Pycnosomes: Condensed Endosomal Structures Secreted by Dictyostelium Amoebae.

Ayman Sabra,Jade Leiba,Mathilde Louwagie,Lauriane Mas,Agnès Journet,Laurence Aubry,Pierre Cosson,Yohann Couté,Thierry Soldati

doi:10.1371/journal.pone.0154875

Abstract

Dictyostelium discoideum has been used largely as a model organism to study the organization and function of the endocytic pathway. Here we describe dense structures present in D. discoideum endocytic compartments, which we named pycnosomes. Pycnosomes are constitutively secreted in the extracellular medium, from which they can be recovered by differential centrifugation. We identified the most abundant protein present in secreted pycnosomes, that we designated SctA. SctA defines a new family of proteins with four members in D. discoideum, and homologous proteins in other protists and eumetazoa. We developed a monoclonal antibody specific for SctA and used it to further characterize secreted and intracellular pycnosomes. Within cells, immunofluorescence as well as electron microscopy identified pycnosomes as SctA-enriched dense structures in the lumen of endocytic compartments. Pycnosomes are occasionally seen in continuity with intra-endosomal membranes, particularly in U18666A-treated cells where intraluminal budding is highly enhanced. While the exact nature, origin and cellular function of pycnosomes remain to be established, this study provides a first description of these structures as well as a characterization of reagents that can be used for further studies.

Highlights

In eukaryotic cells, endocytosed material is rapidly delivered to membrane-delimited endocytic compartments
Pycnosomes are secreted in the extracellular medium where they accumulate and from which they can be recovered by differential centrifugation
These structures were named pycnosomes. We reasoned that they may be secreted in the extracellular medium since D. discoideum endocytic compartments continuously fuse with the plasma membrane

Summary

Introduction

In eukaryotic cells, endocytosed material is rapidly delivered to membrane-delimited endocytic compartments. Endosomes are often filled with membranous material, since their limiting membrane can engage into intralumenal budding. This process can notably be stimulated by a large number of drugs, such as U18666A, the precise mechanism of action of these drugs is poorly characterized [1]. They normally evolve to give rise to lysosomes, endosomes can fuse with the plasma membrane in a regulated manner, and the membranous material released in this process is referred to as exosomes [2]. PLOS ONE | DOI:10.1371/journal.pone.0154875 May 17, 2016

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