Annexin A6 mediates calcium-dependent exosome secretion during plasma membrane repair
Curation statements for this article:-
Curated by eLife
eLife assessment
This compelling study brings together two earlier observations: that Ca2+ influx can trigger exosome release from multivesicular bodies, and that plasma membrane repair after wounding requires Ca2+ and involves Ca2+-binding annexin proteins. This important work takes these earlier findings in an interesting new direction by showing that exosome release from MVBs is also triggered by Ca2+ influx during plasma membrane wounding and requires the annexin isoform ANX6. The study suggests a few possible mechanisms (such as Ca2+-dependent tethering of MVBs to the plasma membrane by ANX6) and raises the interesting possibility that cell injury and repair may contribute to the release of exosomes into biological fluids.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Molecular Biology (eLife)
Abstract
Exosomes are an extracellular vesicle (EV) subtype that is secreted upon the fusion of multivesicular bodies (MVBs) with the plasma membrane. Exosomes may participate in intercellular communication and have utility as disease biomarkers; however, little is known regarding the physiological stimuli that induce their secretion. Ca 2+ influx promotes exosome secretion, raising the possibility that exosomes are secreted during the Ca 2+ -dependent plasma membrane repair of tissues damaged by mechanical stress in vivo. To determine whether exosomes are secreted upon plasma membrane damage, we developed sensitive assays to measure exosome secretion in intact and permeabilized cells. Our results suggest that exosome secretion is coupled to Ca 2+ -dependent plasma membrane repair. We find that annexin A6 (ANXA6), a well-known plasma membrane repair protein, is recruited to MVBs in the presence of Ca 2+ and required for Ca 2+ -dependent exosome secretion, both in intact and in permeabilized cells. ANXA6 depletion stalls MVBs at the cell periphery, and ANXA6 truncations localize to different membranes, suggesting that ANXA6 may serve to tether MVBs to the plasma membrane. We find that cells secrete exosomes and other EVs upon plasma membrane damage and propose that repair-induced secretion may contribute to the pool of EVs present within biological fluids.
Article activity feed
-
eLife assessment
This compelling study brings together two earlier observations: that Ca2+ influx can trigger exosome release from multivesicular bodies, and that plasma membrane repair after wounding requires Ca2+ and involves Ca2+-binding annexin proteins. This important work takes these earlier findings in an interesting new direction by showing that exosome release from MVBs is also triggered by Ca2+ influx during plasma membrane wounding and requires the annexin isoform ANX6. The study suggests a few possible mechanisms (such as Ca2+-dependent tethering of MVBs to the plasma membrane by ANX6) and raises the interesting possibility that cell injury and repair may contribute to the release of exosomes into biological fluids.
-
Reviewer #1 (Public Review):
This is a generally well-written manuscript that elegantly begins to explore the molecular basis of exosome release under conditions of sheer stress or calcium influx. The authors use a sensitive luciferase assay that enables them to monitor the release of exosomes from CD63-tag-expressing cells. Upon SLO pore formation or sheer stress, cells release exosomes in a calcium-dependent manner; MVBs are (indirectly) shown to undergo calcium-dependent plasma membrane fusion in a process that depends on a set of 4 proteins that were identified by an unbiased analysis of proteins that associate with MVBs. One of these is Annexin A6, a protein shown by several other groups to participate in membrane repair. Thus, calcium triggers the binding of 4 proteins to the surface of MVBs, and likely also to the plasma …
Reviewer #1 (Public Review):
This is a generally well-written manuscript that elegantly begins to explore the molecular basis of exosome release under conditions of sheer stress or calcium influx. The authors use a sensitive luciferase assay that enables them to monitor the release of exosomes from CD63-tag-expressing cells. Upon SLO pore formation or sheer stress, cells release exosomes in a calcium-dependent manner; MVBs are (indirectly) shown to undergo calcium-dependent plasma membrane fusion in a process that depends on a set of 4 proteins that were identified by an unbiased analysis of proteins that associate with MVBs. One of these is Annexin A6, a protein shown by several other groups to participate in membrane repair. Thus, calcium triggers the binding of 4 proteins to the surface of MVBs, and likely also to the plasma membrane, driving MVB fusion at the cell surface. The authors also present a semi-intact cell system that will permit functional analysis of the MVB fusion process.
