Prefused lysosomes cluster on autophagosomes regulated by VAMP8

Lysosome–autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes—vesicle-associated membrane protein 8 (VAMP8)—plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.

Rab5 regulates macropinocytosis by recruiting the inositol 5-phosphatases OCRL and Inpp5b that hydrolyse PtdIns(4,5)P2

ABSTRACT Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent endomembrane fusion was necessary for the completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1, prevented macropinosome closure without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete the scission of macropinosomes or to prevent their back-fusion with the plasmalemma.

VAMP3 and VAMP8 regulate the development and functionality of parasitophorous vacuoles housing Leishmania amazonensis

ABSTRACTTo colonize mammalian phagocytic cells, the parasite Leishmania remodels phagosomes into parasitophorous vacuoles that can be either tight-fitting individual or communal. The molecular and cellular bases underlying the biogenesis and functionality of these two types of vacuoles are poorly understood. In this study, we investigated the contribution of host cell Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor proteins in the expansion and functionality of communal vacuoles as well as on the replication of the parasite. The differential recruitment patterns of Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor to communal vacuoles harboring L. amazonensis and to individual vacuoles housing L. major led us to further investigate the contribution of VAMP3 and VAMP8 in the interaction of Leishmania with its host cell. We show that whereas VAMP8 contributes to optimal expansion of communal vacuoles, VAMP3 negatively regulates L. amazonensis replication, vacuole size, as well as antigen cross-presentation. In contrast, neither proteins has an impact on the fate of L. major. Collectively, our data support a role for both VAMP3 and VAMP8 in the development and functionality of L. amazonensis-harboring communal parasitophorous vacuoles.

News about non-secretory exocytosis: mechanisms, properties, and functions

The fusion by exocytosis of many vesicles to the plasma membrane induces the discharge to the extracellular space of their abundant luminal cargoes. Other exocytic vesicles, however, do not contain cargoes, and thus, their fusion is not followed by secretion. Therefore, two distinct processes of exocytosis exist, one secretory and the other non-secretory. The present review deals with the knowledge of non-secretory exocytosis developed during recent years. Among such developments are the dual generation of the exocytic vesicles, initially released either from the trans-Golgi network or by endocytosis; their traffic with activation of receptors, channels, pumps, and transporters; the identification of their tethering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes that govern membrane fusions; the growth of axons and the membrane repair. Examples of potential relevance of these processes for pathology and medicine are also reported. The developments presented here offer interesting chances for future progress in the field.

Endosomal and Phagosomal SNAREs

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein family is of vital importance for organelle communication. The complexing of cognate SNARE members present in both the donor and target organellar membranes drives the membrane fusion required for intracellular transport. In the endocytic route, SNARE proteins mediate trafficking between endosomes and phagosomes with other endosomes, lysosomes, the Golgi apparatus, the plasma membrane, and the endoplasmic reticulum. The goal of this review is to provide an overview of the SNAREs involved in endosomal and phagosomal trafficking. Of the 38 SNAREs present in humans, 30 have been identified at endosomes and/or phagosomes. Many of these SNAREs are targeted by viruses and intracellular pathogens, which thereby reroute intracellular transport for gaining access to nutrients, preventing their degradation, and avoiding their detection by the immune system. A fascinating picture is emerging of a complex transport network with multiple SNAREs being involved in consecutive trafficking routes.

Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17

Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome–lysosome fusion is retained to some extent even in STX17 knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited autophagosome–lysosome fusion partially in wild-type and completely in STX17 KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate autophagosome–lysosome fusion.

Synaptische Transmission im Immunsystem

ZusammenfassungDie Freisetzung von Neurotransmittern an Synapsen gehört zu den wichtigsten Mechanismen im zentralen Nervensystem. In den zurückliegenden Jahrzehnten konnten viele Erkenntnisse über die molekularen Mechanismen, die diesem Prozess zugrunde liegen, gesammelt werden. Die hochregulierte Exozytose, die auf dem SNARE-Komplex („soluble N-ethylmaleimide-sensitive factor attachment protein receptor“) und seinen regulatorischen Molekülen basiert, ist das Merkmal des Nervensystems sowohl in Neuronen als auch in neuroendokrinen Zellen. Zellen des Immunsystems benutzen einen ähnlichen Mechanismus, um zytotoxische Substanzen aus sekretorischen Granulen freizusetzen. Diese Sekretion findet an Kontaktzonen mit Zelle statt, die mit Viren oder Bakterien infiziert sind sowie Krebszellen, um diese Bedrohung zu beseitigen. Diese Kontaktzonen werden als immunologische Synapsen bezeichnet im Hinblick auf die hochspezifische, zielgerichtete Exozytose von Effektormolekülen. Aktuelle Studien haben gezeigt, dass Mutationen in den SNARE oder SNARE-interagierenden Proteinen die Grundlage für zahlreiche schwerwiegende immunologische Erkrankungen sind. Obwohl SNARE-Komplexe ubiquitär vorkommen und eine große Vielfalt an Fusionsereignissen an der Membran vermitteln, ist es überraschend, dass in vielen Fällen die gleichen SNARE – Proteine an der immunologischen Synapse beteiligt sind, die die Regulation der Exozytose von Transmittern und Homonen in Neuronen und neuroendokrinen Zellen vermitteln. Diese Ähnlichkeiten zeigen die Möglichkeit auf, dass Erkenntnisse, die von immunologischen Synapsen erhalten wurden, auch auf neuronale Synapsen zutreffen, insbesondere im Bereich der präsynaptischen Funktion. Da immunologische Synapsen (IS) innerhalb von etwa 30 Minuten gebildet und wieder abgebaut werden, ermöglicht die Verwendung von Immunzellen, die aus humanem Blut gewonnen wurden, nicht nur die Untersuchung der molekularen Mechanismen der synaptischen Transmission in menschlichen Zelle, sondern auch Untersuchungen der Bildung und des Abbaus dieser „Synapsen“ mittels bildgebender Verfahren. In diesem Übersichtartikel vergleichen wir die Ähnlichkeit der Synapsen des Nerven- und Immunsystems und gehen dabei auf unsere Erkenntnisse der Arbeiten der letzten Jahre ein.