Intracellular Sterol Binding Proteins: Cholesterol Transport and Membrane Domains

Atshaves

et al. 2008

Lipids

Self Cite

Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent of dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular sterol trafficking in living organisms. DHE is a naturally-occurring, fluorescent sterol analog that faithfully mimics many of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins, is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock solution to cell culture media is effective, but this process forms an aqueous dispersion of dehydroergosterol crystals which can result in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques can deliver large quantities of monomeric dehydroergosterol with significant distribution into the plasma membrane. The properties and behavior of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging in living cells indicate that this naturally-occurring fluorescent sterol is a useful mimic for probing the properties of cholesterol in these systems. Keywordsdehydroergosterol; cholesterol; ergosterol; fluorescent sterols; microscopy Historical PerspectiveIn order to resolve the dynamics and factors governing cholesterol trafficking within cells, it is important to recognize that the cholesterol molecule has very few polar constituents (Fig. 1A). Consequently, cholesterol is poorly soluble in aqueous environments such as cytosol as evidenced by critical micellar concentrations of cholesterol and other sterols being very low, *To whom correspondence should be addressed: Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 862-1433, FAX: (979) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript near 20-30 nM (1-5). The physiological impact of cholesterol's low aqueous solubility is that spontaneous cholesterol desorption from the cytofacial leaflet of the plasma membrane or lysosomal membrane into the cytosol for transfer to intracellular sites for esterification, oxidation, or secretion is extremely slow (t 1/2 3 hours-days) (6-9). Therefore, it is important to resolve factors...

Section: Physiological Significance Of In Vitro Studies With Dhe and mentioning

confidence: 65%

Section: Summary and Discussionmentioning

confidence: 99%

Section: Plasma Membrane Sterol-rich Microdomains: Real-time Imaging mentioning

confidence: 99%

Section: Advent Of Fluorescent Sterolsmentioning

confidence: 99%

Section: Plasma Membrane Sterol-rich Microdomains: In Vitro Studiesmentioning

confidence: 99%

See 3 more Smart Citations

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Atshaves

et al. 2008

Lipids

Self Cite

Multiphoton Laser-Scanning Microscopy and Spatial Analysis of Dehydroergosterol Distributions on Plasma Membrane of Living Cells

Atshaves

Methods in Molecular Biology

et al. 2007

Multiphoton laser-scanning microscopy (MPLSM) imaging in combination with advanced image analysis techniques provides unique opportunities to visualize the arrangement of cholesterol in the plasma membrane (PM) of living cells. MPLSM makes possible the use of a naturally occurring sterol, dehydroergosterol (DHE), for observing sterol-enriched areas of the PM. Pure DHE has properties similar to cholesterol as observed in model and cellular membranes but with a conjugated double-bond system that fluoresces at ultraviolet wavelengths. MPLSM enables the excitation of DHE at infrared wavelengths that many laser-scanning microscopy systems are able to transmit effectively and that are less harmful to the cell. Thus, with the incorporation of DHE into living cells and the advent of MPLSM, real-time images of the cellular distribution of DHE can be obtained. In juxtaposition, notably the application of newly advanced techniques in image analysis, aids not only the identification and segmentation of sterol-rich regions of the PM of cells, but also the elucidation of the statistical nature of the observed patterns. In studies involving murine L-cell (Larpt-+K-) fibroblasts, DHE is shown to exhibit strong cluster patterns within the PM.

Caveolin, Sterol Carrier Protein-2, Membrane Cholesterol-Rich Microdomains and Intracellular Cholesterol Trafficking

Schroeder

Cholesterol Binding and Cholesterol Transport Proteins:

et al. 2010

While the existence of membrane lateral microdomains has been known for over 30 years, interest in these structures accelerated in the past decade due to the discovery that cholesterol-rich microdomains serve important biological functions. It is increasingly appreciated that cholesterol-rich microdomains in the plasma membranes of eukaryotic cells represent an organizing nexus for multiple cellular proteins involved in transmembrane nutrient uptake (cholesterol, fatty acid, glucose, etc.), cell-signaling, immune recognition, pathogen entry, and many other roles. Despite these advances, however, relatively little is known regarding the organization of cholesterol itself in these plasma membrane microdomains. Although a variety of non-sterol markers indicate the presence of microdomains in the plasma membranes of living cells, none of these studies have demonstrated that cholesterol is enriched in these microdomains in living cells. Further, the role of cholesterol-rich membrane microdomains as targets for intracellular cholesterol trafficking proteins such as sterol carrier protein-2 (SCP-2) that facilitate cholesterol uptake and transcellular transport for targeting storage (cholesterol esters) or efflux is only beginning to be understood. Herein, we summarize the background as well as recent progress in this field that has advanced our understanding of these issues.