Complexin/Synaptotagmin Interplay Controls Acrosomal Exocytosis

Abstract

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. Regulated secretion requires SNARE proteins and, in neurons, also synaptotagmin I and complexin. Recent reports suggest that complexin imposes a fusion block that is released by Ca 2؉ and synaptotagmin I. However, no direct evidence for this model in secreting cells has been provided and whether this complexin/ synaptotagmin interplay functions in other types of secretion is unknown. In this report, we show that the C2B domain of synaptotagmin VI and an anti-complexin antibody blocked the formation of trans SNARE complexes in permeabilized human sperm, and that this effect was reversed by adding complexin. In contrast, an excess of complexin stopped exocytosis at a later step, when SNAREs were assembled in loose trans complexes. Interestingly, this blockage was released by the addition of the synaptotagmin VI C2B domain in the presence of Ca 2؉ . We have previously demonstrated that the activity of this domain is regulated by protein kinase C-mediated phosphorylation. Here, we show that a phosphomimetic mutation in the polybasic region of the C2B domain strongly affects its Ca 2؉ and phospholipids binding properties. Importantly, this mutation completely abrogates its ability to rescue the complexin block. Our results show that the functional interplay between complexin and synaptotagmin has a central role in a physiological secretion event, and that this interplay can be modulated by phosphorylation of the C2B domain.All types of intracellular membrane fusions, including regulated exocytosis (i.e. the stimulus-triggered fusion of secretory vesicles with the plasma membrane), require proteins from the SNARE 3 family (1). SNAREs are small membrane proteins that form stable hetero-oligomeric complexes consisting of a bundle of four parallel helices. Assembly of trans SNARE complexes between SNAREs from two opposing membranes is a key event for membrane fusion. SNAREs residing on the same membrane can form cis complexes that must be disassembled to render free SNAREs competent for membrane fusion. The exocytotic SNAREs involved in neurotransmission are syntaxin1A and SNAP25 in the plasmalemma, and synaptobrevin 2 (also called VAMP2) in secretory vesicles. These proteins are the targets of botulinum and tetanus toxins, a set of highly specific zinc-dependent endoproteases (2). Only when not assembled in tight complexes are SNAREs susceptible to cleavage, making these toxins excellent tools for the diagnosis of SNARE assembly status.Additional proteins that are key for Ca 2ϩ -triggered neurotransmitter release include complexins and synaptotagmins (3). Complexins are small cytosolic proteins that bind with high affinity to SNARE complexes (4), forming an antiparallel ␣ helix that inserts into a groove between the synaptobrevin and syntaxin helices (5). Experiments where complexins were deleted or overexpressed have suggested that they play ...

Complexin-I-deficient sperm are subfertile due to a defect in zona pellucida penetration

“…Their proteins were localized in or around the developing acrosome of spermatids and the acrosomal region of cauda epididymal sperm. This is consistent with a recent study localizing complexins I and II in human sperm, which are, in contrast to mouse sperm, paddle-shaped (Roggero et al 2007). Furthermore, we demonstrated that sperm complexins I and II fractionate as mainly cytosolic proteins.…”

“…Furthermore, antibodies to complexins I and II, when added to permeabilized human sperm, block the acrosome reaction (Roggero et al 2007). Additional data from permeabilized human sperm suggest that the calcium sensor synaptotagmin VI can displace complexins from the SNARE complex, allowing acrosomal exocytosis (Roggero et al 2007). This displacement may be regulated by synaptotagmin phosphorylation (Roggero et al 2007).…”

“…They are encoded by two different genes and differently regulated (Abderrahmani et al 2004); however, they have a similar function, to regulate SNARE-mediated exocytosis (McMahon et al 1995). A recent study using permeabilized sperm suggested that either complexin I or II is necessary to promote acrosomal exocytosis (Roggero et al 2007). By contrast, the observation that sperm contain both complexins I and II but that only complexin I deficiency affects fertility suggests that complexin I has a unique function in acrosomal exocytosis; complexin II cannot compensate for the absence of complexin I during this process, even though complexin II is in the acrosomal region of complexin-I-deficient sperm (Fig.…”

“…Additional data from permeabilized human sperm suggest that the calcium sensor synaptotagmin VI can displace complexins from the SNARE complex, allowing acrosomal exocytosis (Roggero et al 2007). This displacement may be regulated by synaptotagmin phosphorylation (Roggero et al 2007). Our results are consistent with this putative function for complexin I but not complexin II.…”

ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation

“…Purified Rab3A, botulinum neurotoxin C and E (BoNT/C and BoNT/E), tetanus toxin (TeTx), C2B domain of synaptotagmin VI, and GST-RIM-RBD were produced by M. Bustos (IHEM-CONICET, Universidad Nacional de Cuyo, Mendoza, Argentina) as described previously (38,39). The plasmids pET21b-ARF6 and pBB131 were generously provided by Drs.…”