シンポジウム
16 最新可視化技術によるシナプス動態制御の理解促進
16 Back to synapse: visualization of synaptic dynamics
座長：田中（山本） 敬子（KIST）・Kim Jinhyun（KIST） |
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| 2022年7月2日 16:10～16:35 ラグナガーデンホテル 羽衣：中 第9会場
3S09e-01
Real time visualization of exo- and endocytosis in live cells
*Ling-Gang Wu(1)
1. NINDS, NIH, Bethesda, MD 20892, USA
Keyword: exocytosis, endocytosis, STED microscopy, light imaging
Exocytosis mediates synaptic transmission, whreas endocytosis recycles fused vesicles to sustain synaptic transmission. Although being intensely studied for many decades, the membrane transmformation underlying exo- and endocytosis have not been observed directly at synapses. Here I will describe our efforts in using the super-resolution STED microscopy to visualize ex-- and endocytosis membrane transformations. These transformations include fusion pore opening, expansion, constriction and closure, fused vesicle merging with the plasam membrane, and the flat-to-round membrane transformation that produces oval/round vesicles in a secretory cell, adrenal chromaffin cells. I will also dsecribe our efforts in understanding the molecular mechanisms underlying these transformation. | | 2022年7月2日 16:35～17:00 ラグナガーデンホテル 羽衣：中 第9会場
3S09e-02
液－液相分離によるグルタミン酸受容体のシナプス内分離のデュアルカラーSTORMイメージング
Dual-color STORM imaging of subsynaptic segregation of glutamate receptor subtypes regulated by liquid-liquid phase separation
*劉 品吾(1)、細川 智永(1)、林 康紀(2)
1. 名古屋大学大学院理学研究科、2. 京都大学大学院医学研究科
*Pin-Wu Liu(1), Tomohisa Hosokawa(1), Yasunori Hayashi(2)
1. Grad Sch Sci, Nagoya Univ, Aichi, Japan, 2. Grad Sch Med, Kyoto Univ, Kyoto, Japan
Keyword: Synaptic plasticity, Liquid-liquid phase separation, Super-resolution imaging, CaMKII
The regulation of AMPA receptor (AMPARs) at postsynaptic density (PSD) is the major mechanism to maintain long-term potentiation (LTP) of synaptic transmission, the basis for memory formation. Observations with super-resolution microscopies have revealed that AMPARs form segregated clusters, so called nanodomains, in an activity dependent manner. Importantly, the AMPAR nanodomains align with presynaptic vesicle releasing sites to form a trans-synaptic nanocolumn, which regulates the efficacy of synaptic transmission. However, it remained unknown how such segregation is accomplished without demarcating membrane at PSD, and how nanodomains respond to the neuronal activity. Here we found that calcium/calmodulin-dependent protein kinase II (CaMKII) undergoes liquid-liquid phase separation (LLPS) with NMDAR subunit GluN2B through the multivalent interaction contributed by its dodecameric structure in an active conformation-dependent manner. Interestingly, the incorporation of CaMKII into in vitro PSD protein condensate composed of PSD-95, GluN2B and Stargazin, an auxiliary subunit of AMPAR, resulted in the formation of core-shell structure, where CaMKII-GluN2B forms shell phase and PSD-95-Stargazin forms core phase. Furthermore, Neuroligin-1 (NLGN1), a neuronal adhesion molecule, which clusters with presynaptic neurexin, also segregates together with AMPAR. The segregation of AMPAR from NMDAR was also observed in living neuron with dual-color direct stochastic optical reconstruction microscopy (dSTORM). This segregation was disrupted by membrane-permeable CN21 (tat-CN21), a CaMKII inhibitor, which competes and disrupts CaMKII-GluN2B interaction. In addition, the segregation between NLGN1 and NMDAR nanodomains was also reduced by tat-CN21. These results suggest that the incorporation of active CaMKII into PSD and the interaction between CaMKII and GluN2B are critical for the formation of AMPAR nanodomain and the trans-synaptic nanocolumn. We anticipate that calcium-induced and persistent formation of LLPS by CaMKII can be a novel mechanism for synaptic plasticity, and serves as molecular basis of memory by functioning as an activity-dependent crosslinker for postsynaptic proteins. | | 2022年7月2日 17:00～17:25 ラグナガーデンホテル 羽衣：中 第9会場
3S09e-03
Synaptic nanoorganization that controls receptor activation
*Thomas A Blanpied(1)
1. University of Maryland School of Medicine
Keyword: Synaptic transmission, adhesion molecules, glutamate receptors, nanoarchitecture
