Spatiotemporal Visualization of Hyperphosphorylation-induced Tau Conformational Change in Early Stage Tauopathy by FRET-based Biosensor in Living Cells

Published: 11 November 2021| Version 1 | DOI: 10.17632/2z5gjnggmy.1
Sang-Hyun Ahn


Figure 2. TCCB T1 showed a significant increase in the FRET/ECFP emission ratio from 20min and finally a 1.1-fold change at 160 min in soluble ROI like the TCCB T1-SF, but there is no change of FRET/ECFP emission ratio in MT-associated ROI. This indicates that there is no change in MT-associated tau during 160 minutes of conformational change of soluble tau, which is consistent with previous studies that the conformational change occurred in soluble, flexible tau first. Figure 3. Mutant sensors showed no change in the FRET/ECFP emission ratio of soluble tau region and MT-associated tau region, like the DMSO-treated control. These consequently suggest that our TCCB T1 can detect conformational changes due to the interaction between amino acids of the N-terminus (5-15) and a group of amino acids spanning the third repeat domain(312-322) of tau when tau is hyperphosphorylated in the early stage of tauopathy. Figure 4. We identified the approximate time to reach the late stage of tauopathy by checking intensity change after treating Fsk in the cells transfected by Tau-BiFC, one of the aggregation tau sensors. We investigated whether our sensor detects the true early stage compared with Tau-BiFC. These results demonstrate that conformational change of hyperphosphorylated tau occurs at an earlier stage than aggregation in the cell-based tauopathy study. Figure 5. we expected that MT-associated tau did not change significantly in the beginning, but after detaching, conformational change, aggregation proceeded, and MT-depolymerization would also be induced, thereby accelerating the exacerbation. Therefore, we recognized the need to develop a sensor that can track changes in MT-associated tau, named TCCB T2, which was made by taking advantage of the property that the N-terminus and C-terminus of MT-binding tau have an adjacent structure called “paperclip”. We performed live imaging for 80 minutes by treating Nocodazole(Noc) and Paclitaxel(PTX) in TCCB T2 transfected MCF-7 cells. We confirmed that the FRET/ECFP emission ratio gradually decreased compared to the control. In contrast, our TCCB T1, which has been proven to detect conformational changes in hyperphosphorylated tau, did not show any change in FRET/ECFP emission ratio even after MT depolymerization. This suggests that we have developed two biosensors with completely distinct roles (TCCB T1, detecting conformational change caused by hyperphosphorylation in an early stage of tauopathy in the cell-based study; TCCB T2, detecting MT-collapse of neurons occurring during tauopathy). Figure 6. hyperphosphorylation with Fsk showed changes only in the soluble region but did not show in the MT-associated region. To confirm this, TCCB T2 was transfected into MCF-7 cells and live imaging was performed for 160 minutes under the same conditions.


Steps to reproduce

Before imaging, the cells expressing several exogenous proteins were cultured in cover-glass-bottom dishes(100350, SPL, Republic of Korea) and were starved in DMEM/F-12 or RPMI medium containing 0.5% FBS for 6h at 37°C. Then, cells were washed using PBS, and the medium was changed with a CO2-independent medium(18045-088, Gibco, Waltham, MA) containing 0.5% FBS, 4mM L-glutamine, 100unit/ml penicillin, and 100μg/ml streptomycin. Images were obtained by using a Leica Dmi8 microscope equipped with a charge-coupled device(CCD) camera(DFC450C, Leica Germany), a 20X HG PL FLUOTAR objective/numerical aperture(NA) 0.4 dry and a 100X HG PL FLUOTAR objective/NA 1.32 oil immersion. The filter sets for images of tau conformational change biosensors included a 436/20 excitation filter, 455 dichroic mirror, and 480/40 emission filter for detecting ECFP and a 436/20 excitation filter, 455 dichroic mirrors, and 535/30 emission filter for detecting FRET. The Las X software (Leica, Germany) was used to acquire images, compute the emission intensity of ECFP, FRET for FRET biosensor images, and was used to subtract the background of the images. Because the FRET response of the biosensors is different depending on whether it is in the cytoplasm or the microtubule(MT), under epifluorescence microscope setting, the whole cell body of the target cell expressing the FRET biosensor was selected as a region of interest(ROI) to collect signals and conduct quantification. The fluorescence intensity at the background region was selected and quantified to take the signal away from the ROI of the YPet, ECFP, and FRET channels. Quantified values were analyzed GraphPad Prism 7.0(San Diego, CA). The pixel-by-pixel ratio images of FRET/ECFP emission were calculated based on the bg-subtracted fluorescence intensity images of FRET and ECFP as follows: (I(〖FRET〗_ROI )-I(FRET_bg ))/(I(ECFP_ROI )-I(ECFP_bg ) ) Where I represent the intensity of each region from each channel. The ratio images were displayed in the intensity modified display(IMD) mode, where the brightness and color of each pixel are determined by the ECFP intensity and FRET/ECFP emission ratio, respectively. All statistical analysis was performed using GraphPad 7.0. Each representative image and graph were obtained from at least three independent experiments. All results are expressed as the mean ± standard error of the mean (S.E.M) and the statistical evaluation was performed using the unpaired Student’s t-test to determine the statistical significance of the difference between the two mean values. We considered P values at *<0.05, **<0.01, ***<0.001 and ****<0.0001 to be statistically significant.


Pusan National University


Biologic Imaging