Vesicles Of Multivesicular Bodies Research Articles

The Rubicon-WIPI axis regulates exosome biogenesis during ageing.

Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.

Egg multivesicular bodies elicit an LC3-associated phagocytosis-like pathway to degrade paternal mitochondria after fertilization

Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila, we show that special egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.

Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies.

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.

Optimizing Exosome Preparation Based on Size and Morphology: Insights From Electron Microscopy.

Extracellular vesicles (EVs), including exosomes, are crucial in intercellular communication, but differentiating between exosomes and microvesicles is challenging due to their similar morphology and size. This study focuses on multivesicular bodies (MVBs), where exosomes mature, and optimizes exosome isolation using transmission electron microscopy (TEM) for size information. Considering that EVs are nanocolloidal particles, a salt-free Bis-Tris buffer is found to maintain EV integrity better than phosphate-buffered saline (PBS). Dynamic light scattering (DLS) and TEM analysis confirm that intact exosome fractions under the salt-free Bis-Tris buffer condition exhibit polydispersity, including a unique population of <50 nm vesicles resembling intraluminal membrane vesicles (ILVs) in MVBs, alongside larger populations. This <50 nm population disappears in PBS or Bis-Tris buffer with 140 mM NaCl, transforming into a monodisperse population >100 nm. Immunoelectron microscopy also validates the presence of CD63, an exosome biomarker, on approximately 50 nm EVs. These findings provide valuable insights into exosome characterization and isolation, essential for future biomedical applications in diagnostics and drug delivery.

Inter-neuronal signaling mediated by small extracellular vesicles: wireless communication?

Conventional inter-neuronal communication conceptualizes the wired method of chemical synapses that physically connect pre-and post-synaptic neurons. In contrast, recent studies indicate that neurons also utilize synapse-independent, hence "wireless" broadcasting-type communications via small extracellular vesicles (EVs). Small EVs including exosomes are secreted vesicles released by cells and contain a variety of signaling molecules including mRNAs, miRNAs, lipids, and proteins. Small EVs are subsequently absorbed by local recipient cells via either membrane fusion or endocytic processes. Therefore, small EVs enable cells to exchange a "packet" of active biomolecules for communication purposes. It is now well established that central neurons also secrete and uptake small EVs, especially exosomes, a type of small EVs that are derived from the intraluminal vesicles of multivesicular bodies. Specific molecules carried by neuronal small EVs are shown to affect a variety of neuronal functions including axon guidance, synapse formation, synapse elimination, neuronal firing, and potentiation. Therefore, this type of volume transmission mediated by small EVs is thought to play important roles not only in activity-dependent changes in neuronal function but also in the maintenance and homeostatic control of local circuitry. In this review, we summarize recent discoveries, catalog neuronal small EV-specific biomolecules, and discuss the potential scope of small EV-mediated inter-neuronal signaling.

Phosphatidic Acid Accumulates at Areas of Curvature in Tubulated Lipid Bilayers and Liposomes.

Phosphatidic acid (PA) is a signaling lipid that is produced enzymatically from phosphatidylcholine (PC), lysophosphatidic acid, or diacylglycerol. Compared to PC, PA lacks a choline moiety on the headgroup, making the headgroup smaller than that of PC and PA, and PA has a net negative charge. Unlike the cylindrical geometry of PC, PA, with its small headgroup relative to the two fatty acid tails, is proposed to support negatively curved membranes. Thus, PA is thought to play a role in a variety of biological processes that involve bending membranes, such as the formation of intraluminal vesicles in multivesicular bodies and membrane fusion. Using supported tubulated lipid bilayers (STuBs), the extent to which PA localizes to curved membranes was determined. STuBs were created via liposome deposition with varying concentrations of NaCl (500 mM to 1 M) on glass to form supported bilayers with connected tubules. The location of fluorescently labeled lipids relative to tubules was determined by imaging with total internal reflection or confocal fluorescence microscopy. The accumulation of various forms of PA (with acyl chains of 16:0-6:0, 16:0-12:0, 18:1-12:0) were compared to PC and the headgroup labeled phosphatidylethanolamine (PE), a lipid that has been shown to accumulate at regions of curvature. PA and PE accumulated more at tubules and led to the formation of more tubules than PC. Using large unilamellar liposomes in a dye-quenching assay, the location of the headgroup labeled PE was determined to be mostly on the outer, positively curved leaflet, whereas the tail labeled PA was located more on the inner, negatively curved leaflet. This study demonstrates that PA localizes to regions of negative curvature in liposomes and supports the formation of curved, tubulated membranes. This is one way that PA could be involved with curvature formation during a variety of cell processes.

