Fusion of docked membranes requires the armadillo repeat protein Vac8p.

The discovery of molecules required for membrane fusion has revealed a remarkably conserved mechanism that centers upon the formation of a complex of SNARE proteins. However, whether the SNARE proteins or other components catalyze the final steps of membrane fusion in vivo remains unclear. Understanding this last step depends on the identification of molecules that act late in the fusion process. Here we demonstrate that in Saccharomyces cerevisiae, Vac8p, a myristoylated and palmitoylated armadillo repeat protein, is required for homotypic vacuole fusion. Vac8p is palmitoylated during the fusion reaction, and the ability of Vac8p to be palmitoylated appears to be necessary for its function in fusion. Both in vivo and in vitro analyses show that Vac8p functions after both Rab-dependent vacuole docking and the formation of trans-SNARE pairs. We propose that Vac8p may bind the fusion machinery through its armadillo repeats and that palmitoylation brings this machinery to a specialized lipid domain that facilitates bilayer mixing.     

Two Fission Yeast Rab7 Homologs, Ypt7 and Ypt71, Play Antagonistic Roles in the Regulation of Vacuolar Morphology

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe ( S. pombe ) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71 ⁺ exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7 Δ cells. Like ypt7 Δ cells, overexpression of ypt71 ⁺ caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/α3-L7.     

Phagosome fusion vesicles of paramecium. I. Thin-section morphology

Ultrastructural studies of the digestive system of Paramecium caudatum focusing on the first 5 min of digestive-vacuole age reveal a set of vesicles, named phagosome fusion vesicles (PFVs), which fuse with the digestive vacuole just after the vacuoles are released from the cytopharynx and concomitant with vacuole acidification. Serial thin-sections of vacuoles labeled with horseradish peroxidase (HRP) and/or latex beads in pulse-chase studies were observed. PFVs, irregularly shaped, electron-translucent vesicles ranging from a small diameter to over 1 micro, are first seen in the region of the cytopharynx where they bind to the nascent vacuole membrane. Within 30 sec of vacuole release the PFVs fuse with the vacuole where they remain for a brief time connected to the vacuole by a narrow annulus. HRP-reaction product is found in vacuoles but not in PFVs before PFVs fuse with the vacuoles. After fusion with PFVs HRP is quickly inactivated. Tubular extensions of vacuole membrane then form between the fused PFVs. By 3 to 5 min both PFVs and tubules disappear from the vacuole surface and lysosomes appear in their place. We believe the tubules are pinched off as PFV membrane is being added to the vacuole. Microfilaments coat the membrane during all these dynamic events. Since the pH of the vacuole becomes acid during the first few minutes, we are now looking for a direct correlation between PFV fusion and acidification.     

The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP

Many proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions. Vesicles carrying vacuolar cargo bud from the trans-Golgi network are thought to fuse with a pre-vacuolar compartment before being finally transported to the vacuole. In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species. A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t-SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle. However, the biochemical function of these proteins has remained elusive. Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion. This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment. In addition, we have shown that AtPEP12p can bind to alpha-SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE. These preliminary data suggest that AtPEP12p may function jointly with alpha-SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre-vacuolar compartment.     

Involvement of smooth and coated vesicles in the function of the contractile vacuole complex of some cryptophycean flagellates

The contractile vacuole complex of cryptophycean flagellates comprises the contractile vacuole, a pore and a vesicular spongiome. A minority of spongiome vesicles bear a 15-nm coat on the cytoplasmic surface of the membrane. The coat superficially resembles a clathrin coat. The majority of vesicles are smooth surfaced. Both types of vesicles are found at the same time. Smooth vesicles can be seen in profile suggesting vesicle-vesicle and vesicle-vacuole fusion. It is suggested that smooth vesicles are involved in the segregation of fluid from the cytoplasm and in filling the vacuole. Coated elements exist only as independent vesicles and as coated pits in the contractile vacuole membrane. There is no evidence of fusion of coated vesicles. It is suggested that coated vesicles function to retrieve specific membrane components from the contractile vacuole.     

The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex

The regulation of cellular membrane flux is poorly understood. Yeast respond to hypertonic stress by fragmentation of the normally large, low copy vacuole. We used this phenomenon as the basis for an in vivo screen to identify regulators of vacuole membrane dynamics. We report here that maintenance of the fragmented phenotype requires the vacuolar casein kinase I Yck3: when Yck3 is absent, salt-stressed vacuoles undergo fission, but reassemble in a SNARE-dependent manner, suggesting that vacuole fusion is disregulated. Accordingly, when Yck3 is deleted, in vitro vacuole fusion is increased, and Yck3 overexpression blocks fusion. Morphological and functional studies show that Yck3 modulates the Rab/homotypic fusion and vacuole protein sorting complex (HOPS)-dependent tethering stage of vacuole fusion. Intriguingly, Yck3 mediates phosphorylation of the HOPS subunit Vps41, a bi-functional protein involved in both budding and fusion during vacuole biogenesis. Because Yck3 also promotes efficient vacuole inheritance, we propose that tethering complex phosphorylation is a part of a general, switch-like mechanism for driving changes in organelle architecture.     

