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.
Highlights
Regulated neurotransmitter release requires the well orchestrated spatial and temporal actions of many presynaptic proteins [1]
We show that activation of nicotinic acetylcholine receptors, as occurs normally during neurally evoked secretory responses, as well as treatment with lysophosphatidic acid (LPA), activates Rho-GTPase and increases tomosyn-syntaxin 1A complex formation at the plasma membrane in chromaffin cells
Tomosyn Association with a Membrane Fraction Is Enhanced by a Secretory Stimulus and Activation of Rho-GTPase—Interaction of syntaxin 1A with Munc18-1 is essential for regulated exocytosis, whereas its interaction with tomosyn is believed to negatively impact exocytotic activity through the formation of fusion-incompetent SNARE core complexes
Summary
Regulated neurotransmitter release requires the well orchestrated spatial and temporal actions of many presynaptic proteins [1]. Tomosyn Association with a Membrane Fraction Is Enhanced by a Secretory Stimulus and Activation of Rho-GTPase—Interaction of syntaxin 1A with Munc18-1 is essential for regulated exocytosis, whereas its interaction with tomosyn is believed to negatively impact exocytotic activity through the formation of fusion-incompetent SNARE core complexes.
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