Laboratory for Membrane Trafficking, VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium.
Department of Neurosciences, KU Leuven, Leuven, Belgium.
Elife. 2020 Jul 7;9:e56679. doi: 10.7554/eLife.56679.
γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.
γ-分泌酶是一种多亚基酶，其异常活性与阿尔茨海默病和癌症有关。虽然其结构在原子水平上已经解析，但γ-分泌酶在膜中的原位定位主要依赖于生化数据。在这里，我们将γ-分泌酶亚基的荧光标记与成纤维细胞中的超分辨率显微镜相结合。结构光照明显微镜揭示了具有单分散分布和 PSEN1 和 nicastrin 亚基 1:1 化学计量比的单个 γ-分泌酶复合物。在活细胞中，sptPALM 揭示 PSEN1/γ-分泌酶主要具有定向运动，并经常出现在“热点”或高轨迹密度区域，这些区域对 γ-分泌酶抑制剂敏感。我们观察到 γ-分泌酶与淀粉样前体蛋白和 N-钙粘蛋白等底物的结合，但不在细胞表面与它的脱落酶 ADAM10 或 BACE1 结合，这表明不存在预先形成的兆道尔顿复合物。尽管如此，在活细胞中，PSEN1/γ-分泌酶会短暂地访问 ADAM10 热点。我们的结果强调了超分辨率显微镜在研究膜中 γ-分泌酶分布和动力学方面的强大功能。
