Live tissue imaging of functions and dynamics of membrane associated proteins during development of neuronal network formation

神经元网络形成过程中膜相关蛋白的功能和动态的活体组织成像

• 批准号: 14580723
• 负责人: INOUE Akihiro
• 金额: $ 2.3万
• 依托单位: Tokyo Medical and Dental University, Graduate School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580723/
• 关键词: Postsynaptic density; Tissue culture; Synapse formation; GFP; Adenovirus vector; Sindbis virus vector; Two-photon laser microscopy; Cre-loxP system; カドヘリン

Dendritic spines are highly motile structures, but the extent and mode of coordination in motility between spines and presynaptic varicosities with synaptic contacts is not clear. To analyze movements of dendritic spines and axonal varicosities simultaneously, we labeled CA1 pyramidal cells with green fluorescent protein and CA3 pyramidal cells with rhodamine-dextran in hippocampal slice cultures. Varicosities and spines were visualized using two-photon microscopy to detect close association of two components. Time-lapse imaging revealed that they performed rapid morphological changes without losing their contacts. The extent of overall structural changes between varicosities and spines was correlated, while the direction of short-term volume changes was regulated independently. Furthermore, alterations of dendritic morphology induced by strong electrical stimulation had little effects on their association. These results indicate the presence of local regulatory mechanisms to coordinat … More e presynaptic and postsynaptic motility.N-methyl-d-aspartate (NMDA) receptors regulate structural plasticity by modulating actin organization within dendritic spines. Herein, we report identification and characterization of p250GAP, a novel GTPase-activating protein for Rho family proteins that interacts with the GluRepsilon2 (NR2B) subunit of NMDA receptors in vivo. The p250GAP mRNA was enriched in brain, with high expression in cortex, corpus striatum, hippocampus, and thalamus. Within neurons, p250GAP was highly concentrated in the postsynaptic density and colocalized with the GluRepsilon2 (NR2B) subunit of NMDA receptors and with postsynaptic density-95. p250GAP promoted GTP hydrolysis of Cdc42 and RhoA in vitro and in vivo. When overexpressed in neuroblastoma cells, p250GAP suppressed the activities of Rho family proteins, which resulted in alteration of neurite outgrowth. Finally, NMDA receptor stimulation led to dephosphorylation and redistribution of p250GAP in hippocampal slices. Together, p250GAP is likely to be involved in NMDA receptor activity-dependent actin reorganization in dendritic spines. Less

树突棘是高度运动的结构，但棘与突触前曲张之间的运动协调程度和方式与突触接触尚不清楚。为了同时分析树突棘的运动和轴突的静脉曲张，我们用绿色荧光蛋白标记了海马切片培养的CA1锥体细胞，用罗丹明-葡聚糖标记了CA3锥体细胞。使用双光子显微镜观察静脉曲张和脊柱，以检测两种成分的密切联系。延时成像显示，它们在没有失去接触的情况下进行了快速的形态变化。整体结构变化的程度与椎体之间存在相关性，而短期体积变化的方向是独立调节的。此外，强电刺激引起的树突形态改变对它们的关联影响不大。这些结果表明存在局部调节机制来协调突触前和突触后的运动。n -甲基-d-天冬氨酸（NMDA）受体通过调节树突棘内的肌动蛋白组织来调节结构可塑性。在此，我们报告了p250GAP的鉴定和表征，p250GAP是一种新的Rho家族蛋白的gtpase激活蛋白，可与体内NMDA受体的GluRepsilon2 （NR2B）亚基相互作用。p250GAP mRNA在大脑中富集，在皮层、纹状体、海马和丘脑中高表达。在神经元内，p250GAP高度集中于突触后密度，并与NMDA受体的GluRepsilon2 （NR2B）亚基共定位，突触后密度为-95。p250GAP在体外和体内均促进Cdc42和RhoA的GTP水解。当p250GAP在神经母细胞瘤细胞中过表达时，会抑制Rho家族蛋白的活性，从而导致神经突生长的改变。最后，NMDA受体刺激导致海马切片中p250GAP的去磷酸化和重新分布。总之，p250GAP可能参与了树突棘中NMDA受体活性依赖的肌动蛋白重组。少

项目成果

Synchronized formation and remodeling of postsynaptic densities : long-term visualization of hippocampal neurons expressing postsynaptic density proteins tagged with GFP.

突触后密度的同步形成和重塑：表达 GFP 标记的突触后密度蛋白的海马神经元的长期可视化。

• 发表时间: 2003
• 期刊: Journal of Neuroscience 23
• 作者: Ebihara;T.;Kawabata;I.;Usui;S.;Sobue;K.;S.Okabe.
• 通讯作者: S.Okabe.

Synaptic targeting of PSD-Zip45 (Homer 1c) and its involvement in the synaptic accumulation of F-actin

• DOI: 10.1074/jbc.m210802200
• 发表时间: 2003-03-21
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Usui, S;Konno, D;Sobue, K
• 通讯作者: Sobue, K

Okabe S.: "Birth growth and elimination of a single synapse"Anatomical Science International. 77. 229-236 (2002)

Okabe S.：“单个突触的出生生长和消除”国际解剖科学。

The dynamic organization fo postsynaptic proteins : translocating molecules regulate synaptic function.

突触后蛋白质的动态组织：易位分子调节突触功能。

• 发表时间: 2003
• 期刊: Current Opinion in Neurobiology 13
• 作者: Inoue A;Okabe S
• 通讯作者: Okabe S

Usui, S.: "Synaptic targeting of PSD-Zip45(Homer 1c)and its involvement in the synaptic accumulation of F-actin."Journal of Biological Chemistry. 278. 10619-10628 (2003)

Usui, S.：“PSD-Zip45（Homer 1c）的突触靶向及其参与 F-肌动蛋白的突触积累。”生物化学杂志。