用不同大小的荧光探针对突触和非突触 AMPA 受体进行超分辨率成像。
Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.
机构信息
Department of Physics, Center for Biophysics, and Quantitative Biology, and Center for the Physics of Living Cells, University of Illinois, Urbana-Champaign, Champaign, United States.
Department of Neurobiology, University of Chicago and the Marine Biological Laboratory, Chicago, United States.
出版信息
Elife. 2017 Jul 27;6:e27744. doi: 10.7554/eLife.27744.
Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 min or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5-10% of bQD-AMPARs were in PSDs and 90-95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD 'slots', our findings suggest that AMPARs rapidly enter stable 'nanodomains' in PSDs with lifetime 15 min, and do not accumulate in extrasynaptic membranes.
先前的研究追踪了 AMPA 受体（AMPAR）在突触处的扩散，观察到了一个大的可移动的突触外 AMPAR 池。我们使用超分辨率显微镜，研究了荧光团的大小和光稳定性如何影响大鼠突触后密度（PSD）内外的 AMPAR 转运。有机荧光染料（≈4nm）、量子点，小的（≈10nm 直径；sQDs）或大的（>20nm；bQDs），通过不同大小的接头与 AMPAR 偶联。我们发现，用荧光染料或 sQDs 标记的>90%的 AMPAR 在 PSDs 中的受限纳米域中扩散，这些纳米域稳定 15 分钟或更长时间。少于 10%的 sQD-AMPAR 是突触外的，并且高度移动。相比之下，正如之前观察到的那样，5-10%的 bQD-AMPAR 位于 PSDs 中，90-95%位于突触外。与 AMPAR 进入受开放 PSD“插槽”占据限制的假设相反，我们的研究结果表明，AMPAR 可以快速进入 PSD 中具有 15 分钟寿命的稳定“纳米域”，并且不会在突触外膜中积累。
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