Rab39 and its effector UACA regulate basolateral exosome release from polarized epithelial cells.

Exosomes are small extracellular vesicles that originate from the intraluminal vesicles of multivesicular bodies (MVBs). We previously reported that polarized Madin-Darby canine kidney (MDCK) epithelial cells secrete two types of exosomes, apical and basolateral exosomes, from different MVBs. However, how these MVBs are selectively targeted to the apical or basolateral membrane remained unknown. Here, we analyze members of the Rab family small GTPases and show that different sets of Rabs mediate asymmetrical exosome release. Rab27, the best-known regulator of MVB transport for exosome release, is specifically but partially involved in apical exosome release, and Rab37, a close homolog of Rab27, is an additional apical exosome regulator. By contrast, Rab39 functions as a specific regulator of basolateral exosome release. Mechanistically, Rab39 interacts with its effector UACA, and UACA then recruits Lyspersin, a component of BLOC-1-related complex (BORC). Our findings suggest that the Rab39-UACA-BORC complex specifically mediates basolateral exosome release.

A Subunit of ESCRT-III, MoIst1, Is Involved in Fungal Development, Pathogenicity, and Autophagy in Magnaporthe oryzae.

The culprit of rice blast, Magnaporthe oryzae, is a filamentous fungus that seriously affects the yield and quality of rice worldwide. MoIst1, a subunit of ESCRT-III, is involved in identified ubiquitinated proteins and transports them into the intraluminal vesicles of multivesicular bodies (MVBs) for degradation in lysosomes. Here, we identify and characterize MoIst1 in M. oryzae. Disruption of MoIst1 leads to a significant decrease in sporulation and formation of appressoria, defects in response to oxidative stress, cell wall stress, hyperosmotic stress, and reduced pathogenicity. Deletion of MoIst1 also caused the decreased Pmk1 phosphorylation levels, appressorium formation, the delayed translocation and degradation of lipid droplets and glycogen, resulting in a decreased appressorium turgor. In addition, deletion of MoIst1 leads to an abnormal autophagy. In summary, our results indicate that MoIst1 is involved in sporulation, appressorium development, plant penetration, pathogenicity, and autophagy in M. oryzae.

In situ and ex situ imaging of plant extracellular vesicles as nanovectors for cross‐domain communication

AbstractExtracellular vesicles (EVs) are nanometric, lipid membrane‐bound vesicles released outside the cell. Plants have not been traditionally regarded to have EVs due to the presence of a cell wall. However, since the mid‐1960s, EVs have been reported in various plant species. They are often shown as vesicles in multivesicular bodies (MVBs) that later fuse with the plasma membrane (PM). Plant EVs have been commonly visualized using electron microscopy. Using membrane‐spanning domains, plant EVs can be fluorescence‐detected. They were observed near the infection sites in host tissues attacked by pathogens or at the interfaces with symbiotic fungi. Plant EVs could be taken up by bacteria and fungi. Proteins, lipids and nucleic acids, including small ribonucleic acids (RNAs), were found in plant EVs. Plant EVs have been isolated from the symplast and apoplast of various organs and purified after density gradient ultracentrifugation. Improved protocols and technologies for EV visualization provide a basis for the future applications of EVs in plant pathology.

