内源性标记揭示突触前活性区的纳米级 RIM 簇。
Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging.
机构信息
https://ror.org/00fbnyb24 Department of Neurophysiology, Institute of Physiology, University of Würzburg, Würzburg, Germany.
Department of Neurology, Leipzig University Medical Center, Leipzig, Germany.
出版信息
Life Sci Alliance. 2023 Sep 11;6(12). doi: 10.26508/lsa.202302021. Print 2023 Dec.
Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of remains elusive. We used CRISPR/Cas9-assisted genome engineering of the locus to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rim but not in rim In addition, rim synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, we detect ∼10 RIM subclusters with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.
化学突触传递涉及神经递质从突触前活性区（AZ）释放。AZ 蛋白 Rab-3 相互作用分子（RIM）对于正常的 Ca 触发释放很重要。然而，其在谷氨酸能神经肌肉接头的 AZ 中的精确定位仍不清楚。我们使用 CRISPR/Cas9 辅助的基因组工程来修饰 基因座，以纳入用于靶向超分辨率成像的小表位标签。一个来自猿猴病毒 5 的 V5 标签和一个来自人类流感病毒的 HA 标签分别被 N 端融合到 RIM 的锌指上。虽然这两种变体都与核心 AZ 支架 Bruchpilot 共表达，但电生理特性表明，AP 诱发的突触释放在 rim 中受到干扰，而在 rim 中没有受到干扰。此外，rim 突触显示完整的突触前同型激活增强。通过结合超分辨率定位显微镜和层次聚类，我们检测到每个 AZ 有大约 10 个 RIM 亚簇，直径约为 13nm，在突触前同型激活增强时变得更加紧凑且数量增加。
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