Proteome analysis of actin filament-associated proteins in the postnatal rat cerebellum
Proteome analysis of actin filament-associated proteins in the postnatal rat cerebellum
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DOI：
10.1016/j.neuroscience.2012.09.050
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发表时间：
2012-12
期刊：
影响因子：
3.3
通讯作者：
N. Shi;C. Tian;X. Liang;P. Jiang;L. Liang;L. Zhou;Y. Shu;P. Chen;Y. Wang
中科院分区：
文献类型：
--
作者：
N. Shi;C. Tian;X. Liang;P. Jiang;L. Liang;L. Zhou;Y. Shu;P. Chen;Y. Wang
小脑包含的神经元比所有其他大脑区域的总和还要多，这些细胞在出生后表现出复杂的回路发育和树突状细胞的发育。神经发育、细胞形态发生和突触可塑性依赖于肌动蛋白结合蛋白对肌动蛋白细胞骨架的动态调节。肌动蛋白丝相互作用组的鉴定，包括出生后小脑中发育调节的蛋白质，可以帮助确定发育中小脑神经元肌动蛋白细胞骨架动力学的重要调节因子。利用F-actin柱亲和纯化小脑蛋白质，结合质谱分析，共鉴定出434个大鼠小脑肌动蛋白相关蛋白。此外，还利用半定量RT-PCR技术筛选了与大鼠小脑（P0-P56）肌动蛋白动力学和膜运输有关的生后发育调控基因。其结果是，9个基因编码的小脑F-肌动蛋白相互作用体的成员在转录水平上的发育调控，其中至少有5个表现出类似的模式在蛋白质表达水平的Western印迹分析。进一步的荧光免疫组织化学观察表明，肌动蛋白相关蛋白Lethal（2）giant larvae protein homolog 1（LLGL 1）和metastasis suppressor 1（MTSS 1）在轴突和树突的形态发生过程中在颗粒神经元和浦肯野细胞中特异性上调。这项工作定义了一个临时肌动蛋白丝相互作用组在大鼠出生后小脑，并确定了几个候选蛋白，可能参与出生后小脑的发育。
The cerebellum contains more neurons than all other brain regions combined and these cells exhibit complex circuit development and dendritic elaboration during the postnatal period. Neural development, cellular morphogenesis, and synaptic plasticity are dependent on the dynamic regulation of the actin cytoskeleton by actin-binding proteins. The identification of the actin filament interactome, including proteins developmentally regulated in the postnatal cerebellum, could help define important regulators of actin cytoskeletal dynamics in developing cerebellar neurons. Affinity purification of cerebellar proteins on F-actin columns, combined with mass spectrometry, in total, 434 actin filament-associated proteins in postnatal rat cerebellum (P7) were identified. Furthermore, semi-quantitative RT-PCR was performed to screening postnatal developmentally regulated genes involved in actin dynamics and membrane trafficking in rat cerebellum (P0–P56). As the result, nine genes encoding members of the cerebellar F-actin interactome were developmentally regulated in the transcriptional level and at least five of them exhibited a similar pattern at the protein expression level by Western blot analysis. Further fluorescent immunohistochemical observations demonstrated that the actin-associated proteins Lethal(2) giant larvae protein homolog 1 (LLGL1) and metastasis suppressor 1 (MTSS1) were specifically upregulated in granule neurons and Purkinje cells during morphogenesis of axons and dendrites. This work defines a provisional actin filament interactome in rat postnatal cerebellum and identifies several candidate proteins that may be involved in the postnatal development of the cerebellum.