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Involvement of complexin 2 in docking, locking and unlocking of different SNARE complexes during sperm capacitation and induced acrosomal exocytosis.

Tsai PS, Brewis IA, van Maaren J, Gadella BM - PLoS ONE (2012)

Bottom Line: By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes.These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2.The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+) which are all involved in AE make sperm an ideal model for studying exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Farm Animal Health, Graduate School of Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

ABSTRACT
Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca(2+)-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+) which are all involved in AE make sperm an ideal model for studying exocytosis.

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Identification of the complexin-SNAREpin complex in the non-raft fractions.(A) A Q-SNARE complex containing syntaxin 3 and SNAP 23 was detected on Western blots of the high sucrose gradient fractions (#11–13, indicated with number signs). This complex was found to lack the complementary R-SNARE VAMP 2 and thus enabled us to distinguish two different SNARE containing complexes in the porcine sperm. (B) The previously identified SNARE complex responsible for acrosome to plasma membrane docking (containing syntaxin 1B/SNAP 23/VAMP 3 [8]) was not observed in the same fractionated DRM sample at the high M.W. position (at 75 kDa) indicating the dissociation of this SNARE complex upon AE. Arrowheads indicate monomeric form of SNARE proteins present in these fractions. The arrows indicate the expected molecular weight of protein on the Western blot.
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pone-0032603-g005: Identification of the complexin-SNAREpin complex in the non-raft fractions.(A) A Q-SNARE complex containing syntaxin 3 and SNAP 23 was detected on Western blots of the high sucrose gradient fractions (#11–13, indicated with number signs). This complex was found to lack the complementary R-SNARE VAMP 2 and thus enabled us to distinguish two different SNARE containing complexes in the porcine sperm. (B) The previously identified SNARE complex responsible for acrosome to plasma membrane docking (containing syntaxin 1B/SNAP 23/VAMP 3 [8]) was not observed in the same fractionated DRM sample at the high M.W. position (at 75 kDa) indicating the dissociation of this SNARE complex upon AE. Arrowheads indicate monomeric form of SNARE proteins present in these fractions. The arrows indicate the expected molecular weight of protein on the Western blot.

Mentions: The 40 kDa complexin positive band reflects the germ-line specific stable dimeric form of complexin (Fig. 2C). We speculate that the 79 kDa complexin positive complex represents the recently reported pre-fusion SNAREpin-complexin complex that consists of two Q-SNARE proteins (syntaxin, SNAP) and complexin [22]. To investigate this possibility, we performed Western-blotting of the sucrose gradient fractions from sperm samples with antibodies against potential SNAREpin forming candidates. We indeed observed the same 79 kDa syntaxin 3 and SNAP 23 positive protein complexes in the non-DRM fractions (Fig. 5A, marked with number signs). More importantly, this 79 kDa protein complex had no affinity for anti-VAMP 2 antibodies. With these data we were able to discriminate between the SNAREpin-complexin complex (syntaxin 3/SNAP 23/complexin 2) and the trimeric form of SNARE complex (syntaxin3/SNAP 23/VAMP2) and showed the bicarbonate-dependent formation of both protein complexes in porcine sperm. In line with our earlier observation on the isolated MVs (Fig. 2), in Ca2+ ionophore treated (AE) sperm, we found a major bicarbonate dependent shift of the protein complex from 79 kDa to 95 kDa (Fig. 4C, Fig. 5A). The presence of this 95 kDa complexin-containing complex not only supported the findings of Figure 2, but also demonstrated that complexin was indeed interacting with the trimeric syntaxin 3/SNAP 23/VAMP 2 (to form the 95 kDa complex) and was equally efficient in binding syntaxin 3/SNAP 23 complex (to form a 79 kDa complexin-SNAREpin) prior to attracting VAMP 2 for the 95 kDa complex when Ca2+ ionophore was added. All together this data indicated the additional participation of the complementary VAMP 2 with the SNAREpin-complexin complex upon the rise of intracellular calcium levels that initiate AE. Interestingly, we were not able to recover previously identified SNARE protein complexes involved in acrosome to plasma membrane docking [8] in the same fractionated DRM samples, where only the monomeric form of syntaxin 1B and VAMP 3 were detected (Fig. 5B). The presence of these monomeric SNARE proteins (syntaxin 1B, VAMP 3) in the raft fraction and the absence of high M.W. docking SNARE complex suggests the dissociation of this “docking” SNARE complex during the formation of the SNARE complex that drives AE fusions and contains syntaxin3/SNAP 23/VAMP2.


