Mazloom-Farsibaf Hanieh, Kanagy William K, Lidke Diane S, Lidke Keith A
Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA.
Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Sci Data. 2025 Apr 3;12(1):562. doi: 10.1038/s41597-025-04782-7.
Membrane protein dynamics regulates cell functions by initiating downstream signaling cascades. The cell membrane is compartmentalized into nanodomains by actin structures, restricting lateral protein diffusion. Single-particle tracking offers high spatiotemporal resolution for studying protein dynamics in living cells. However, directly observing actin filaments that form barriers of nanodomains for fast protein diffusion is challenging due to their size being below the diffraction limit. Single-molecule localization microscopy resolves these structures but requires imaging in fixed cells. We integrated fast single-particle tracking with single-molecule localization microscopy to generate a dataset of membrane protein dynamics and actin filaments within the same cells at the nanoscales. Optimizing the fixation protocol enabled transition from live-cell tracking to fixed-cell super-resolution imaging. Data for the high-affinity IgE receptor, a transmembrane protein, and the GPI-anchored protein, an outer leaflet protein, was collected at 490 Hz. After fixation, actin filaments were imaged using dSTORM. The treatment of actin structures with phalloidin and PMA generated a dataset of distinct actin architectures for studying their potential influence on membrane protein dynamics.
膜蛋白动力学通过启动下游信号级联反应来调节细胞功能。细胞膜由肌动蛋白结构分隔成纳米结构域，限制了蛋白质的侧向扩散。单粒子追踪为研究活细胞中的蛋白质动力学提供了高时空分辨率。然而，由于肌动蛋白丝的尺寸低于衍射极限，直接观察形成纳米结构域屏障以实现蛋白质快速扩散的肌动蛋白丝具有挑战性。单分子定位显微镜可以分辨这些结构，但需要在固定细胞中成像。我们将快速单粒子追踪与单分子定位显微镜相结合，以生成同一细胞内纳米尺度下膜蛋白动力学和肌动蛋白丝的数据集。优化固定方案实现了从活细胞追踪到固定细胞超分辨率成像的转变。以490Hz的频率收集了跨膜蛋白高亲和力IgE受体和外叶蛋白糖基磷脂酰肌醇锚定蛋白的数据。固定后，使用直接随机光学重建显微镜（dSTORM）对肌动蛋白丝进行成像。用鬼笔环肽和佛波酯处理肌动蛋白结构，生成了不同肌动蛋白结构的数据集，用于研究它们对膜蛋白动力学的潜在影响。
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