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

• Published in: Electrophoresis Vol. 23; no. 13; pp. 2117 - 2129
• Main Authors: Ellinger, Isabella, Klapper, Herbert, Courtoy, Pierre J., Vaerman, Jean-Pierre, Fuchs, Renate
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag GmbH 01.07.2002; WILEY‐VCH Verlag GmbH
• Subjects: Animals; Asialoglycoproteins - analysis; Bile - secretion; Biological Transport; Cell Fractionation - methods; Electrophoresis - methods; Endosomes; Endosomes - physiology; Endosomes - ultrastructure; Fluorescein-5-isothiocyanate; Free-flow electrophoresis; Hepatocytes; Hepatocytes - cytology; Hepatocytes - physiology; Isolated perfused rat liver; Liver - cytology; Liver - physiology; Lysosomes - physiology; Lysosomes - ultrastructure; Orosomucoid - analogs & derivatives; Orosomucoid - analysis; Perfusion; Rats; Spectrometry, Fluorescence - methods; Temperature; Temperature sensitivity
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/1522-2683(200207)23:13<2117::AID-ELPS2117>3.0.CO;2-Z

Endocytosis at reduced temperature has been used to define and characterize endosome subpopulations. Thus, the temperature sensitivity of endosome subpopulations involved in transport to lysosomes and transcytosis in rat hepatocytes was analyzed applying endosome labeling in the isolated perfused rat liver with route‐specific ligands in combination with temperature shift protocols. Free‐flow electrophoresis (FFE) that separates membranes and organelles based on their surface charge was then applied to isolate functional endosomes. Using asialoorosomucoid (ASOR) and polymeric immunoglobulin A (pIgA) as specific ligands of the lysosomal and transcytotic route, respectively, two distinct endosome subpopulations along either pathway were separated by FFE. Upon a short (1–3 min) internalization at 37°C, 125I‐ASOR and fluorescein isothiocyanate (FITC)‐pIgA were colocalized in common early endosomes. Following a 5–10 min chase of the ligands at 37°C endosomes labeled with 125I‐ASOR were separated from endosomes labeled with FITC‐pIgA, indicative of two distinct late compartments along the lysosomal and transcytotic route. Internalization at 16°C resulted in accumulation of both ligands in common early endosomes and, consequently, in inhibition of transport to lysosomes and transcytosis. When 125I‐ASOR or 125I‐pIgA were first chased into late compartments at 37°C and the temperature was subsequently lowered to 16°C, biliary secretion of 125I‐ASOR‐derived counts was arrested, while biliary output of 125I‐pIgA continued. In summary, ASOR en route to lysosomes can be blocked in early as well as in late endosomes at 16°C, while biliary secretion of pIgA cannot be prevented by temperature reduction once the ligand had been transferred from early to late compartments.