Reconstitution of calcium-mediated exocytosis of dense-core vesicles

Abstract

Regulated exocytosis is a process by which neurotransmitters, hormones, and secretory proteins are released from the cell in response to elevated levels of calcium. In cells, secretory vesicles are targeted to the plasma membrane, where they dock, undergo priming, and then fuse with the plasma membrane in response to calcium. The specific roles of essential proteins and how calcium regulates progression through these sequential steps are currently incompletely resolved. We have used purified neuroendocrine dense-core vesicles and artificial membranes to reconstruct in vitro the serial events that mimic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent membrane docking and fusion during exocytosis. Calcium recruits these vesicles to the target membrane aided by the protein CAPS (calcium-dependent activator protein for secretion), whereas synaptotagmin catalyzes calcium-dependent fusion; both processes are dependent on phosphatidylinositol 4,5-bisphosphate. The soluble proteins Munc18 and complexin-1 are necessary to arrest vesicles in a docked state in the absence of calcium, whereas CAPS and/or Munc13 are involved in priming the system for an efficient fusion reaction.

Fig. 1. Reconstitution of DCV membrane fusion.

(A) Western blots of the postnuclear supernatant (PNS), postmitochondrial supernatant (PMS), and individual fractions of the iodixanol gradient. Fraction 9 containing the DCV content marker secretogranin was collected as DCVs. DCVs are also enriched in the calcium-interacting proteins syt-1, syt-9, CAPS-1, and CAPS-2. Contamination by mitochondria [succinate-ubiquinone oxidoreductase (SQR)] and endoplasmic reticulum (calnexin) is minimally detected in the DCV fraction. (B) Fusion of purified DCVs with liposomes containing syntaxin-1a (residues 183 to 288):SNAP-25 at a lipid/protein ratio of 500 (black) and a lipid composition of porcine brain PC (bPC):porcine brain PE (bPE):porcine brain PS (bPS):cholesterol (Chol):PI:PI(4,5)P₂:Rh-DOPE:NBD-DOPE (23.5:23.5:15:30:4:1:1.5:1.5). Lipid mixing (NBD dequenching) traces shown are averages of four repeated experiments. The presence of synaptobrevin-2 (residues 1 to 96) (syb96) inhibitor peptide (2 mM) abolishes lipid mixing (red). See Materials and Methods for expanded definition of the abbreviated lipid names used here. (C) Single fusion event of NPY-Ruby–labeled DCV with a planar supported bilayer containing syntaxin-1a (syx) (residues 183 to 288):SNAP-25 (lipid/protein = 3000). The black line is an intensity trace of NPY-Ruby fluorescence. The initial increase is caused by binding of the DCV to the planar bilayer within the total internal reflection fluorescence (TIRF) field of the microscope. This intensity is constant during docking; fusion begins with an abrupt decrease, sharp increase, and then following decay in fluorescence to baseline. As described in detail in fig. S1 and the two-step fusion/diffusion model in the Supplementary Materials, this profile reflects collapse of the DCV into the planar bilayer, where NPY-Ruby initially moves forward in the TIRF field and then diffuses away beneath the bilayer. (D) Docking of DCVs to planar supported bilayers that differ in target SNARE (t-SNARE) content in the absence (black) and presence (red) of Ca²⁺. Syb96 inb refers to the presence of the inhibitor peptide. The total number of events in each case is shown in table S1. dSN25 refers to dodecylated SNAP-25. (E) Docking as a function of [Ca²⁺] (black) or [Mg²⁺] (blue) in the single-vesicle planar supported bilayer assay with a K_1/2 [Ca²⁺] of 236 ± 46 μM. Table S2 contains a summary of the total number of docking and fusion events. (F) Delay time between docking and fusion (Δt_D) at different [Ca²⁺] (black, 100 μM EDTA; red, 50 μM Ca²⁺; green, 100 μM Ca²⁺; cyan, 150 μM Ca²⁺; blue, 200 μM Ca²⁺) shown as cumulative distribution functions of single DCV fusion events normalized to the fusion probability. The kinetics were fit with a parallel reaction model N(t) = N(1 − e^−kt)^m, where N is the fusion probability, k is the rate constant, and m is the number of parallel reactions occurring (40). Summary of fit values is shown in table S3. Cumulative distribution functions for additional (higher) [Ca²⁺] and [Mg²⁺] are shown in fig. S3. (G) The fusion probabilities (black) and rate constants (red) for parallel reactions are shown as functions of [Ca²⁺] with K_1/2 [Ca²⁺] of 60 ± 8 μM and 61 ± 8 μM, respectively. For membranes containing syntaxin-1a (residues 183 to 288):SNAP-25 (C to G), the lipid composition was bPC:bPE:bPS:Chol:PI:PI(4,5)P₂ (25:25:15:30:4:1) for all experiments. (H) The effect on docking and fusion of DCVs when PI(4,5)P₂ is increased or decreased in the planar bilayers containing bPC:bPE:bPS:Chol:[PI + PI(4,5)P₂] (25:25:15:30:5) or in the absence of charged lipids [bPC:bPE:Chol (35:35:30)]. Summaries of docking and fusion events and fitting results of fusion kinetics are shown in tables S5 and S6, respectively.

