Cholesterol Reporter Molecules

Gimpl, Gerald; Gehrig‐Burger, Katja

doi:10.1007/s10540-007-9060-1

Cited by 99 publications

(88 citation statements)

References 153 publications

(185 reference statements)

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“…6b), suggesting that membrane cholesterol was required for the Gp140-dependent transmembrane phosphorylation. We also saw an increase in cholesterol resistance to detergents upon antibody ligation as seen with filipin, a compound from Streptomyces filipinensis that binds to membrane cholesterol [46] (Fig. 7).…”

Section: Evidence That Drm and Tem Components Are Required For Phosphmentioning

confidence: 78%

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

Alvares

Dunn

Brown

et al. 2008

Biochimica et Biophysica Acta (BBA) - General Subjects

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Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and DetergentResistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals-possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior.

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Section: Evidence That Drm and Tem Components Are Required For Phosphmentioning

confidence: 78%

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

Alvares

Dunn

Brown

et al. 2008

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…24 A newer probe for unesterified cholesterol, BC, shows a similar staining pattern. 25 For filipin labeling, sections were washed 2 ϫ 10 minutes in PBS and 2 ϫ 10 minutes in wash solution (0.02% saponin in PBS).…”

Section: Immunoperoxidase and Histochemical Proceduresmentioning

confidence: 93%

Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

Zhou

Davidson

McGlynn

et al. 2011

The American Journal of Pathology

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Niemann-Pick disease type C (NPC) is a severe neurovisceral lysosomal storage disorder caused by defects in NPC1 or NPC2 proteins. Although numerous studies support the primacy of cholesterol storage, neurons of double-mutant mice lacking both NPC1 and an enzyme required for synthesis of all complex gangliosides (␤1,4GalNAc transferase) have been reported to exhibit dramatically reduced cholesterol sequestration. Here we show that NPC2-deficient mice lacking this enzyme also exhibit reduced cholesterol, but that genetically restricting synthesis to only a-series gangliosides fully restores neuronal cholesterol storage to typical disease levels. Examining the subcellular locations of sequestered compounds in neurons lacking NPC1 or NPC2 by confocal microscopy revealed that cholesterol and the two principal storage gangliosides (GM2 and GM3) were not consistently colocalized within the same intracellular vesicles. To determine whether the lack of GM2 and GM3 co-localization was due to differences in synthetic versus degradative pathway expression, we generated mice lacking both NPC1 and lysosomal ␤-galactosidase, and therefore unable to generate GM2 and GM3 in lysosomes. Double mutants lacked both gangliosides, indicating that each is the product of endosomal/lysosomal processing. Unexpectedly, GM1 accumulation in double mutants increased compared to single mutants consistent with a direct role for NPC1 in ganglioside salvage. These studies provide further evidence that NPC1 and NPC2 proteins participate in endosomal/lysosomal processing of both sphingolipids and cholesterol.

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“…Since cholesterol itself has very few structural/chemical features suitable for UV/VIS or optical imaging, the advent of DHE with many properties mimicking or very similar to those of cholesterol, has proven to be a major asset for probing/elucidating real-time sterol dynamics by video and MPLSM imaging in living cells. For additional insights, the reader is referred to earlier reviews on DHE, synthetic fluorescent sterols, spin labeled sterols, photoreactive sterols, and chemical tags of cholesterol (65)(66)(67)(68).…”

Section: Advent Of Fluorescent Sterolsmentioning

confidence: 99%

“…It is important to note that not only the quantity, but also the chemical structure of the sterol and the presence of cholesterol oxidation products are known to significantly affect the structure and/or function of model membranes (rev. in (54,55,70,72,73), biological membranes (13,(74)(75)(76)(77)(78)(79)(80), lipid raft/caveolae (58,(81)(82)(83)(84)(85)(86)(87)(88), and cholesterol-requiring plasma membrane receptors (66,(89)(90)(91). Because of this sensitivity it is essential that fluorescent sterols such as DHE not contain other sterols (e.g.…”

Section: Effect Of Sterol Structure and Purity On Sterol Architecturementioning

confidence: 99%

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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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...

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Cholesterol Reporter Molecules

Cited by 99 publications

References 153 publications

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

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