A Methodology for Specific Disruption of Microtubules in Dendritic Spines.
A Methodology for Specific Disruption of Microtubules in Dendritic Spines.
复制标题
树突棘微管特异性破坏的方法。
DOI：
10.1101/2024.03.04.583370
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Dent,ErikW
中科院分区：
文献类型：
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作者：
Holland,ElizabethD;Miller,HannahL;Millette,MatthewM;Taylor,RussellJ;Drucker,GabrielleL;Dent,ErikW
树突棘是兴奋性神经元树突轴沿着延伸的蘑菇状结构，对突触功能至关重要，是神经发育和神经退行性疾病中最早破坏的神经元结构之一。微管（MT）聚合成树突棘是能够影响棘形状和功能的活性依赖性过程。研究表明，MT聚合成棘特别发生在经历塑性变化的棘。然而，识别MT入侵树突棘的功能需要特异性抑制MT聚合成棘，同时在树突轴、突触连接的轴突和相关胶质细胞中保持MT动力学完整。这是不可能的与无限制的，浴应用的药理化合物。为了特异性地破坏MT进入棘，我们将Efa 6蛋白的MT消除结构域（MTED）与肌动蛋白抑制剂结合肽LifeAct偶联。选择LifeAct是因为肌动蛋白丝高度集中在刺中，并且是MT入侵所必需的。这种LifeAct-MTED构建的时间控制表达抑制MT进入树突棘，同时保留树突轴中的典型MT动力学。该构建体的表达将允许确定MT侵袭棘的功能，并且更广泛地，辨别MT-肌动蛋白相互作用如何影响细胞过程。
Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.