We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces p...

Supplemental Experimental Procedures Confocal UV Laser Scanning Microscopy of Filipin-Sterol Fluorescence Dot-Blot Detection of Filipin-Sterol Fluorescence Analysis of Arabidopsis roots was performed by employing a Nikon Sterols (Sigma) and 4-cholesten-3-one (Sigma) in chloroform at 20 Diaphot 300 inverted microscope, connected to a Nikon RCM 8000 mM were spotted onto Transblot nitrocellulose m...

The discovery of an acylated steryl glucoside in plant tissue in 1964 (13) raised the number of known forms of sterols in plants to four. These are the free sterols, sterol esters, steryl glycosides, and acylated steryl glycosides. Kiribuchi et al. (12) analyzed the sterol content of soybean and found that sterol ester was present in very small amount and that the sterol was mostly in the form ...

The mechanism by which cholera toxin (CT) is internalized from the plasma membrane before its intracellular reduction and subsequent activation of adenylyl cyclase is not well understood. Ganglioside GM1, the receptor for CT, is predominantly clustered in detergent-insoluble glycolipid rafts and in caveolae, noncoated, cholesterol-rich invaginations on the plasma membrane. In this study, we use...

Filipin at low concentrations rapidly hemolyzes bovine erythrocytes. Of the compounds tested, which were known a constituents of cell membranes, only cholesterol and phosphatidyl-choline prevented hemolysis. A specific complex was formed only with cholesterol. Filipin treatment caused a release of cholesterol from erythrocytes. Since all cell sterol is located in the cell membrane, the hemolyti...

The alterations in the absorption and fluorescence spectra observed for the polyene antibiotics filipin and nystatin in the presence of cholesterol are due to an exciton interaction (polyene aggregates) and cannot be attributed to a specific sterol-antibiotic complex. Filipin and nystatin molecules partition into the sterol aggregates, these structures being very efficient to induce exciton int...

Filipin binding to membrane sterols induces deformations of the membrane that are detected by freeze-fracture either as 20- to 25-nanometer protuberances or as pits on the fracture faces. By using the filipin probe in pancreatic acinar cells, it was found that the polarity of filipin-induced deformations in the membrane limiting the Golgi condensing vacuoles is opposite that in the membrane lim...

Specialized areas within broad, close, cell-substratum contacts seen with reflection interference contrast microscopy in cultures of Xenopus embryonic muscle cells were studied. These areas usually contained a distinct pattern of light and dark spots suggesting that the closeness of apposition between the membrane and the substratum was irregular. They coincided with areas containing acetylchol...

Among the polyene antibiotics, many, like filipin, cannot be used clinically because they are toxic; amphotericin B, however, is useful in therapy of human fungal infections because it is less toxic. Both the toxicity of filipin and the therapeutic value of amphotericin B can be rationalized at the cellular and molecular level by the following observations: (i) these polyene antibiotics showed ...

We have isolated and characterized a Triton-insoluble floating fraction (TIFF) from Dictyostelium. Ten major proteins were consistently detected in TIFF, and six species were identified by mass spectrometry as actin, porin, comitin, regulatory myosin light chain, a novel member of the CD36 family, and the phospholipid-anchored cell adhesion molecule gp80. TIFF was enriched with many acylated pr...