Extracellular vesicles in spontaneous preterm birth.

Feto-maternal communication helps to maintain pregnancy and contributes to parturition at term and preterm. Endocrine and immune factor are well-reported communication mediators. Recent advances in extracellular vesicle (EV) biology have introduced them as major communication channels between the mother and fetus. EVs are round structures with a lipid bilayer membrane. EVs are generally categorized based on their size and mode of biogenesis. The most commonly reported EVs are exosomes with a size range of 30-160nm that are formed inside the intraluminal vesicles of multivesicular body. Microvesicles (MVs) are larger than>200nm and formed by outward budding of plasma membrane. Vesicles are released from all cells and carry various factors that reflect the physiologic state of cell at the time of their release. Analysis of vesicle provides a snapshot of origin cell. Recent studies in perinatal medicine have shown that exosomes are key communicators between feto-maternal units, and they can cross placenta. Fetal-derived exosomes released under term labor-associated conditions can cause parturition-associated changes in maternal uterine tissues. Exosomes carrying inflammatory cargo can cause preterm birth in animal models suggesting their functional role in parturition. A few reports have profiled differences between exosome cargos from term and preterm pregnancies and indicated their biomarker potential to predict high-risk pregnancy status. There are hardly any reports on MVs and their functional roles in reproduction. Herein, we review of EVs and MVs, their characteristics, function, and usefulness predicting adverse pregnancy complications such as preterm birth.

The Beginning of the End: Initial Steps in the Degradation of Plasma Membrane Proteins.

The plasma membrane (PM), as border between the inside and the outside of a cell, is densely packed with proteins involved in the sensing and transmission of internal and external stimuli, as well as transport processes and is therefore vital for plant development as well as quick and accurate responses to the environment. It is consequently not surprising that several regulatory pathways participate in the tight regulation of the spatiotemporal control of PM proteins. Ubiquitination of PM proteins plays a key role in directing their entry into the endo-lysosomal system, serving as a signal for triggering endocytosis and further sorting for degradation. Nevertheless, a uniting picture of the different roles of the respective types of ubiquitination in the consecutive steps of down-regulation of membrane proteins is still missing. The trans-Golgi network (TGN), which acts as an early endosome (EE) in plants receives the endocytosed cargo, and here the decision is made to either recycled back to the PM or further delivered to the vacuole for degradation. A multi-complex machinery, the endosomal sorting complex required for transport (ESCRT), concentrates ubiquitinated proteins and ushers them into the intraluminal vesicles of multi-vesicular bodies (MVBs). Several ESCRTs have ubiquitin binding subunits, which anchor and guide the cargos through the endocytic degradation route. Basic enzymes and the mode of action in the early degradation steps of PM proteins are conserved in eukaryotes, yet many plant unique components exist, which are often essential in this pathway. Thus, deciphering the initial steps in the degradation of ubiquitinated PM proteins, which is the major focus of this review, will greatly contribute to the larger question of how plants mange to fine-tune their responses to their environment.

Reduced Glutathione Protects Subcellular Compartments From Pb-Induced ROS Injury in Leaves and Roots of Upland Cotton (Gossypium hirsutum L.).

