Microtubule density and landing rates as parameters to analyze tau protein in the MT-kinesin “gliding” assay
Microtubule density and landing rates as parameters to analyze tau protein in the MT-kinesin “gliding” assay
复制标题
微管密度和着陆率作为参数来分析 MT-驱动蛋白“滑动”测定中的 tau 蛋白
DOI：
10.1016/j.snb.2016.07.082
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
R. Yokokawa
中科院分区：
文献类型：
--
作者：
S. Subramaniyan Parimalam;M. C. Tarhan;S. L. Karsten;H. Fujita;H. Shintaku;H. Kotera;R. Yokokawa
微管相关蛋白（MAP）tau是一大组年龄相关的神经退行性疾病（统称为tau蛋白病）的公认标志。在病理条件下，tau与MT结合的平衡受到干扰，或者是由于特定tau亚型表达水平的失调，或者是由于MAPT基因突变。临床前检测脑脊液（CSF）中这种失调的tau蛋白对于神经变性的鉴别诊断和有效预后是理想的。常规的tau蛋白检测方法利用tau同种型特异性抗体。这种基于免疫的方案，包括酶联免疫吸附测定（ELISA）和蛋白质印迹具有适当的灵敏度和特异性，但通常显示出高变异性并且耗时。在这里，我们建立了一个非免疫tau蛋白检测方法，利用微管（MT）-驱动蛋白“滑行”试验。研究了在人脑中表达的所有六种tau亚型（0 N3 R、1 N3 R、2N 3R、0 N4 R、1 N4 R和2N 4 R）和五种MAPT基因突变体（V248 L、G272 V、P301 L、V337 M和R406 W）。着陆率，结合密度和滑行速度的MT相对于每一个tau类型被确定，并建议作为tau检测参数。检测参数描述了与MT结合的tau的类型。此外，MT着陆率和密度被发现是上级的滑动速度在区分tau亚型和突变体。对3R与4 R亚型、它们的混合物、野生型与突变型2N 4 R和特异性突变体进行了区分。我们的数据表明，MT-驱动蛋白滑动试验提供了一个方便的，实验室上的芯片（XNUMX）兼容和抗体的tau蛋白分析协议。
Microtubule-associated protein (MAP) tau is a well-established hallmark of a large group of age related neurodegenerative diseases collectively called tauopathies. Under pathological conditions the equilibrium of tau binding to the MTs is perturbed, either by misregulation in the expression levels of specific tau isoforms or byMAPTgene mutations. Preclinical detection of such misregulated tau proteins in cerebrospinal fluid (CSF) is desirable for differential diagnosis and effective prognosis of neurodegeneration. Conventional tau protein detection methods utilize tau isoform-specific antibodies. Such immuno-based protocols, including enzyme-linked immunosorbent assay (ELISA) and Western blots have appropriate sensitivity and specificity, but often show high variability and are time consuming. Here, we established a non-immuno tau protein detection method utilizing microtubule (MT)-kinesin “gliding”assay. All the six tau isoforms expressed in the human brain (0N3R, 1N3R, 2N3R, 0N4R, 1N4R and 2N4R) and fiveMAPTgene mutants (V248L, G272V, P301L, V337M and R406W) were studied. The landing rate, binding density and gliding velocity of MTs with respect to each tau type were determined and are proposed as tau detection parameters. The detection parameters depicted the type of tau bound to the MTs. Furthermore, MT landing rate and density were found to be superior to gliding velocity in differentiating tau isoforms and mutants. The 3Rvs.4R isoforms, their admixtures, wildvs.mutant 2N4R and specific mutants were differentiated. Our data show that MT-kinesin gliding assay provides a convenient, lab-on-a-chip (LOC) compatible and antibody-free protocol for tau protein analysis.