Involvement of complexin 2 in docking, locking and unlocking of different SNARE complexes during sperm capacitation and induced acrosomal exocytosis.

Tsai PS, Brewis IA, van Maaren J, Gadella BM - PLoS ONE (2012)

Identification of the complexin-SNAREpin complex in the non-raft fractions.(A) A Q-SNARE complex containing syntaxin 3 and SNAP 23 was detected on Western blots of the high sucrose gradient fractions (#11–13, indicated with number signs). This complex was found to lack the complementary R-SNARE VAMP 2 and thus enabled us to distinguish two different SNARE containing complexes in the porcine sperm. (B) The previously identified SNARE complex responsible for acrosome to plasma membrane docking (containing syntaxin 1B/SNAP 23/VAMP 3 [8]) was not observed in the same fractionated DRM sample at the high M.W. position (at 75 kDa) indicating the dissociation of this SNARE complex upon AE. Arrowheads indicate monomeric form of SNARE proteins present in these fractions. The arrows indicate the expected molecular weight of protein on the Western blot.
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Related In: Results  -  Collection

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pone-0032603-g005: Identification of the complexin-SNAREpin complex in the non-raft fractions.(A) A Q-SNARE complex containing syntaxin 3 and SNAP 23 was detected on Western blots of the high sucrose gradient fractions (#11–13, indicated with number signs). This complex was found to lack the complementary R-SNARE VAMP 2 and thus enabled us to distinguish two different SNARE containing complexes in the porcine sperm. (B) The previously identified SNARE complex responsible for acrosome to plasma membrane docking (containing syntaxin 1B/SNAP 23/VAMP 3 [8]) was not observed in the same fractionated DRM sample at the high M.W. position (at 75 kDa) indicating the dissociation of this SNARE complex upon AE. Arrowheads indicate monomeric form of SNARE proteins present in these fractions. The arrows indicate the expected molecular weight of protein on the Western blot.
Mentions: The 40 kDa complexin positive band reflects the germ-line specific stable dimeric form of complexin (Fig. 2C). We speculate that the 79 kDa complexin positive complex represents the recently reported pre-fusion SNAREpin-complexin complex that consists of two Q-SNARE proteins (syntaxin, SNAP) and complexin [22]. To investigate this possibility, we performed Western-blotting of the sucrose gradient fractions from sperm samples with antibodies against potential SNAREpin forming candidates. We indeed observed the same 79 kDa syntaxin 3 and SNAP 23 positive protein complexes in the non-DRM fractions (Fig. 5A, marked with number signs). More importantly, this 79 kDa protein complex had no affinity for anti-VAMP 2 antibodies. With these data we were able to discriminate between the SNAREpin-complexin complex (syntaxin 3/SNAP 23/complexin 2) and the trimeric form of SNARE complex (syntaxin3/SNAP 23/VAMP2) and showed the bicarbonate-dependent formation of both protein complexes in porcine sperm. In line with our earlier observation on the isolated MVs (Fig. 2), in Ca2+ ionophore treated (AE) sperm, we found a major bicarbonate dependent shift of the protein complex from 79 kDa to 95 kDa (Fig. 4C, Fig. 5A). The presence of this 95 kDa complexin-containing complex not only supported the findings of Figure 2, but also demonstrated that complexin was indeed interacting with the trimeric syntaxin 3/SNAP 23/VAMP 2 (to form the 95 kDa complex) and was equally efficient in binding syntaxin 3/SNAP 23 complex (to form a 79 kDa complexin-SNAREpin) prior to attracting VAMP 2 for the 95 kDa complex when Ca2+ ionophore was added. All together this data indicated the additional participation of the complementary VAMP 2 with the SNAREpin-complexin complex upon the rise of intracellular calcium levels that initiate AE. Interestingly, we were not able to recover previously identified SNARE protein complexes involved in acrosome to plasma membrane docking [8] in the same fractionated DRM samples, where only the monomeric form of syntaxin 1B and VAMP 3 were detected (Fig. 5B). The presence of these monomeric SNARE proteins (syntaxin 1B, VAMP 3) in the raft fraction and the absence of high M.W. docking SNARE complex suggests the dissociation of this “docking” SNARE complex during the formation of the SNARE complex that drives AE fusions and contains syntaxin3/SNAP 23/VAMP2.

Bottom Line: By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes.These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2.The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+) which are all involved in AE make sperm an ideal model for studying exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Farm Animal Health, Graduate School of Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

ABSTRACT
Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca(2+)-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+) which are all involved in AE make sperm an ideal model for studying exocytosis.

Show MeSH
Related in: MedlinePlus