Abstract
The endosomal sorting complex required for transport (ESCRT) orchestrates cell membrane-remodeling mechanisms in eukaryotes, including endocytosis. However, ESCRT functions in phagocytosis (ingestion of ≥250 nm particles), has been poorly studied. In macrophages and amoebae, phagocytosis is required for cell nutrition and attack to other microorganisms and cells. In Entamoeba histolytica, the voracious protozoan responsible for human amoebiasis, phagocytosis is a land mark of virulence. Here, we have investigated the role of ESCRT-III in the phagocytosis of E. histolytica, using mutant trophozoites, recombinant proteins (rEhVps20, rEhVps32, rEhVps24, and rEhVps2) and giant unilamellar vesicles (GUVs). Confocal images displayed the four proteins located around the ingested erythrocytes, in erythrocytes-containing phagosomes and in multivesicular bodies. EhVps32 and EhVps2 proteins co-localized at the phagocytic cups. Protein association increased during phagocytosis. Immunoprecipitation and flow cytometry assays substantiated these associations. GUVs revealed that the protein assembly sequence is essential to form intraluminal vesicles (ILVs). First, the active rEhVps20 bound to membranes and recruited rEhVps32, promoting membrane invaginations. rEhVps24 allowed the detachment of nascent vesicles, forming ILVs; and rEhVps2 modulated their size. The knock down of Ehvps20 and Ehvps24genes diminished the rate of erythrophagocytosis demonstrating the importance of ESCRT-III in this event. In conclusion, we present here evidence of the ESCRT-III participation in phagocytosis and delimitate the putative function of proteins, according to the in vitro reconstruction of their assembling.
Highlights
In eukaryotic cells, endocytosis is the universal process to ingest nutrients and separate proteins for digestion and recycling pathways
The ancient endosomal sorting complex required for transport (ESCRT) machinery is critical for several central cellular processes; among them, endocytosis, multivesicular bodies (MVBs) biogenesis, viral budding, cytokinesis, autophagy, exosomes secretion and others based on membrane deformation and scission (Carlton and Martin-Serrano, 2007; Filimonenko et al, 2007; Lee et al, 2007; Rusten et al, 2007)
EhVps32 and EhADH are involved in pinocytosis of dextran and phagocytosis of latex-coated beads (Avalos-Padilla et al, 2015; Castellanos-Castro et al, 2016) which implies that ESCRT-III proteins participate in different types of endocytosis
Summary
Endocytosis is the universal process to ingest nutrients and separate proteins for digestion and recycling pathways. The endosomal sorting complex required for transport (ESCRT) orchestrates several important cell membrane-remodeling mechanisms. Endocytosis involves the ingestion of small particles (≤250 nm) and of large particles (≥250 nm), including cells. In the latter case, one usually speaks of phagocytosis, a mechanism used as an attack strategy against bacteria and other target cells. Even though the ingestion of small and large particles has many common features, phagocytosis involves a more complex molecular machinery (including contractil actin myosin ring).
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