Seidenthal Marius, Jánosi Barbara, Rosenkranz Nils, Schuh Noah, Elvers Nora, Willoughby Miles, Zhao Xinda, Gottschalk Alexander
Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt, Germany.
Institute of Biophysical Chemistry, Department of Biochemistry, Chemistry and Pharmacy, Goethe University, Frankfurt, Germany.
Front Cell Neurosci. 2023 Mar 31;17:1120651. doi: 10.3389/fncel.2023.1120651. eCollection 2023.
pH-sensitive fluorescent proteins are widely used to study synaptic vesicle (SV) fusion and recycling. When targeted to the lumen of SVs, fluorescence of these proteins is quenched by the acidic pH. Following SV fusion, they are exposed to extracellular neutral pH, resulting in a fluorescence increase. SV fusion, recycling and acidification can thus be tracked by tagging integral SV proteins with pH-sensitive proteins. Neurotransmission is generally activated by electrical stimulation, which is not feasible in small, intact animals. Previous approaches depended on distinct (sensory) stimuli, thus limiting the addressable neuron types. To overcome these limitations, we established an all-optical approach to stimulate and visualize SV fusion and recycling. We combined distinct pH-sensitive fluorescent proteins (inserted into the SV protein synaptogyrin) and light-gated channelrhodopsins (ChRs) for optical stimulation, overcoming optical crosstalk and thus enabling an all-optical approach. We generated two different variants of the pH-sensitive optogenetic reporter of vesicle recycling (pOpsicle) and tested them in cholinergic neurons of intact nematodes. First, we combined the red fluorescent protein pHuji with the blue-light gated ChR2(H134R), and second, the green fluorescent pHluorin combined with the novel red-shifted ChR ChrimsonSA. In both cases, fluorescence increases were observed after optical stimulation. Increase and subsequent decline of fluorescence was affected by mutations of proteins involved in SV fusion and endocytosis. These results establish pOpsicle as a non-invasive, all-optical approach to investigate different steps of the SV cycle.
pH 敏感型荧光蛋白被广泛用于研究突触小泡（SV）的融合与循环利用。当这些蛋白靶向定位于突触小泡腔时，其荧光会被酸性 pH 淬灭。突触小泡融合后，它们会暴露于细胞外中性 pH 环境中，导致荧光增强。因此，通过用 pH 敏感型蛋白标记突触小泡整合蛋白，就可以追踪突触小泡的融合、循环利用和酸化过程。神经传递通常由电刺激激活，而这在小型完整动物中是不可行的。以往的方法依赖于不同的（感觉）刺激，因此限制了可研究的神经元类型。为了克服这些限制，我们建立了一种全光学方法来刺激和可视化突触小泡的融合与循环利用。我们将不同的 pH 敏感型荧光蛋白（插入到突触小泡蛋白突触结合蛋白中）与光门控通道视紫红质（ChRs）相结合用于光学刺激，克服了光学串扰，从而实现了全光学方法。我们生成了两种不同变体的突触小泡循环利用的 pH 敏感型光遗传学报告基因（pOpsicle），并在完整线虫的胆碱能神经元中对其进行了测试。首先，我们将红色荧光蛋白 pHuji 与蓝光门控的 ChR2(H134R)相结合，其次，将绿色荧光蛋白 pHluorin 与新型红移通道视紫红质 ChrimsonSA 相结合。在这两种情况下，光学刺激后均观察到荧光增强。荧光的增强及随后的减弱受到参与突触小泡融合和内吞作用的蛋白质突变的影响。这些结果确立了 pOpsicle 作为一种非侵入性的全光学方法来研究突触小泡循环的不同步骤。
