Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.

TLDR

Endocytic sorting separates recycling receptors from lysosomal cargo, with membrane-associated molecules directed to recycling while luminal ligands are targeted to lysosomes. The study seeks to determine whether recycling membrane receptors follow bulk membrane flow or are actively sorted based on specific sequences or structures. Using quantitative fluorescence microscopy, the authors tracked bulk membrane lipids to map their endocytic route and kinetics. C6‑NBD‑SM and other fluorescent lipids traffic as bulk membrane, displaying kinetics indistinguishable from transferrin, sorting from lysosomal cargo in peripheral endosomes, accumulating in a peri‑centriolar recycling compartment, and exiting the cell, indicating that bulk membrane flow drives recycling while fluid phase is directed to lysosomes in a geometrically based sorting mechanism.

Abstract

A central question in the endocytic process concerns the mechanism for sorting of recycling components (such as transferrin or low density lipoprotein receptors) from lysosomally directed components; membrane-associated molecules including receptors are generally directed towards the recycling pathway while the luminal content of sorting endosomes, consisting of the acid-released ligands, are lysosomally targeted. However, it is not known whether recycling membrane receptors follow bulk membrane flow or if these proteins are actively sorted from lysosomally directed material because of specific protein sequences and/or structural features. Using quantitative fluorescence microscopy we have determined the endocytic route and kinetics of traffic of the bulk carrier, membrane lipids, to address this issue directly. We show that N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]- sphingosylphosphorylcholine (C6-NBD-SM) in endocytosed as bulk membrane, and it transits the endocytic system kinetically and morphologically identically to fluorescently labeled transferrin in a CHO cell line. With indistinguishable kinetics, the two labeled markers sort from lysosomally destined molecules in peripherally located sorting endosomes, accumulate in a peri-centriolar recycling compartment, and finally exit the cell. Other fluorescently labeled lipids, C6-NBD-phosphatidylcholine and galactosylceramide also traverse the same pathway. The constitutive nature of sorting of bulk membrane towards the recycling pathway and the lysosomal direction of fluid phase implies a geometric basis of sorting.