Heavy metals-based changes in the plants and their alleviation through eco-friendly agents including reduced glutathione (GSH) have been widely studied. In the present experiment, we tested the alleviatory role of reduced glutathione (GSH) in seedlings of upland cotton cultivar, TM-1 under lead (Pb) toxicity. Plants were grown in the Hoagland solution containing Pb (0 μM), Pb (500 μM), GSH (50 μM), and GSH + Pb (50 μM + 500 μM). Lead exposure exacerbated hydrogen peroxide (H2O2) and hydroxyl radical (OH•) levels, induced lipid peroxidation (MDA), and decreased the activities of catalase (CAT) and ascorbate peroxidase (APX) in the terminal and median leaves of 28-days old cotton seedlings stressed for 10 days. However, in the primary and secondary roots, CAT activity was increased but APX decreased. Similarly, peroxidase (POD) and superoxide dismutase (SOD) activities were enhanced in the median leaves but a declining trend was observed in the terminal leaves, primary roots and secondary roots. Glutathione reductase (GR) activity, ascorbic acid (AsA) contents and GSH concentrations were increased in all parts except AsA in the median leaves. Transmission electron micrographs of Pb-treated plants exhibited deformed cell wall and cell membrane, disfigured chloroplasts and irregularly shaped mitochondria in the terminal and median leaves. Further, cell membrane, mitochondria, nucleus and other cell organelles in root cells were severely affected by the Pb. Thus their identification was little bit difficult through ultramicroscopy. External GSH stabilized leaf and root ultramorphology by stabilizing cell membranes, stimulating formation of multivesicular body vesicles, and by maintaining structural integrity of other organelles. Evidently, GSH played major alleviatory role against Pb toxicity in upland cotton.

UNC93B1 recruits syntenin-1 to dampen TLR7 signalling and prevent autoimmunity.

At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self-RNA or DNA, respectively. Despite the structural and functional similarities between these receptors, their contribution to autoimmune diseases such as systemic lupus erythematosus (SLE) can be quite different. For example, TLR7 and TLR9 have opposing effects in mouse models of SLE; disease is exacerbated in TLR9-deficient mice but attenuated in TLR7-deficient mice1. However, mechanisms of negative regulation that differentiate between TLR7 and TLR9 have not been described. Here we report a new function for the TLR trafficking chaperone Unc93b1 that specifically limits signaling of TLR7, but not TLR9, and prevents TLR7-dependent autoimmunity in mice. We find that mutations in Unc93b1 leading to enhanced TLR7 signaling also disrupt binding to Syntenin-1, a protein implicated in exosome biogenesis. Both Unc93b1 and TLR7 are detectable in exosomes, suggesting that Unc93b1 recruitment of Syntenin-1 facilitates sorting of TLR7 into intralumenal vesicles of multivesicular bodies which terminates signaling. Syntenin-1 binding requires phosphorylation of Unc93b1, providing a mechanism for dynamic regulation of TLR7 activation and signaling. Thus, Unc93b1 not only enables proper trafficking of nucleic acid-sensing TLRs but also sets the activation threshold of potentially self-reactive TLR7.

Lysosomal N-acetyltransferase interacts with ALIX and is detected in extracellular vesicles

Heparan acetyl CoA: α-glucosaminide N-acetyltransferase (HGSNAT) is a lysosomal multi-pass transmembrane protein whose deficiency may lead to an accumulation of heparan sulphate and the neurodegenerative lysosomal storage disorder mucopolysaccharidosis (MPS) IIIC. In this study, HGSNAT activity was detected in extracellular vesicles isolated from both human urine and culture medium conditioned with HEK 293T cells. We also demonstrate that HGSNAT co-immunoprecipitates with antibodies to ALIX, which is associated with the endosomal sorting complexes required for transport (ESCRT) proteins, and is implicated in the targeting of proteins to intraluminal vesicles of multivesicular bodies, the origin of exosomes. Furthermore, mutation of a putative LYPXnL-based binding site within HGSNAT for the V-domain of ALIX ablated association of HGSNAT with ALIX, post-translational maturation, and transport through the endo-lysosomal network. Unexpectedly, however, a mutation within the V-domain of ALIX demonstrated enhanced HGSNAT association, perhaps due to the actual involvement of other binding sites in this interaction. Indeed, HGSNAT still co-immunoprecipitates with truncations of ALIX lacking the V-domain. Interestingly, CRISPR/Cas9 mediated knock-down of ALIX did not inhibit HGSNAT trafficking through the endo-lysosomal network, suggesting that there is an alternative pathway for trafficking HGSNAT that does not require ALIX. Nonetheless, the targeting of HGSNAT to extracellular vesicles may provide a mechanism to subsequently transfer this enzyme extracellularly to provide a foundation for a therapy for MPS IIIC patients.

