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

树突棘是一种高度运动性的结构，但棘突与突触接触的突触前曲张之间的运动协调程度和方式尚不清楚。为了同时分析树突棘和轴突曲张的运动，我们在海马片培养中用绿色荧光蛋白标记CA1锥体细胞和用罗丹明-葡聚糖标记CA3锥体细胞。使用双光子显微镜观察静脉曲张和脊柱，以检测两种成分之间的紧密联系。时间推移成像显示，它们在没有失去接触的情况下进行了快速的形态变化。静脉曲张和脊柱之间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，强电刺激引起的树枝状形态的改变对它们之间的联系几乎没有影响。这些结果表明存在协调…的地方监管机制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的可塑性。在这里，我们报道了一种新的Rho家族蛋白的GTPase激活蛋白p250GAP的鉴定和鉴定，该蛋白与NMDA受体的GluRepsilon2(NR2B)亚基相互作用。P250GAP基因在脑内表达丰富，在大脑皮层、纹状体、海马区和丘脑中高表达。在神经元内，p250GAP高度集中于突触后密度，并与NMDA受体的GluRepsilon2亚单位(NR2B)共存，突触后密度为-95。P250GAP在体内外均能促进GTP对Cdc42和RhoA的降解。当p250GAP在神经母细胞瘤细胞中过表达时，抑制了Rho家族蛋白的活性，从而导致轴突生长的改变。最后，刺激NMDA受体导致p250GAP在海马片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。较少

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