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Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.

ABSTRACT: Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming ?1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1's lipid and sugar components and CaV2.3's ?1E and ?2? subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

SUBMITTER: Cohen R

PROVIDER: S-EPMC3947087 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.

Cohen Roy R Buttke Danielle E DE Asano Atsushi A Mukai Chinatsu C Nelson Jacquelyn L JL Ren Dongjun D Miller Richard J RJ Cohen-Kutner Moshe M Atlas Daphne D Travis Alexander J AJ

Developmental cell 20140201 3

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong s ...[more]

PMID: 24525187

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Project description:Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP2, and that it is phosphorylated during acrosomal exocytosis.

Project description:?-SNAP has an essential role in membrane fusion that consists of bridging cis SNARE complexes to NSF. ?-SNAP stimulates NSF, which releases itself, ?-SNAP, and individual SNAREs that subsequently re-engage in the trans arrays indispensable for fusion. ?-SNAP also binds monomeric syntaxin and NSF disengages the ?-SNAP/syntaxin dimer. Here, we examine why recombinant ?-SNAP blocks secretion in permeabilized human sperm despite the fact that the endogenous protein is essential for membrane fusion. The only mammalian organism with a genetically modified ?-SNAP is the hyh mouse strain, which bears a M105I point mutation; males are subfertile due to defective sperm exocytosis. We report here that recombinant ?-SNAP-M105I has greater affinity for the cytosolic portion of immunoprecipitated syntaxin than the wild type protein and in consequence NSF is less efficient in releasing the mutant. ?-SNAP-M105I is a more potent sperm exocytosis blocker than the wild type and requires higher concentrations of NSF to rescue its effect. Unlike other fusion scenarios where SNAREs are subjected to an assembly/disassembly cycle, the fusion machinery in sperm is tuned so that SNAREs progress uni-directionally from a cis configuration in resting cells to monomeric and subsequently trans arrays in cells challenged with exocytosis inducers. By means of functional and indirect immunofluorescense assays, we show that recombinant ?-SNAPs--wild type and M105I--inhibit exocytosis because they bind monomeric syntaxin and prevent this SNARE from assembling with its cognates in trans. Sequestration of free syntaxin impedes docking of the acrosome to the plasma membrane assessed by transmission electron microscopy. The N-terminal deletion mutant ?-SNAP-(160-295), unable to bind syntaxin, affects neither docking nor secretion. The implications of this study are twofold: our findings explain the fertility defect of hyh mice and indicate that assembly of SNAREs in trans complexes is essential for docking.

Dataset Information

Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.

Publications

Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.

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