Free‐flow electrophoretic analysis of endosome subpopulations of rat hepatocytes

Abstract

The separation of functional early and late endosomes from other cellular compartments by free-flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells. Here, using 125I-labeled anti-secretory component antibodies ([125I]SC Ab) and FITC-labeled asialoorosomucoid (FITC-ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16 degrees C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20 degrees C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37 degrees C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC-ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e. early endosomes) and two distinct endosome subpopulations (i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.

Fluid‐phase marker transport in rat liver: Free‐flow electrophoresis separates distinct endosome subpopulations

“…Our results suggest that the endosome populations labeled by the fluid-phase marker dextran are early endosomes and late endosomes since they colocalize with typical markers for the recycling and lysosomal pathway, respectively. Although we have shown that transcytotic endosomes can be separated from early and late endosomes [37], no fluid-phase marker peak was observed at a position corresponding to transcytotic vesicles. This result does not exclude localization of FITCdextran in the endosome population involved in transcytosis, but indicates that only a minor fraction of endocytosed marker enters the transcytotic pathway.…”

Different temperature sensitivity of endosomes involved in transport to lysosomes and transcytosis in rat hepatocytes: Analysis by free-flow electrophoresis

“…Endosomes were labeled in the isolatedperfused rat liver using pulse-chase conditions based on the transport kinetics of ASOR and pIgA, as determined above and in previous investigations [9,20,35,[43][44][45].…”

“…Kinetically early endosomes labeled with 125 I-pIgA (1 min; peak fraction #42) could be resolved from late transcytotic endosomes labeled with FITC-pIgA (10 min; peak fraction #44), the latter being thus shifted slightly more towards the anode. A minor peak of pIgA, found at fraction #34, presumably represents pIgA still bound to the plasma membrane [35].…”

“…Although transcytosis of anti-pIgR antibodies into bile is prevented by low-temperature perfusion [35], inhibition of receptor-mediated transcytosis of the natural ligands ASOR and pIgA due to low temperature has not been demonstrated. Consequently, the influence of low temperature perfusion on transcytosis of 125 I-pIgA and 125 I-ASOR was studied.…”

“…Since characterization of endosome subcompartments is based on the time-dependent internalization of the respective ligand, the isolated-perfused liver offers several advantages as compared to in vivo labeling in the intact animal. Free-flow electrophoresis (FFE) separates membranes and organelles based on their surface charge and has been applied to isolate highly purified and functional endosome subpopulations from nonpolarized [31][32][33] and polarized cells [34][35][36]. As the procedure is nondestructive and fractions obtained retain full in vitro function, organelles such as endosomes isolated by FFE can be subsequently analyzed for e.g., their acidification properties [34].…”