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Proceedings Paper

Fluorescence recovery after photobleaching measured by confocal microscopy as a tool for the analysis of vesicular lipid transport and plasma membrane mobility
Author(s): Gerd Schmitz; Alexandra Goetz; Evelyn Orso; Gregor Rothe
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Paper Abstract

The vesicular transport of lipids from the endoplasmic reticulum via the Golgi apparatus affects the composition of the plasma membrane. The purpose of our study was to develop an in vitro test system for characterization of vesicular lipid transport kinetics by using confocal microscopy and fluorescence recovery after photobleaching (FRAP). Fibroblasts from two patients homozygous for the hypercatabolic HDL deficiency syndrome Tangier disease and 4 control subjects were pulsed with the C6-NBD-ceramide for 30 minutes. Chase incubation at room temperature resulted in the metabolic accumulation of fluorescent C6-NBD-sphingolyelin and C6-NBD-glycosylceramides in the medial- and trans-Golgi region. Cells were analyzed with an inverted Leica TCS microscope. Calibration was performed through the analysis of diffusion of 50 nm microparticles embedded in media of different viscosity. An acousto optical tunable filter (AOTF) was used for the selective bleaching of the medial- and trans- Golgi region followed by analysis of the fluorescence recovery for 4 minutes. Post-bleach fluorescence recovery through the trans-Golgi-oriented transport of NBD-sphingomyelin was calculated from 2-dimensional scans. Tangier fibroblasts displayed a retarded recovery of fluorescence in the trans- Golgi region. This suggests that the vesicular transport of sphingomyelin and cholesterol is disturbed in Tangier disease confirming data from our laboratory generated with radiometabolites on whole cells. Our data suggest that FRAP analysis allows a sensitive kinetic and spatially resolved analysis of disturbances of vesicular lipid transport.

Paper Details

Date Published: 29 April 1998
PDF: 9 pages
Proc. SPIE 3260, Optical Investigations of Cells In Vitro and In Vivo, (29 April 1998); doi: 10.1117/12.307088
Show Author Affiliations
Gerd Schmitz, Institute for Clinical Chemistry and Laboratory Medicine/Univ. Regensburg (Germany)
Alexandra Goetz, Institute for Clinical Chemistry and Laboratory Medicine/Univ. Regensburg (Germany)
Evelyn Orso, Institute for Clinical Chemistry and Laboratory Medicine/Univ. Regensburg (Germany)
Gregor Rothe, Institute for Clinical Chemistry and Laboratory Medicine/Univ. Regensburg (Germany)


Published in SPIE Proceedings Vol. 3260:
Optical Investigations of Cells In Vitro and In Vivo
Robert C. Leif; Daniel L. Farkas; Robert C. Leif; Bruce J. Tromberg, Editor(s)

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