Recycling Compartments and the Internal Vesicles of Multivesicular Bodies Harbor Most of the Cholesterol Found in the Endocytic Pathway

TLDR

We used a novel immuno‑electron microscopic technique to map cholesterol distribution in the endocytic pathway of human B lymphocytes. The method allowed us to distinguish six endocytic compartment categories based on morphology, BSA gold uptake kinetics, and organelle marker analysis. Cholesterol was predominantly found in multivesicular bodies (63 % of total), especially within their internal membrane vesicles, with 20 % in recycling tubulo‑vesicles and almost none in lysosomes, revealing a highly differential distribution across membrane domains.

Abstract

We employed our recently developed immuno‐electron microscopic method (W. Möbius, Y. Ohno‐Iwashita, E. G. van Donselaar, V. M. Oorschot, Y. Shimada, T. Fujimoto, H. F. Heijnen, H. J. Geuze and J. W. Slot, J Histochem Cytochem 2002; 50: 43–55) to analyze the distribution of cholesterol in the endocytic pathway of human B lymphocytes. We could distinguish 6 categories of endocytic compartments on the basis of morphology, BSA gold uptake kinetics and organelle marker analysis. Of all cholesterol detected in the endocytic pathway, we found 20% in the recycling tubulo‐vesicles and 63% present in two types of multivesicular bodies. In the multivesicular bodies, most of the cholesterol was contained in the internal membrane vesicles, the precursors of exosomes secreted by B cells. Cholesterol was almost absent from lysosomes, that contained the bulk of the lipid bis(monoacylglycero)phosphate, also termed lysobisphosphatidic acid. Thus, cholesterol displays a highly differential distribution in the various membrane domains of the endocytic pathway .