A novel live-cell imaging assay reveals regulation of endosome maturation.

Maria Podinovskaia,Anne Spang,Dominik P Buser,Cristina Prescianotto-Baschong

doi:10.7554/elife.70982

Abstract

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

Highlights

Endosomes are central organelles in orchestrating cell interactions with the extracellular environment, whether by regulating the composition of signaling molecules at the plasma membrane or by facilitating uptake and digestion of certain nutrients or degrading toxic or no longer needed material
As these sorting endosomes mature into late endosomes, mainly containing cargo destined for degradation, they no longer accept cargo from the cell surface and acquire properties necessary for their interaction with lysosomes
The ability of the enlarged endosomes to recruit mediators of recycling, Snx1 and Rab11 vesicles, suggests that our assay can recapitulate recycling pathways. To further probe this possibility, we monitored the fate of three cargoes: TfR-G FP for recycling to the plasma membrane, cation-dependent mannose-6-phosphate receptor (GFP-C DMPR) for recycling to the trans-G olgi network (TGN) and GalT-G FP, which we have shown above to remain in the endosome to be transported to the lysosome

Summary

Introduction

Endosomes are central organelles in orchestrating cell interactions with the extracellular environment, whether by regulating the composition of signaling molecules at the plasma membrane or by facilitating uptake and digestion of certain nutrients or degrading toxic or no longer needed material Their wide range of functions is accomplished through a sequential and highly regulated process known as endosome maturation (Huotari and Helenius, 2011; Podinovskaia and Spang, 2018; Spang, 2016). These lipids serve as organelle identity molecules, facilitating recruitment of components, such as sorting and tethering factors, necessary for endosomal function (Schink et al, 2013) Endosomal acidification is another essential change that must take place for endosomes to mature, with pH ~6.5, 5.5 and 4.5 characterising early endosomes, late endosomes and lysosomes, respectively (Casey et al, 2010). Fusing GalT to ratiometric pHlemon (Burgstaller et al, 2019) (GalT-p Hlemon) allowed us to follow the degradation pathway to the lysosome and to demonstrate that acidification is slowed down when Rab conversion is blocked, suggesting that Rab conversion is required for efficient acidification during endosome maturation

Results

Discussion

Materials and methods

Funding Funder