Digital fluorescence imaging of trafficking of endosomes containing low-density lipoprotein in brain astroglial cells

We have used digital fluorescence microscopy to examine transport of LDL-containing endosomes in rat brain astroglial cells to show that individual middle endosomes undergo rapid transitions between forward/backward movements and immobile states over short distances. The population of rapidly moving endosomes (>0.04 mu m/sec) was 35.9%, and the remaining endosomes were slowly moving or temporarily immobile (<0.04 mu m/sec). The averaged motion was, however, a very slow perinuclear motion with a velocity of 3.25 mu m/h. This small velocity is mainly due to frequent changing of directions in movements, requiring 6 h for a significant concentration around the circumference of the cell nuclei. The application of both anti-dynein antibodies and vanadate in permeabilized cells resulted in peripherally concentrated distribution of endosomes, probably due to inhibition of perinuclear motion by dynein-like motor proteins. These results imply that both dynein-like and kinesin-like proteins bind to the same endosome resulting in both perinuclear and peripherally directed movements. (C) 2000 Academic Press.