Receptor-mediated Regulation of Tomosyn-Syntaxin 1A Interactions in Bovine Adrenal Chromaffin Cells

Abstract

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.Regulated neurotransmitter release requires the well orchestrated spatial and temporal actions of many presynaptic proteins (1). Although the primary molecular entities in the release pathway have been identified, the exact mechanics of synaptic vesicle fusion and its precise regulation are still not established. Central to the fusion process is the transient formation of SNARE 4 core complexes that include the target membrane SNARE proteins syntaxin 1A and SNAP25 and the vesicle SNARE protein synaptobrevin/VAMP (2-4). A SNARE core complex is a highly stable, four-␣-helix parallel bundle consisting of one SNARE motif from each of syntaxin 1A and synaptobrevin/VAMP, and two SNARE motifs from SNAP25 (5, 6). Although these proteins alone are sufficient to induce a slow fusion when reconstituted into liposomes (7), additional proteins are necessary to establish the properties that describe fast, Ca 2ϩ -dependent neurotransmitter release (8). For example, assembly of SNARE core complexes is subject to temporal and spatial regulation by a variety of protein families, including Rab-GTPases (9 -13), Sec/Munc18s (14 -16), exocyst tethering complexes (17-20), and Munc13s (21-24). In addition, recent evidence suggests that the temporal and spatial availability...

Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity

“…1C, p Ͼ 0.05) and also similar to the value calculated for syntaxin previously (17,27). This finding, together with the Förster (fluorescence) resonance energy transfer (FRET) between syntaxin and tomosyn (26), suggests that at the PM tomosyn moves with a dynamics similar to that of syntaxin. As for the obtained binding constants, they reflect a rather fast association/dissociation rate of tomosyn with PM syntaxin, with time constants of 5.5 and 7.5 s, respectively.…”

“…The 0.9 -1.1 ratio pixels represent 15.5 Ϯ 1.8% of all double-labeled pixels measured from the cells. This apparent FRET efficiency is severalfold greater than the background FRET level between citrine-tomosyn-⌬CT and CFP-syntaxin in these cells (1.6 Ϯ 0.06%), although the value is lower than that previously reported in HEK293 cells and in bovine adrenal chromaffin cells (26). This is likely the result of the different intracellular milieu in PC12 cells.…”

“…However, when tomosyn was coexpressed with syntaxin, it demonstrated clear colocalization with syntaxin in the PM region (Fig. 1A), as reported previously (26). To ensure that tomosyn indeed localizes at the PM in the presence of syntaxin and not in submembranal regions, we compared its staining with the membranal staining of the FM dye FM5-95 and observed full overlap between tomosyn and the FM dye (data not shown).…”

“…On the PM, tomosyn interacts directly with syntaxin (26). However, it is not clear whether this interaction involves SNAP25.…”

“…binary or ternary) (26). To investigate whether the FRET signal between citrinetomosyn and CFP-syntaxin requires the presence of SNAP25, we performed experiments using the light chain of BoNT-E.…”