Extracellular vesicles direct migration by synthesizing and releasing chemotactic signals.

Chemotactic signals are relayed to neighboring cells through the secretion of additional chemoattractants. We previously showed in Dictyostelium discoideum that the adenylyl cyclase A, which synthesizes the chemoattractant cyclic adenosine monophosphate (cAMP), is present in the intraluminal vesicles of multivesicular bodies (MVBs) that coalesce at the back of cells. Using ultrastructural reconstructions, we now show that ACA-containing MVBs release their contents to attract neighboring cells. We show that the released vesicles are capable of directing migration and streaming and are central to chemotactic signal relay. We demonstrate that the released vesicles not only contain cAMP but also can actively synthesize and release cAMP to promote chemotaxis. Through proteomic, pharmacological, and genetic approaches, we determined that the vesicular cAMP is released via the ABCC8 transporter. Together, our findings show that extracellular vesicles released by Ddiscoideum cells are functional entities that mediate signal relay during chemotaxis and streaming.

Exosomal secretion of truncated cytosolic lysyl-tRNA synthetase induces inflammation during cell starvation.

Previous work by Kim, et al. (2017) unveiled that lysyl-tRNA synthetase (KRS) is secreted through a mechanism involving syntenin-containing exosomes. They described how KRS, commonly known as part of the translational machinery in the cytoplasm, is secreted into the extracellular space where it induces inflammation. First, KRS secretion is triggered by starvation conditions. The increase in caspase-8 levels during starvation is responsible for proteolysis and generation of the N-terminal truncated form of KRS, and this event is required for KRS dissociation from the multi-synthetase complex (MSC). N-terminal cleavage of KRS eventually leads to a conformational change that allows its interaction with the C-terminal PDZ binding motif of syntenin and subsequent exosome biogenesis. The KRS-syntenin complex translocates to multivesicular bodies (MVBs) that originate from endosomes involved with intraluminal vesicle (ILVs). MVBs are transporters for the secretion of cellular contents into the extracellular space. Syntenin localizes intraluminal vesicles within endosomal membranes. The KRS-syntenin complex transfers on to intraluminal vesicles in MVBs. MVBs are translocated to the plasma membrane for ILV secretion mediated by Rab family proteins. Once KRS exosomes are secreted, their membranes are eventually ruptured by proteases and KRS is released from the exosomes where it can act as an inflammatory cytokine in the extracellular space. Secreted KRS triggers macrophage/neutrophil migration and induces inflammation.

Cellular Evidence of Exosomes in the Reproductive Tract of Chinese Soft-Shelled Turtle Pelodiscus sinensis.

The oviduct is a location of egg production, fertilization, and sperm storage. While its secretions have broadly attributes toward different physiological functions. We examined the ultrastructure of oviduct epithelium and glands in relation to the secretions, particularly with exosomes origin in Chinese soft-shelled turtle Pelodiscus sinensis using immunohistochemistry and transmission electron microscopy. The ciliated epithelial and gland cells were involved in the release of exosomes and secretions into lumen throughout the year. The exosomes were either released directly from epithelium or in relation with multivesicular body (MVB). The average size of the particles varies between 50 and 130 nm. These exosomes were also widely distributed in the epithelial ciliated cells and pericytoplasm of glands lumen. Intracellular MVB was characterized by membrane-bounded exosomes of different sizes. Exosomes were also found in close contact with the cilia and sperm membrane in the lumen, which is suggestive of their fusogenic properties. Immunohistochemistry results showed strong to moderate positive expression of exosomes, in ciliated and gland cells, during January, September, and December, as it is the time of sperm storage in this turtle, whereas they showed moderate to weak level of expression during breeding season (May). This is first study about identification of the exosomes in female turtles. Epithelial and glandular exosomes, intracellular MVB, secretions, and secretory vesicles give this turtle specie a unique secretory morphology and a potential model for investigating the secretory nature of the oviduct.

