Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells.
While plasma membrane cholesterol-rich microdomains play a role in cholesterol trafficking, little is known about the appearance and dynamics of cholesterol through these domains in living cells. The fluorescent cholesterol analog 6-dansyl-cholestanol (DChol), its biochemical fractionation, and confocal imaging of L-cell fibroblasts contributed the following new insights: i) fluorescence properties of DChol were sensitive to microenvironment polarity and mobility; (ii) DChol taken up by L-cell fibroblasts was distributed similarly as cholesterol and preferentially into cholesterol-rich vs. -poor microdomains resolved by affinity chromatography of purified plasma membranes; iii) DChol reported similar polarity (dielectric constant near 18) but higher mobility near phospholipid polar head group region for cholesterol in purified cholesterol-rich versus -poor microdomains; and iv) real-time confocal imaging, quantitative colocalization analysis, and fluorescence resonance energy transfer with cholesterol-rich and -poor microdomain markers confirmed that DChol preferentially localized in plasma membrane cholesterol-rich microdomains of living cells. Thus, DChol sensed a unique, relatively more mobile microenvironment for cholesterol in plasma membrane cholesterol-rich microdomains, consistent with the known, more rapid exchange dynamics of cholesterol from cholesterol-rich than -poor microdomains.
Huang
Huan
H
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843, USA.
McIntosh
Avery L
AL
Atshaves
Barbara P
BP
Ohno-Iwashita
Yoshiko
Y
Kier
Ann B
AB
Schroeder
Friedhelm
F
D000818:Animals; D001692:Biological Transport; D015415:Biomarkers / Q000378:metabolism; D002021:Buffers; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D002777:Cholestanols / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D002846:Chromatography, Affinity; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism*; D007739:L Cells; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D013261:Sterols / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D053835:Unilamellar Liposomes / Q000378:metabolism; D014867:Water / Q000378:metabolism
C473152:6-dansylcholestanol; D015415:Biomarkers; D002021:Buffers; D002777:Cholestanols; D005456:Fluorescent Dyes; D013261:Sterols; D053835:Unilamellar Liposomes; D014867:Water; D002784:Cholesterol