Use of quantitative image microfluorometry to follow fluorescent ricin internalization in single living cells.
We used microspectrofluorometry and videomicrofluorometry to follow the binding and internalization of fluorescein-labeled toxic lectin ricin in living Zajdela hepatoma cells. Microspectrofluorometry showed that when ricin was specifically labeled on its B-chain with one molecule of fluorescein (ABF), its fluorescence spectrum did not alter during its binding to the cell surface and subsequent internalization. This enabled us to use image analysis to follow cell internalization of labeled ricin. Accordingly, we measured the appropriate fluorescent cell parameters, comprising total fluorescence intensity, cell surface area, mean fluorescence intensity and its standard error, and used the measurements for mono- and biparametric studies of cell fluorescence distribution. The results showed that (a) ricin binds two different subpopulations of Zajdela hepatoma cells, (b) Zajdela hepatoma cells internalize ricin rapidly and after a relatively stable period of 1-2 hr, internalization starts again at 4 hr, and (c) the distribution of intracellular fluorescence is heterogeneous and ABF accumulates in certain cellular localizations. Our results demonstrate that quantitative microfluorometry is an effective and interesting approach for real-time studies of macromolecule internalization in living cells.
Frénoy
J P
JP
CNRS X 0036, Etats Liés Moléculaires, UFR Biomédicale des Saints-Pères, Université René Descartes, Paris, France.
Emmanuel
F
F
Salmon
J M
JM
D000818:Animals; D003592:Cytophotometry / Q000379:methods*; D019793:Fluorescein; D005452:Fluoresceins; D007091:Image Processing, Computer-Assisted; D008297:Male; D010784:Photomicrography; D051381:Rats; D017207:Rats, Sprague-Dawley; D012276:Ricin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D005452:Fluoresceins; D012276:Ricin; D019793:Fluorescein