Effects of Lipid Membrane Curvatures on Binding, Secondary Structure, and Aggregation of Amyloid-Beta Protein

Conversion of amyloid-beta (Abeta) protein from a non-toxic monomer into the toxic aggregates is the possible pathogenic pathways in Alzheimer's disease. Recent studies have suggested that lipid membranes play key roles in protein aggregation. It has been demonstrated that Abeta accumulated and formed amyloid fibrils in specific intra- or extracellular membrane structures such as intraluminal vesicles of multivesicular bodies (MVBs) and exosomes with a diameter that ranges from 30 to 80 nm. These studies raise the possibility that Abeta preferentially binds to small-sized intra- or extracellular vesicles with a high membrane curvature, although the presence of specific lipid components might also influence the interaction. Indeed, it has been reported that small lipid vesicles (≤50 nm) composed of PC accelerated Abeta fibrillation. However, the binding modes and the mechanisms of Abeta aggregation on lipid vesicles of high curvature are not fully understood. Here, we observed that a high positive curvature of lipid vesicles with diameters of ∼30 nm enhanced the binding of Abeta with anionic PG membranes in the liquid crystalline phase and with zwitterionic PC membranes in the gel phase. The binding modes of Abeta to these membranes differ in terms of the depth of the protein in the membrane and of the protein secondary structure. Amyloid fibril formation of Abeta was accelerated in the presence of the vesicles and at high protein-to-lipid ratios. Under these conditions, the protein highly accumulated on the surfaces, as demonstrated by high (∼10ˆ7 Mˆ-1) binding constants. Our findings suggest that packing defects on membranes with high curvatures might result in the accumulation of toxic protein aggregates.

Protocols for Exosome Isolation and RNA Profiling.

Exosomes are small extracellular vesicles of multivesicular bodies derived from the cell endosome. Molecules have a characteristic composition both in the membrane and in the carried load. These particles are located in the culture medium of cells in vitro and in vivo body fluids, being synthesized by almost all kind of cells. The most characteristic molecule transported within is the RNA with a large clinical potential. In this chapter, the methodology for isolation of exosomes and subsequent purification of the RNA is reviewed.

ESCRT (Endosomal Sorting Complex Required for Transport) Machinery Is Essential for Acrosomal Exocytosis in Human Sperm.

The sperm acrosome reaction is a unique, regulated exocytosis characterized by the secretion of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal and plasma membranes. In previous reports, we have shown that inward invaginations of the acrosomal membrane delineate ring-shaped membrane microdomains that contact the plasma membrane. We have postulated that the opening and expansion of fusion pores along these rings trigger acrosomal exocytosis. The invaginations of the acrosomal membrane topologically resemble the deformations of the endosomal membrane leading to the assembly of luminal vesicles in multivesicular bodies. In fact, intra-acrosomal vesicles are also formed during acrosomal exocytosis. Endosomal sorting complex required for transport (ESCRT) participates in the organization of membrane microdomains that are invaginated and released as intraluminal vesicles in endosomes. We report here that members of ESCRT I (TSG101), ESCRT III (CHMP4), and the AAA ATPase VPS4 are present in the acrosomal region of the human sperm. Perturbing the function of these factors with antibodies or recombinant proteins inhibited acrosomal exocytosis in permeabilized cells. A similar effect was observed with a dominant-negative mutant of VPS4A cross-linked to a cell-penetrating peptide in nonpermeabilized sperm stimulated with a calcium ionophore. When the function of ESCRTs was inhibited, acrosomes showed abnormal deformation of the acrosomal membrane, and SNARE proteins that participate in acrosomal exocytosis failed to be stabilized in neurotoxin-resistant complexes. However, the growing of membrane invaginations was not blocked, and numerous intra-acrosomal vesicles were observed. These observations indicate that ESCRT-mediated processes are essential for acrosomal secretion, implicating these multifunctional complexes in an exocytic event crucial for sperm-egg fusion.