SNAREs (soluble NEM-sensitive factor attachment protein receptors) mediate the fusion of synaptic vesicles at the plasma membrane. The v-SNARE synaptobrevin is localised primarily to synaptic vesicles, although it redistributes onto the plasma membrane during vesicle recycling (Sankaranarayanan & Ryan 2000). Inversely, the t-SNARE syntaxin is thought to be anchored in the target plasma membrane. However, a cofractionation study showed that syntaxin-1 is also present in vesicular membrane (Walch-Solimena et al. 1995). We have investigated whether the subcellular distribution of syntaxin-1A is regulated during synaptic vesicle recycling.
Neurons were dissociated from hippocampal CA1-CA3 regions of 2-to 4-day-old Sprague-Dawley rats, which were killed humanely, and maintained in culture for 2-3 weeks prior to experiments. To monitor the distribution of syntaxin, we expressed super-ecliptic pHluorin (SE; Sankaranarayanan et al. 2000) fused to the C-terminus of syntaxin-1A (stx-SE). Data are presented as means ± S.E.M.
Under steady-state conditions, stx-SE was localised in somatic, dendritic and axonal regions. We quantified the subcellular localisation of stx-SE by monitoring fluorescence changes in stx-SE during either low pH or NH4Cl application. We found that 9 ± 3 % (n = 9) of stx-SE at synaptic boutons (identified by FM 4-64 loading) was localised in an acidic compartment, which may correspond to synaptic vesicles (Walch-Solimena et al. 1995), while the remainder was on the presynaptic surface. At non-synaptic axonal regions, 100 ± 2 % (n = 9) of stx-SE was present on the plasma membrane.
We next investigated whether intracellular presynaptic syntaxin was localised to synaptic vesicles that recycle during action potentials (AP). Repetitive AP stimulation at 10-100 Hz failed to induce accumulation or redistribution of stx-SE on the plasma membrane. Similar results were found when the signal-to-noise level was enhanced using prior photobleaching of baseline fluorescence and application of bafilomycin-A1, which blocks reacidification of recycling vesicles thereby trapping recycling stx-SE in a fluorescent form. These data indicate that syntaxin-1A is present in an internal compartment that does not participate in AP-evoked vesicle recycling.
Our results show that most syntaxin-1A is localised to the plasma membrane and remains excluded from synaptic vesicles during endocytosis. These data suggest that sorting of synaptic vesicle cargo from an abundant plasma membrane component operates with high fidelity.
SJM is in receipt of a Wellcome Trust International Research Fellowship.