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.     

The Yeast ATP-binding Cassette (ABC) Transporter Ycf1p Enhances the Recruitment of the Soluble SNARE Vam7p to Vacuoles for Efficient Membrane Fusion

The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd²⁺ transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1p^K669M) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion.     

LC3 and GATE-16 N termini mediate membrane fusion processes required for autophagosome biogenesis

Autophagy is a unique membrane trafficking pathway describing the formation and targeting of double membrane autophagosomes to the vacuole/lysosome. The biogenesis of autophagosomes and their delivery to the vacuole/lysosome depend on multiple membrane fusion events. Using a cell-free system, we have investigated the ability of LC3 and GATE-16, two mammalian Atg8 orthologs, to mediate membrane fusion. We found that both proteins promote tethering and membrane fusion, mediated by the proteins' N-terminal α helices. We further show that short, 10 amino acid long synthetic peptides derived from the N terminus of LC3 or GATE-16 are sufficient to promote membrane fusion. Our data indicate that the fusion activity of LC3 is mediated by positively charged amino acids, whereas the activity of GATE-16 is mediated by hydrophobic interactions. Finally, we demonstrate that LC3 and GATE-16 N termini in general and specific residues needed for the fusion activity are essential for the proteins role in autophagosome biogenesis.     

V-ATPase, ScNhx1p and yeast vacuole fusion

Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.     

Stimulation of actin polymerization by vacuoles via Cdc42p-dependent signaling

We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.     

Cdc42p and Rho1p are sequentially activated and mechanistically linked to vacuole membrane fusion

Small monomeric GTPases act as molecular switches, regulating many biological functions via activation of membrane localized signaling cascades. Activation of their switch function is controlled by GTP binding and hydrolysis. Two Rho GTPases, Cdc42p and Rho1p, are localized to the yeast vacuole where they regulate membrane fusion. Here, we define a method to directly examine vacuole membrane Cdc42p and Rho1p activation based on their affinity to probes derived from effectors. Cdc42p and Rho1p showed unique temporal activation which aligned with distinct subreactions of in vitro vacuole fusion. Cdc42p was rapidly activated in an ATP-independent manner while Rho1p activation was kinetically slower and required ATP. Inhibitors that are known to block vacuole membrane fusion were examined for their effect on Cdc42p and Rho1p activation. Rdi1p, which inhibits the dissociation of GDP from Rho proteins, blocked both Cdc42p and Rho1p activation. Ligands of PI(4,5)P₂ specifically inhibited Rho1p activation while pre-incubation with U73122, which targets Plc1p function, increased Rho1p activation. These results define unique activation mechanisms for Cdc42p and Rho1p, which may be linked to the vacuole membrane fusion mechanism.     

Piecemeal microautophagy of the nucleus requires the core macroautophagy genes

Autophagy is a diverse family of processes that transport cytoplasm and organelles into the lysosome/vacuole lumen for degradation. During macroautophagy cargo is packaged in autophagosomes that fuse with the lysosome/vacuole. During microautophagy cargo is directly engulfed by the lysosome/vacuole membrane. Piecemeal microautophagy of the nucleus (PMN) occurs in Saccharomyces cerevisiae at nucleus-vacuole (NV) junctions and results in the pinching-off and release into the vacuole of nonessential portions of the nucleus. Previous studies concluded macroautophagy ATG genes are not absolutely required for PMN. Here we report using two biochemical assays that PMN is efficiently inhibited in atg mutant cells: PMN blebs are produced, but vesicles are rarely released into the vacuole lumen. Electron microscopy of arrested PMN structures in atg7, atg8, and atg9 mutant cells suggests that NV-junction–associated micronuclei may normally be released from the nucleus before their complete enclosure by the vacuole membrane. In this regard PMN is similar to the microautophagy of peroxisomes (micropexophagy), where the side of the peroxisome opposite the engulfing vacuole is capped by a structure called the “micropexophagy-specific membrane apparatus” (MIPA). The MIPA contains Atg proteins and facilitates terminal enclosure and fusion steps. PMN does not require the complete vacuole homotypic fusion genes. We conclude that a spectrum of ATG genes is required for the terminal vacuole enclosure and fusion stages of PMN.     