Fine-tuning the protein content and organization of brain glutamatergic synapses underlies faithful neurotransmission and permits the large dynamic range of response magnitude at single contacts which is necessary to convey and modulate information across neural circuits. A key yet poorly understood aspect of synaptic organization is seemingly simple: the distribution of postsynaptic receptors with respect to the sites of presynaptic glutamate release. For many types of glutamate receptors, their position within the synapse strongly influences the likelihood of activation, including both AMPA receptors and GluN2B-containing NMDA receptors. This implies that the mechanisms which determine the nanometer-scale positioning of glutamate receptors likely help regulate not only synaptic strength but the induction and expression of synaptic plasticity. Thus, our efforts are directed to understanding both pre- and post-synaptic molecular organization, and transsynaptic protein links that communicate or synergize molecular patterns in the opposing neurons. Using localization microscopy, we have revealed the nanocolumn clustering of AMPA receptors under release sites, and identified the adhesion molecule LRRTM2 as required for ongoing maintenance of this transsynaptic architecture. I will discuss recent work to expand on this and test the organization and regulation of NMDA receptor positioning. | | 2022年7月2日 17:25～17:50 ラグナガーデンホテル 羽衣：中 第9会場
3S09e-04
Nanoscale imaging of the synaptic microenvironment using super-resolution shadow imaging (SUSHI)
*Valentin Nagerl(1)
1. University of Bordeaux
Keyword: SYNAPTIC STRUCTURE AND FUNCTION, BRAIN EXTRACELLULAR SPACE, SUPER_RESOLUTION MICROSCOPY
The extracellular space (ECS) forms an important but understudied frontier in neuroscience. It consists of the narrow gaps that surround all brain cells, which are filled with interstitial fluid and extracellular matrix molecules, occupying around one fifth of the volume of the brain. It likely provides the molecular cues and physical rails that incite and guide morphogenic processes, including physical interactions between neurons and glia cells. However, mapping the dynamic landscape of the ECS with enough spatial resolution has been impossible to accomplish until now for lack of appropriate tools. In my presentation, I will review our technical progress in imaging the structure and function of the ECS vis-à-vis fluorescently labeled neurons and glia cells in living brain slices using the recent super-resolution shadow imaging technique (SUSHI). | | 2022年7月2日 17:50～18:10 ラグナガーデンホテル 羽衣：中 第9会場
3S09e-05
Visualization of synaptic convergence
*Jinhyun Kim(1)
1. Brain Science Institute, Korea Institute of Science and Technology, Seoul, Korea
Keyword: synaptic connectivity, mGRASP, convergence
Information is processed in the brain by a vast number of diverse neurons that are extensively intermingled and interconnected mainly through synapses. Because the synapse is the primary unit of information processing, detailed descriptions of connectivity converging from multiple individual neurons at the synapse-level as well as region-level has strengthened our understanding of fine-scale organization of synaptic input profiles governing global and subcellular signal computations. For synapse-level connectivity mapping, genetically encoded synaptic detectors have been developed, such as GFP reconstitution across synaptic partners (GRASP) technology. This is based on functional complementation between two non-fluorescent split-GFP fragments targeted to the synaptic membranes of the synaptic cleft. Our mGRASP is developed for improved accuracy, efficacy, and specificity to detect synapses in complex circuits (Kim et al., 2012; Druckmann et al., 2014; Kwon et al., 2018). Combination of mGRASP and optogenetics demonstrated that functional measures of synaptic strength correspond strongly with mGRASP-based structural measures of synapse size, which enabled high-resolution functional connection mapping (Song et al., 2018). Further, we generated improved versions of mGRASP called mGRASPi, a multiplex toolbox for mapping convergent circuits. Our mGRASPi-based mapping of convergent synaptic connectivity defines and quantifies complex, multi-synaptic inputs, providing vital information about the synaptic organization of neural circuitry. | | | |
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