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

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.