A detergent-free method for purifying caveolae membrane from tissue culture cells.

Smart, Elizabeth; Ying, Yun Shu; Mineo, Chieko; Anderson, Richard G. W.

doi:10.1073/pnas.92.22.10104

Cited by 707 publications

(720 citation statements)

References 26 publications

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“…Techniques differ in the efficiency with which they can detect short-lived transient dimers as well as in the ability to distinguish true functional receptor dimers from groups of receptors in close proximity due to colocalization in membrane domains (25)(26)(27)(28). Partitioning of EGFR in cholesterol-rich microdomains has been reported in several studies (29)(30)(31)(32); we have shown recently that EGFR partitions into cholesterol-dependent as well as cholesterol-independent domains and interacts with the cytoskeleton (33).…”

Section: Introductionmentioning

confidence: 93%

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

Yavas

Macháň

Wohland

2016

Biophysical Journal

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The epidermal growth factor receptor (EGFR) is a prototypical receptor tyrosine kinase involved in cell growth and proliferation and associated with various cancers. It is commonly assumed that after activation by binding of epidermal growth factor to the extracellular domain it dimerizes, followed by autophosphorylation of tyrosine residues at the intracellular domain. However, its oligomerization state before activation is controversial. In the absence of ligands, EGFR has been found in various, inconsistent amounts of monomeric, inactive dimeric, and oligomeric forms. In addition, evidence suggests that the active conformation is not a simple dimer but contains higher oligomers. As experiments in the past have been conducted at different conditions, we investigate here the influence of cell lines (HEK293, COS-7, and CHO-K1), temperature (room temperature and 37 C), and membrane localization on the quantitation of preformed dimers using SW-FCCS, DC-FCCS, quasi PIE-FCCS, and imaging FCCS. While measurement modality, temperature, and localization on upper or lower membranes have only a limited influence on the dimerization amount observed, the cell line and location to periphery versus center of the cell can change dimerization results significantly. The observed dimerization amount is strongly dependent on the expression level of endogenous EGFR in a cell line and shows a strong cell-to-cell variability even within the same cell line. In addition, using imaging FCCS, we find that dimers have a tendency to be found at the periphery of cells compared to central positions.

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Section: Introductionmentioning

confidence: 93%

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

Yavas

Macháň

Wohland

2016

Biophysical Journal

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“…CM were fractionated to isolate caveolae-rich domains using a detergent-free method [23]. Cells from a 10-cm 2 plate were washed twice in ice-cold PBS, scraped into 3 ml of 150 mM Na 2 CO 3 with 1 mM EDTA (pH 11.0), homogenized with a tissue grinder with three 10-sec bursts, and then sonicated with three cycles of 20-sec bursts interspersed with 1 min of incubation on ice.…”

Section: Sucrose Density Membrane Fractionationmentioning

confidence: 99%

Caveolin-3 expression and caveolae are required for isoflurane-induced cardiac protection from hypoxia and ischemia/reperfusion injury

Horikawa

Patel

Tsutsumi

et al. 2008

Journal of Molecular and Cellular Cardiology

102

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Volatile anesthetics protect the heart from ischemia/reperfusion injury but the mechanisms for this protection are poorly understood. Caveolae, sarcolemmal invaginations, and caveolins, scaffolding proteins in caveolae, localize molecules involved in cardiac protection. We tested the hypothesis that caveolae and caveolins are essential for volatile anesthetic-induced cardiac protection using cardiac myocytes (CM) from adult rats and in vivo studies in caveolin-3 knockout mice (Cav-3 −/− ). We incubated CM with methyl-β-cyclodextrin (MβCD) or colchicine to disrupt caveolae formation, and then exposed the myocytes to the volatile anesthetic isoflurane (30 min, 1.4%), followed by simulated ischemia/reperfusion (SI/R). Isoflurane protected CM from SI/R [23.2±1.6% vs. 71.0±5.8% cell death (assessed by trypan blue exclusion), P<0.001] but this protection was abolished by MβCD or colchicine (84.9±5.5% and 64.5±6.1% cell death, P<0.001). Membrane fractionation by sucrose density gradient centrifugation of CM treated with MβCD or colchicine revealed that buoyant (caveolae-enriched) fractions had decreased phosphocaveolin-1 and caveolin-3 compared to control CM. Cardiac protection in vivo was assessed by measurement of infarct size relative to the area at risk and cardiac troponin levels. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. expression of caveolin-3. We conclude that caveolae and caveolin-3 are critical for volatile anesthetic-induced protection of the heart from ischemia/reperfusion injury. NIH Public Access

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“…Because of the tremendous interest of biologists and membraneologists in cholesterol-rich microdomains (also called lipid rafts or caveolae) over the past 15 years (rev. in (54)(55)(56)(57)(58)(59)(60)(61)(62)(63), DHE has emerged as a popular, potentially less perturbing fluorescent sterol probe since it does not contain additional bulky fluorescent groups added to the sterol structure. Although this ultraviolet (UV) light absorbing and fluorescent sterol (i.e.…”

Section: Advent Of Fluorescent Sterolsmentioning

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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A detergent-free method for purifying caveolae membrane from tissue culture cells.

Cited by 707 publications

References 26 publications

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

Caveolin-3 expression and caveolae are required for isoflurane-induced cardiac protection from hypoxia and ischemia/reperfusion injury

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

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