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

树突棘是高度运动性的结构，但棘和突触前静脉曲张与突触接触之间的运动协调的程度和模式尚不清楚。为了同时分析树突棘和轴突静脉曲张的运动，我们在海马切片培养中用绿色荧光蛋白标记CA 1锥体细胞，用罗丹明-葡聚糖标记CA 3锥体细胞。使用双光子显微镜观察静脉曲张和棘，以检测两种成分的密切相关性。延时成像显示，他们进行了快速的形态变化，而不会失去他们的联系。静脉曲张和脊柱之间的整体结构变化的程度是相关的，而短期体积变化的方向是独立调节的。此外，由强电刺激引起的树突形态的改变对它们的关联几乎没有影响。这些结果表明，局部调节机制的存在，以协调 ...更多信息 N-甲基-D-天冬氨酸（NMDA）受体通过调节树突棘内肌动蛋白的组织来调节结构可塑性。在这里，我们报告的鉴定和表征p250 GAP，一种新的GTP酶激活蛋白的Rho家族蛋白，相互作用的GluRepsilon 2（NR 2B）亚基的NMDA受体在体内。p250 GAP mRNA在脑组织中富集，在皮质、纹状体、海马和丘脑中有高表达。在神经元内，p250 GAP高度集中在突触后密度和共定位与谷氨酸Repsilon 2（NR 2B）亚基的NMDA受体和突触后密度-95。p250 GAP在体内外均能促进Cdc 42和RhoA的GTP水解。当p250 GAP在神经母细胞瘤细胞中过表达时，抑制Rho家族蛋白的活性，从而导致神经突起生长的改变。最后，NMDA受体刺激导致海马脑片中p250 GAP的去磷酸化和重新分布。总之，p250 GAP可能参与树突棘中NMDA受体活性依赖性肌动蛋白重组。少

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