In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau

Abstract

The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer’s disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated the conformations of two different isoforms of Tau, Tau40 and k19, in mammalian cells. Combined with immunofluorescence imaging and western blot analyses, we found that the isotope-enriched Tau, which was delivered into the cultured mammalian cells by electroporation, is partially colocalized with MT and actin filaments (F-actin). We acquired the NMR spectrum of Tau in human embryonic kidney 293 (HEK-293T) cells, and compared it with the NMR spectra of Tau added with MT, F-actin, and a variety of crowding agents, respectively. We found that the NMR spectrum of Tau in complex with MT best recapitulates the in-cell NMR spectrum of Tau, suggesting that Tau predominantly binds to MT at its MT-binding repeats in HEK-293T cells. Moreover, we found that disease-associated phosphorylation of Tau was immediately eliminated once phosphorylated Tau was delivered into HEK-293T cells, implying a potential cellular protection mechanism under stressful conditions. Collectively, the results of our study reveal that Tau utilizes its MT-binding repeats to bind MT in mammalian cells and highlight the potential of using in-cell NMR to study protein structures at the residue level in mammalian cells.

NMR Studies of Tau Protein in Tauopathies

“…In-cell NMR has over the years evolved into a well-established, valuable tool to study proteins in close-to-native conditions ( Serber and Dötsch, 2001 ; Montheith and Pielak., 2014 ; Theillet et al, 2016 ; Luchinat and Banci., 2018 ). Until now, only a single study has been published on in-cell NMR experiments of tau protein, focusing on a shorter fragment ( Zhang et al, 2018 ). In this work, the authors acquired 2D HSQC spectra of the 15 N-labelled K19 fragment in several buffer conditions and subsequently, upon electroporating the protein into HEK-293 cells.…”

Studying protein folding in health and disease using biophysical approaches

“…Time-resolved in-cell NMR demonstrated that Tyr125 phosphorylation was selectively impaired by the C-terminal methionine oxidation, suggesting that alteration of the cellular environment can selectively affect post-translational modifications of α-synuclein and, thus, potentially control its conformation, conformational landscape, and aggregation propensity [ 42 ]. Isotope-enriched Tau was delivered into HEK-293T cells by electroporation and the NMR spectrum of Tau in living cells was acquired [ 43 ]. It was found that Tau predominantly binds to microtubules (MT) at its MT-binding repeats in HEK-293T cells.…”

“…It was found that Tau predominantly binds to microtubules (MT) at its MT-binding repeats in HEK-293T cells. It was also found that disease-associated phosphorylation of Tau was immediately eliminated once phosphorylated Tau was delivered into HEK-293 T cells, implying a potential cellular protection mechanism under stressful conditions [ 43 ].…”

Characteristics of pulmonary microvascular structure in postnatal yaks

“…With the development of modern biotechnology, increasing numbers of researchers are conducting molecular biology research pertaining to the environmental adaptability of this mammal. In the last few years, high-throughput studies on yaks, such as genome and miRNA sequencing and transcriptome analysis, have become popular 13 – 15 . In fact, there are still many unexplored areas of basic research in this animal.…”