Miura Shuto, Ishibashi Eisuke, Nakamichi Takuto, Uwai Koji, Kuragano Masahiro, Tokuraku Kiyotaka
Graduate School of Engineering, Muroran Institute of Technology, Hokkaido, 050-8585, Japan.
Biochem Biophys Rep. 2025 Jun 27;43:102118. doi: 10.1016/j.bbrep.2025.102118. eCollection 2025 Sep.
We previously reported that microtubule-associated protein (MAP) 4 was detected in the cytoplasm as abnormal "puncta" in post-ischemic mouse cardiomyocytes. MAP4, a member of the MAP superfamily, has a repeat region consisting of multiple microtubule-binding sequences in its microtubule-binding domain (MBD), like tau. The tau forms aggregates composed of amyloid fibrils and grows into neurofibrillary tangles in neurons. Therefore, we hypothesized that MAP4 also forms amyloid fibrils in cells. Here, we observed whether MAP4 forms aggregates composed of amyloid fibrils using fluorescence microscopy and transmission electron microscopy with quantum dot (QD) nanoprobes. Since we had previously succeeded in real-time 3D imaging of tau MBD fragment aggregate formation using QD nanoprobes, we attempted to observe aggregates using human MAP4 MBD fragments under the same conditions. Fluorescence microscopy showed that 10 μM MAP4 formed aggregates at a rate similar to that of tau. Time-laps 3D imaging by confocal laser microscopy revealed that MAP4 aggregate grains were smaller in size and the deposits were thinner than tau aggregates. Transmission electron microscopy of the MAP4 aggregates revealed that they consisted of helical filaments with a width of 22.6 ± 2.8 nm and a helical pitch length of 48.2 ± 8.4 nm. The helical filaments of MAP4 were shorter in width and longer in helical pitch than those of tau. Furthermore, MAP4 aggregates did not increase the fluorescence intensity of thioflavin T (ThT), and the circular dichroism (CD) spectrum slightly differed from that of tau. These findings suggest that while MAP4 forms aggregates composed of helical filaments similar to those of tau, the structural properties of these filaments are somewhat distinct.
我们之前报道过，在缺血后小鼠心肌细胞的细胞质中，微管相关蛋白（MAP）4以异常的“斑点”形式被检测到。MAP4是MAP超家族的成员之一，其微管结合结构域（MBD）中有一个由多个微管结合序列组成的重复区域，与tau蛋白类似。tau蛋白会形成由淀粉样原纤维组成的聚集体，并在神经元中发展为神经原纤维缠结。因此，我们推测MAP4在细胞中也会形成淀粉样原纤维。在这里，我们使用荧光显微镜和量子点（QD）纳米探针的透射电子显微镜观察MAP4是否形成由淀粉样原纤维组成的聚集体。由于我们之前成功地使用QD纳米探针实时三维成像tau MBD片段聚集体的形成，因此我们试图在相同条件下观察人MAP4 MBD片段的聚集体。荧光显微镜显示，10 μM的MAP4以与tau蛋白相似的速率形成聚集体。共聚焦激光显微镜的时间推移三维成像显示，MAP4聚集体颗粒尺寸较小，沉积物比tau聚集体更薄。MAP4聚集体的透射电子显微镜显示，它们由宽度为22.6±2.8 nm、螺旋节距长度为48.2±8.4 nm的螺旋丝组成。MAP4的螺旋丝宽度比tau的更窄，螺旋节距更长。此外，MAP4聚集体没有增加硫黄素T（ThT）的荧光强度，并且圆二色性（CD）光谱与tau的略有不同。这些发现表明，虽然MAP4形成了与tau类似的由螺旋丝组成的聚集体，但这些丝的结构特性有些不同。
