accessible and inaccessible pools of cholesterol since their binding to cholesterol-containing vesicles is insensitive to the presence of SM or other phospholipids ( Liu et al, 2017 ). To avoid the complications associated with using these probes to measure membrane cholesterol accessibility ( Buwaneka et al, 2021 ; Courtney et al, 2018 ; Steck and Lange, 2018 ), we sought to measure outer leaflet accessible cholesterol using a completely independent method. Our TIRFM assay uses the TdTomato-PFOD4H probe to simply monitor the cholesterol content of the inner leaflet; cholesterol accessibility in the outer leaflet is assessed by the rate of cholesterol transfer from the membrane to MβCD.…”

ABCA1 causes an asymmetric cholesterol distribution to regulate intracellular cholesterol homeostasis

“…Furthermore, a decrease in the IPM cholesterol level was also examined by TIRF microscopy using PFO-D4H, which has a higher affinity for cholesterol than wild-type PFO-D4 (Maekawa and Fairn, 2015). To observe a change in the IPM cholesterol level, we established HeLa cells expressing a proper level of GFP-D4H, because the expression level of the cholesterol probe greatly affects its translocation to the PM (Buwaneka et al, 2021). When the cells were transiently transfected with ABCA1-mCherry and cultured in the presence of PSC-833, GFP-D4H gradually dissociated from the PM of the cells expressing ABCA1-mCherry after removing PSC-833 to exert ABCA1 activity, but in other cells without ABCA1 expression, GFP-D4H stayed at the PM (Figure 3b).…”

“…However, very recently, Cho's group showed that the IPM cholesterol level is much lower than the OPM cholesterol level by quantitative imaging analysis using two different types of ratiometric cholesterol sensors that are not appreciably affected by changes in the lipid environmental (Buwaneka et al, 2021). Some IPM cholesterol molecules are sequestered by membrane proteins such as Caveolin-1.…”

“…On the other hand, we recently reported that ABCA1 not only exports cholesterol, but also translocates (flops) it from the inner (IPM) to the outer leaflet of the PM (OPM) (Liu et al, 2017;Ogasawara et al, 2019;Okamoto et al, 2020). IPM cholesterol is maintained at around 3 mol% in various cell lines, while OPM cholesterol is 30~50 mol% (Buwaneka et al, 2021). This asymmetric cholesterol distribution allows cholesterol to function as an intramembrane signaling molecule (Liu et al, 2017;Ogasawara et al, 2020), but its physiological importance is not fully understood.…”