Pathways and Mechanisms of Cellular Cholesterol Efflux-Insight From Imaging

Abstract

Cholesterol is an essential molecule in cellular membranes, but too much cholesterol can be toxic. Therefore, mammalian cells have developed complex mechanisms to remove excess cholesterol. In this review article, we discuss what is known about such efflux pathways including a discussion of reverse cholesterol transport and formation of high-density lipoprotein, the function of ABC transporters and other sterol efflux proteins, and we highlight their role in human diseases. Attention is paid to the biophysical principles governing efflux of sterols from cells. We also discuss recent evidence for cholesterol efflux by the release of exosomes, microvesicles, and migrasomes. The role of the endo-lysosomal network, lipophagy, and selected lysosomal transporters, such as Niemann Pick type C proteins in cholesterol export from cells is elucidated. Since oxysterols are important regulators of cellular cholesterol efflux, their formation, trafficking, and secretion are described briefly. In addition to discussing results obtained with traditional biochemical methods, focus is on studies that use established and novel bioimaging approaches to obtain insight into cholesterol efflux pathways, including fluorescence and electron microscopy, atomic force microscopy, X-ray tomography as well as mass spectrometry imaging.

Keywords: ABC transporter; HDL; LDL; apoprotein A1 (Apo A1); cholesterol; extracellular vesicles; niemann pick disease; oxysterol.

FIGURE 1

Regulation of cholesterol synthesis and uptake - an overview. LDL binds to the LDL receptor (LDLR) at the cell surface and becomes internalized by clathrin-mediated endocytosis. Upon arrival in the sorting endosomes (SE), LDL dissociates from the LDLR and is retained in the lumen of SE which gradually mature into late endosomes (LE). The LDLR recycles back to the cell surface, either directly from the SE or by passing through the endocytic recycling compartment (ERC). The maturation process of SE into LE, and the further conversion of LE into lysosomes (LYS), involves the formation of internal vesicles in LE, acquisition of LBPA, a drop in pH, and import of proteases, lipases, and other lysosomal enzymes from the Golgi. When the cell is rich in cholesterol, SREBP will be retained in the ER by SCAP and Insig. Cholesterol will bind to SCAP, whereas oxysterol will bind to Insig (green arrows). LXR will be activated by oxysterols, which together with RXR can induce the transcription of genes involved in protecting the cells from becoming overloaded with cholesterol. During low cholesterol conditions, the SCAP-SREBP complex will be released from Insig in the ER and move to the Golgi for cleavage and activation (nSREBP). In the nucleus, nSREBP can activate the transcription of genes involved in cholesterol biosynthesis and uptake. The expression of PCSK9, which promotes the degradation of the LDL-receptor by interfering with its recycling, is also under the control of the nSREBP system. Intracellular degradation of LDLR is promoted by the E3 ubiquitin ligase IDOL, whose expression is stimulated by LXR under conditions of high cellular cholesterol. See the text for more details. ABAC1 (ATP-binding cassette A1), Chol (cholesterol), ER (endoplasmic reticulum), ERC (endocytic recycling compartment), HMG-CoA (3-hydroxy-3-methylglutaryl-Coenzyme A), IDOL (inducible degrader of the LDL receptor), LDL (low-density lipoprotein), LDLR (LDL-receptor), LXR (liver-X receptor), PCSK9 (proprotein convertase substilisin/kexin type 9), RXR (retinoid X receptor) SCAP (SREBP-Cleavage-Activating protein), SE (sorting endosomes), SREBP (sterol regulatory element-binding protein), Ub (ubiquitin).

FIGURE 2

Passive and active protein-mediated cholesterol efflux from mammalian cells. (A) passive efflux does not require hydrolysis of ATP but depends on the difference of chemical potentials of cholesterol in the acceptor, here HDL $\left({\mu }_{Chol}^{HDL}\right)$ and PM $\left({\mu }_{Chol}^{PM}\right)$ . Cholesterol efflux requires, that ${\mu }_{Chol}^{HDL}$ < ${\mu }_{Chol}^{PM}$ , and is shown here for scavenger receptor BI (SR-BI). (B) Active efflux, illustrated here for the ABC transporter ABCG1, depends on the binding of two ATP molecules, which stabilize an outward-open conformation for efflux of cholesterol and phospholipids to e.g., HDL. ATP-hydrolysis causes the transition of the protein two an outward-closed conformation, allowing for entrance of new substrate on the cytosolic side of the PM. The full cycle is more complex and involves at least four steps; see (Skarda et al., 2021).

FIGURE 3

Visualization of extracellular vesicles and particles during cholesterol efflux. (A) Spinning disk microscopy image of extracellular vesicles from a NPC2-deficient fibroblasts treated with NPC2 protein, tagged with a red Alexa546 fluorophore (red), and labeled additionally with TopFluor-cholesterol (green) (Juhl et al., 2021). (B) Confocal microscopy image of migrasomes from L929 cell transfected with TSPAN4-GFP (Chen et al., 2018). Scalebar 10 µm. (C) SEM of nanoSIMS of macrophage after incubation with [¹⁵N]ALO-D4. Scalebar 2 µm (He et al., 2018). (D,D′,D′′) examples of extracellular vesicles from NPC2 deficient and healthy fibroblasts imaged with cryo-SXT. Scalebar 0.5 µm for (D) and 0.15 µm in (D′,D′′) (Juhl et al., 2021). Red arrows point to microvesicles.

FIGURE 4

Summary of discussed cholesterol efflux mechanisms. ABCA1 can efflux cholesterol to ApoA1 as the main sterol acceptor. Passive cholesterol exchange between the cell and HDL is mediated by SR-BI. HDL can also gain cholesterol upon endocytosis, passage through endosomes and recycling back to the PM. Additionally, cholesterol might leave the cell by the release of exosomes, upon LE/LYSs fusion with the plasma membrane, or by shedding as microvesicles and/or as migrasomes during cell migration. The LE/LYSs are drawn in different sizes to show the proteins and intraluminal organelles and not to illustrate different populations. ABAC1 (ATP-binding cassette A1), ApoA1 (apolipoprotein A1), EE (early endosomes), HDL (high density lipoprotein), ILV (intraluminal vesicle), LBPA (lysobisphosphatidic acid), LE/LYS (late endosome/lysosome), SR-BI (scavenger receptor BI).

FIGURE 5

Uptake and efflux of fluorescent oxysterols in human fibroblasts. (A) uptake and efflux of cholestatrienol (CTL) and 27-hydroxycholestatrienol (27-OH-CTL) in human fibroblasts studied by UV-sensitive fluorescence microscopy. (B) structures of cholesterol and its fluorescent analogue CTL. (C) 27-hydroxycholesterol and its fluorescent analogue 27-OH-CTL. Extra double bonds in the fluorescent analogs are the only modifications compared to the natural sterols and are shown in blue. Figure adapted and reproduced from (Szomek et al., 2020) with permission.