-
Reviewer #2 (Public Review):
The authors improved significantly a previously published luminescence-based assay for the detection of MVB-derived exosome secretion, by using a membrane-impermeable Nluc inhibitor to make sure only intact vesicles and not cellular debris are quantified. Using this improved assay they confirmed prior reports that exposure to the Ca2+ ionophore ionomycin triggers exosome release. They then build on this by showing that exosomes are also released when Ca2+ influx is caused by plasma membrane (PM) wounding, using pore-forming toxins or mechanical stress. Investigating possible molecular mechanisms involved in Ca2+-regulated MVB exocytosis/exosome release, the authors use proteomics to identify proteins recruited to purified MVBs in an ionomycin-dependent fashion. One of these proteins is ANX6, which …
Reviewer #2 (Public Review):
The authors improved significantly a previously published luminescence-based assay for the detection of MVB-derived exosome secretion, by using a membrane-impermeable Nluc inhibitor to make sure only intact vesicles and not cellular debris are quantified. Using this improved assay they confirmed prior reports that exposure to the Ca2+ ionophore ionomycin triggers exosome release. They then build on this by showing that exosomes are also released when Ca2+ influx is caused by plasma membrane (PM) wounding, using pore-forming toxins or mechanical stress. Investigating possible molecular mechanisms involved in Ca2+-regulated MVB exocytosis/exosome release, the authors use proteomics to identify proteins recruited to purified MVBs in an ionomycin-dependent fashion. One of these proteins is ANX6, which interestingly was previously implicated in the repair of PM wounds in other cell types. The paper then explores the possible role of ANX6, showing that ionophore-dependent exosome secretion is inhibited in ANX6-depleted intact cells, or in permeabilized cells reconstituted with cytosol in the presence of anti-ANX6 antibodies. These results are convincing and very consistent with prior findings from other groups. The interesting advance is the demonstration that Ca2+ influx through PM lesions also triggers exocytosis of MVBs, and not only mature lysosomes as previously described. This reveals that PM injury, a frequent event in vivo, could play a role in the extensively documented detection of extracellular exosomes in biological fluids.
They also present some imaging data suggesting that ANX6 inhibition stalls MVBs at the cell surface and that ANX6 may promote MVB exocytosis and exosome release by tethering different intracellular membranes. These results are consistent with the author's interpretation but less compelling since they are based on limited confocal imaging without markers for specific compartments such as the PM and without quantification.
Another limitation of the study is that most experiments were performed using 30 min of cell exposure to micromolar concentrations of ionomycin, and the kinetics of exosome secretion after shorter times of ionophore exposure is not shown. The improved luminescence assay is described as sensitive and linear, but a linear time course over 24 h is only shown for constitutive exosome release, not for cells treated with ionomycin. Nocodazole experiments led to the conclusion that microtubules are required for 'sustained' exosome release, but this is somewhat misleading since ionophores markedly enhance exocytosis, raising questions as to whether the process is still linear after 30 min in the presence of ionomycin. The permeabilized-cell reconstitution assay apparently detected a requirement for ANX6 after just 2 min, which is reassuring but also raises the possibility that exosome release may not be sustained up to 30 min. PM resealing is a rapid process, completed in 1-2 min, so if one of the goals was to explore a connection between MVB exocytosis and PM repair, shorter time points would make more sense. This is particularly important since prolonged exposure to micromolar concentrations of ionomycin is known to cause extensive cytotoxicity, including actin cytoskeleton alterations, changes in ATP levels, and apoptosis (the authors perform only one limited control for apoptosis, a western that did not detect PARP cleavage).
Overall, this is an interesting study that brings together earlier observations but places them in a new context - that Ca2+-dependent exosome release from MVBs may occur in the context of PM wounding, and thus might play a role in PM resealing. Strong evidence was presented for the ANX6 requirement in ionophore-induced exosome release. However, since most previous studies implicating ANX6 in PM repair in other cell types involved a non-physiological form of laser wounding, it is still unclear if ANX6 is required for PM resealing after mechanical wounding, in the cells used in this study.
-
Reviewer #3 (Public Review):
The authors report that the secretion of endosome-derived exosomes is enhanced by a calcium-dependent response to damage to the plasma membrane of cells. The authors present convincing evidence that in response to the influx of calcium that follows damage to the plasma membrane annexin A6 is recruited to multivesicular bodies (MVBs) and likely serves to tether the MVBs to the plasma membrane causing a concomitant release of exosomes. Although it is not directly addressed in the Discussion, I am left with the impression that the authors are hinting that exosome secretion is more a byproduct of plasma membrane repair rather than a means of intercellular communication. In other words, the cell needs the membrane material from the MVB to patch and repair holes in the plasma membrane and exosome ejection from the …
Reviewer #3 (Public Review):
The authors report that the secretion of endosome-derived exosomes is enhanced by a calcium-dependent response to damage to the plasma membrane of cells. The authors present convincing evidence that in response to the influx of calcium that follows damage to the plasma membrane annexin A6 is recruited to multivesicular bodies (MVBs) and likely serves to tether the MVBs to the plasma membrane causing a concomitant release of exosomes. Although it is not directly addressed in the Discussion, I am left with the impression that the authors are hinting that exosome secretion is more a byproduct of plasma membrane repair rather than a means of intercellular communication. In other words, the cell needs the membrane material from the MVB to patch and repair holes in the plasma membrane and exosome ejection from the cell is a secondary (perhaps even irrelevant) consequence. Obviously, these two possibilities are not mutually exclusive. The authors are encouraged to speculate about which possibility they favor and how their findings might change our understanding of the cell biology of exosome secretion.
-
-
