Cavender Chapin E, Gottipati Manoj K, Malarkey Erik B, Parpura Vladimir
Department of Neurobiology, University of Alabama at Birmingham.
Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham.
Inquiro. 2013;7:48-52.
Astrocytes provide a principal pathway for glutamate uptake in the mammalian brain, a task accomplished by the powerful action of excitatory amino acid transporters (EAAT) 1 and 2. These transporters are synthesized within the endoplasmic reticulum and are then trafficked to the plasma membrane. The characteristics of their intracellular traffic within astrocytes have not been investigated. We monitored the trafficking of secretory vesicles laden with the recombinant fluorescent protein chimera of EAAT2 in cultured astrocytes. Such vesicles appeared as fluorescent puncta, and their trafficking parameters were obtained using original algorithms, which we describe here in detail. We determined the maximal displacement, average instantaneous speed, and trajectory angle of individual puncta/vesicles, with angles near 0° indicating radial movement directly away from or toward the nucleus and angles near 90° indicating tangential movement. Analysis of these trafficking parameters demonstrated that trafficking of EAAT2-laden vesicles has typical characteristics expected of the trafficking of secretory vesicles in cultured astrocytes. The distribution of trajectory angles for directional vesicles, i.e. those with a maximal displacement greater than 1 μm within the 40-s time-lapse imaging, was found to be unimodal, with angles near 0° being the most prominent (mode 7°). These measurements are in good agreement with previous measurements of trajectory angles of similar trafficking vesicles carrying cannabinoid receptor 1, evidencing the validity and robustness of our analytical approach and algorithms.
星形胶质细胞为哺乳动物大脑中的谷氨酸摄取提供了主要途径，这一任务是通过兴奋性氨基酸转运体（EAAT）1和2的强大作用来完成的。这些转运体在内质网中合成，然后被运输到质膜。它们在星形胶质细胞内的细胞内运输特征尚未得到研究。我们监测了在培养的星形胶质细胞中装载有EAAT2重组荧光蛋白嵌合体的分泌囊泡的运输情况。这些囊泡表现为荧光斑点，其运输参数是使用我们在此详细描述的原始算法获得的。我们确定了单个斑点/囊泡的最大位移、平均瞬时速度和轨迹角度，角度接近0°表示直接远离或朝向细胞核的径向运动，角度接近90°表示切向运动。对这些运输参数的分析表明，装载EAAT2的囊泡的运输具有培养的星形胶质细胞中分泌囊泡运输所预期的典型特征。发现定向囊泡（即在40秒延时成像中最大位移大于1μm的囊泡）的轨迹角度分布是单峰的，角度接近0°最为突出（众数7°）。这些测量结果与先前对携带大麻素受体1的类似运输囊泡的轨迹角度测量结果非常一致，证明了我们分析方法和算法的有效性和稳健性。
