Visualizing the microtubule-associated protein tau in the nucleus

Abstract

Although tau is mainly known as an axonal microtubule-associated protein, many studies indicate that it is not restricted to this subcellular compartment. Assessing tau's subcellular distribution, however, is not trivial as is evident from transgenic mouse studies. When human tau is over-expressed, it can be immunohistochemically localized to axons and the somatodendritic domain, modeling what is found in neurodegenerative diseases such as Alzheimer's disease. Yet, in wild-type mice, despite its abundance, tau is difficult to visualize even in the axon. It is even more challenging to detect this protein in the nucleus, where tau has been proposed to protect DNA from damage. To establish a framework for future studies into tau's nuclear functions, we compared several methods to visualize endogenous nuclear tau in cell lines and mouse brain. While depending on the fixation and permeabilization protocol, we were able to detect nuclear tau in SH-SY5Y human neuroblastoma cells, we failed to do so in N2a murine neuroblastoma cells. As a second method we used subcellular fractionation of mouse tissue and found that in the nucleus tau is mainly present in a hypophosphorylated form. When either full-length or truncated human tau was expressed, both accumulated in the cytoplasm, but were also found in the nuclear fraction. Because subcellular fractionation methods have their limitations, we finally isolated nuclei to probe for nuclear tau and found that the nuclei were free of cytoplasmic contamination. Together our analysis identifies several protocols for detecting tau in the nucleus where it is found in a less phosphorylated form.

Connecting the dots between tau dysfunction and neurodegeneration

“…While the presence of tau in the nucleus has been the subject of some debate, recent work supports the idea that nuclear tau exists and demonstrates that visualization of nuclear tau is dependent upon several factors, including antibody, experimental system, and the staining protocol [74]. Disease associated TG-3 and Alz-50 positive tau colocalize with heterochromatin in human Alzheimer’s disease brain based on electron microscopy [75], and tau colocalizes with pericentromeric heterochromatic DNA in cultured cells based on immunofluorescence [76].…”

Early multidomain intervention to stave off Alzheimer’s disease

“…甲 醛 参 与 DNA 、 RNA、 组蛋白等甲基化 [27] . 因此, 甲醛代谢失调, 可以 通过影响表观遗传学, 以及蛋白质的异常修饰和分子 聚集, 从而造成包括学习记忆在内的认知损伤 [28,29] .…”

14-3-3 λ protein interacts with ADF1 to regulate actin cytoskeleton dynamics in Arabidopsis

“…ADF1 protein was immunopreciptitated from the total protein by incubating with anti-Flag agarose. For the phosphatase treatment, the immunopreciptitated protein was treated with  phosphatase (Promega, USA) at 30C for 15 min [35]. Immunoblotting was performed with anti-Flag antibody or anti-phosphor-Ser/Thr antibody (Sigma-Aldrich).…”