Fig. 2. Molecular origin of DCV calcium response.

The role of synaptotagmin (A and B) and CAPS (C and D) on DCV docking and fusion in the presence of 100 μM EDTA (black) or 100 μM Ca²⁺ (red). Wild-type preparations of DCVs were compared either to those that were treated with function-blocking antibodies or to those that were purified from cells subjected to shRNA-mediated knockdown (KD). In the latter case, expression of RNAi-resistant syt1 or CAPS-1 was used as a control. Black and red bar data were obtained in the absence and presence of Ca²⁺, respectively. Table S7 contains a summary of events, and table S8 contains fitting results. As indicated in Materials and Methods, docking values for preparations of DCVs from wild-type, knockdown, and RNAi rescue cell lines were individually normalized to the value obtained in the presence of 100 μM EDTA, enabling comparison of the relative effects elicited by calcium among the different preparations. This strategy does not enable us to rule out the possible effects of syt or CAPS knockdown on docking in the absence of calcium. For antibody-treated samples, we observed no significant effect on docking in the absence of calcium.

Fig. 3. Effects of Munc18, complexin, and the regulatory H_abc domain of syntaxin-1a on docking and fusion.

Docking and fusion probability for DCVs to planar supported bilayers containing truncated syntaxin-1a (residues 183 to 288):SNAP-25 (A and B) or full-length syntaxin-1a (1 to 288):SNAP-25 (C and D) in the presence of 0.5 μM Munc18, 2 μM complexin-1 (cpx1), or both. Black and red bar data were obtained in the absence and presence of Ca²⁺, respectively. Summary of events and fitting results are presented in tables S9 and S10. Western blots show that Munc18 and complexin-1 were not detectable in DCV-enriched fractions prepared by centrifugation (fig. S6).

Fig. 4. Calcium-triggered DCV fusion.

(A) Intensity trace of a single DCV calcium-triggered fusion event (black line) for a DCV docked to a planar supported bilayer in the presence of 0.5 μM Munc18 and 2 μM complexin-1. Fusion was triggered with a buffer [120 mM potassium glutamate, 20 mM potassium acetate, and 20 mM Hepes (pH 7.4)] containing Ca²⁺. A soluble fluorescent dye (Cy5) was added to the buffer as an indicator for Ca²⁺ arrival (red line). (B) Cumulative distribution functions for the time delay of fusion following the arrival of calcium (Δt_Ca) at different [Ca²⁺]. Summary of the data is shown in table S11. The probability of triggering DCV fusion as a function of [Ca²⁺] with K_1/2 [Ca²⁺] of 40 μM is shown in fig. S9. (C) Effect of PI(4,5)P₂ and the absence of membrane anionic charge on spontaneous fusion of DCVs in the absence of Ca²⁺ (black bars) or upon triggering of fusion with 100 μM Ca²⁺ (red bars). (D) The cumulative distribution function for calcium-triggered fusion as a function of PI(4,5)P₂ is shown with the summary of data for spontaneous and triggered fusion in tables S12 and S13, respectively. (E) Effects on spontaneous fusion (black bars) or fusion triggered by 100 μM Ca²⁺ (red bars) of RNAi-mediated knockdowns of syt and CAPS, of corresponding knockdown/rescue using RNAi-resistant constructs, and of addition of recombinant Munc13-derived C1C2-MUN or the MUN domain alone. Summary of data for spontaneous and triggered fusion of knockdowns is shown in tables S14 and S15, respectively. (F) Cumulative distribution functions of fusion probability beginning at the time of Ca²⁺ arrival for DCVs from wild-type (wt), CAPS knockdown, and CAPS knockdown rescued by the two Munc13 constructs.

Fig. 5. Model of DCV docking and fusion during exocytosis.

Calcium-dependent release of DCV content requires an acceptor complex consisting of syntaxin-1a, SNAP-25, Munc18, and complexin as well as PI(4,5)P₂ in the plasma membrane. The complex interacts with synaptobrevin-2 (syb) and synaptotagmin-1/9 (syt) in the vesicle membrane as well as CAPS that may or may not be vesicle-associated (I). DCVs are able to dock to the complex in the absence of calcium in a SNARE-dependent fashion (IIa). The presence of complexin “clamps” the resulting pre-fusion (trans-SNARE) complex, preventing progression to an open fusion pore, and allows priming of the fusion machinery. Priming depends on CAPS [and/or Munc13 (not shown)] and PI(4,5)P₂ and might involve a spatial organization of multiple copies of trans-SNARE complexes and accessory proteins as well as the organization of a specific local nanoscale lipid environment. The primed intermediate state resembles granules in the readily releasable pool in situ. Calcium influx triggers fusion pore opening catalyzed by syts (III) and eventually the collapse of the vesicle membrane into the plasma membrane (IV). Calcium also facilitates CAPS-dependent docking of DCVs to the plasma membrane (IIb). These granules proceed through some intermediates that might resemble the priming steps in the absence of calcium to a fusion pore (III) and eventually to the complete merger of the two membranes (IV).