Enhanced membrane fusion in sterol-enriched vacuoles bypasses the Vrp1p requirement

Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Delta growth defect selective for vacuole function. VRP1 encodes verprolin, an actin-binding protein that colocalizes to vacuoles. The vrp1Delta mutant has fragmented vacuoles in vivo and isolated vacuoles do not fuse in vitro, indicative of a Vrp1p requirement for membrane fusion. ERG6 overexpression rescues vrp1Delta vacuole fusion in a cytosol-dependent manner. Cytosol prepared from the vrp1Delta strain remains active; therefore, cytosol is not resupplying Vrp1p. Las17p (Vrp1p functional partner) antibodies, which inhibit wild-type vacuole fusion, do not inhibit the fusion of vacuoles from the vrp1Delta-ERG6 overexpression strain. Vacuole-associated actin turnover is decreased in the vrp1Delta strain, but recovered by ERG6 overexpression linking sterol enrichment to actin remodeling. Therefore, the Vrp1p/Las17p requirement for membrane fusion is bypassed by increased sterols, which promotes actin remodeling as part the membrane fusion mechanism.     

Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking

Rho GTPases, which control polarized cell growth through cytoskeletal reorganization, have recently been implicated in the control of endo- and exocytosis. We now report that both Rho1p and Cdc42p have a direct role in mediating the docking stage of homotypic vacuole fusion. Vacuoles prepared from strains with temperature-sensitive alleles of either Rho1p or Cdc42p are thermolabile for fusion. RhoGDI (Rdi1p), which extracts Rho1p and Cdc42p from the vacuole membrane, blocks vacuole fusion. The Rho GTPases can not fulfill their function as long as priming and Ypt7p-dependent tethering are inhibited. However, reactions that are reversibly blocked after docking by the calcium chelator BAPTA have passed the point of sensitivity to Rdi1p. Extraction and removal of Ypt7p, Rho1p and Cdc42p from docked vacuoles (by Gdi1p, Gyp7p and Rdi1p) does not impede subsequent membrane fusion, which is still sensitive to GTPgammaS. Thus, multiple GTPases act in a defined sequence to regulate the docking steps of vacuole fusion.     

Cdc42p Is Activated during Vacuole Membrane Fusion in a Sterol-dependent Subreaction of Priming

Cdc42p is a Rho GTPase that initiates signaling cascades at spatially defined intracellular sites for many cellular functions. We have previously shown that Cdc42p is localized to the yeast vacuole where it initiates actin polymerization during membrane fusion. Here we examine the activation cycle of Cdc42p during vacuole membrane fusion. Expression of either GTP- or GDP-locked Cdc42p mutants caused several morphological defects including enlarged cells and fragmented vacuoles. Stimulation of multiple rounds of fusion enhanced vacuole fragmentation, suggesting that cycles of Cdc42p activation, involving rounds of GTP binding and hydrolysis, are required to propagate Cdc42p signaling. We developed an assay to directly examine Cdc42p activation based on affinity to a probe derived from the p21-activated kinase effector, Ste20p. Cdc42p was rapidly activated during vacuole membrane fusion, which kinetically coincided with priming subreaction. During priming, Sec18p ATPase activity dissociates SNARE complexes and releases Sec17p, however, priming inhibitors such as Sec17p and Sec18p ligands did not block Cdc42p activation. Therefore, Cdc42p activation seems to be a parallel subreaction of priming, distinct from Sec18p activity. Specific mutants in the ergosterol synthesis pathway block both Sec17p release and Cdc42p activation. Taken together, our results define a novel sterol-dependent subreaction of vacuole priming that activates cycles of Cdc42p activity to facilitate membrane fusion.     

VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

Arabidopsis VPS18 plays an important role in regulating pollen tube growth through mediating the late endocytic trafficking and secretion of pectin and associated enzymes to the cell wall.     

Vac8p release from the SNARE complex and its palmitoylation are coupled and essential for vacuole fusion

Activated fatty acids stimulate budding and fusion in several cell-free assays for vesicular transport. This stimulation is thought to be due to protein palmitoylation, but relevant substrates have not yet been identified. We now report that Vac8p, a protein known to be required for vacuole inheritance, becomes palmitoylated when isolated yeast vacuoles are incubated under conditions that allow membrane fusion. Similar requirements for Vac8p palmitoylation and vacuole fusion, the inhibition of vacuole fusion by antibodies to Vac8p and the strongly reduced fusion of vacuoles lacking Vac8p suggest that palmitoylated Vac8p is essential for homotypic vacuole fusion. Strikingly, palmitoylation of Vac8p is blocked by the addition of antibodies to Sec18p (yeast NSF) only. Consistent with this, a portion of Vac8p is associated with the SNARE complex on vacuoles, which is lost during Sec18p- and ATP-dependent priming. During or after SNARE complex disassembly, palmitoylation occurs and anchors Vac8p to the vacuolar membrane. We propose that palmitoylation of Vac8p is regulated by the same machinery that controls membrane fusion.     

LegC3, an Effector Protein from Legionella pneumophila,Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.     

Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface

The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze-fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed.