以消散场成像和单微粒跟踪技术揭示GLUT4囊泡和分泌囊泡的不同动态过程

葡萄糖转运子蛋白4(glucose transporter 4,GLUT4)在维持体内葡萄糖动态平衡的过程中起着至关重要的作用。GLUT4贮存囊泡(GLUT4 storage vesicle,GSV)和神经内分泌细胞中的分泌囊泡含有许多相同的蛋白。研究证明这些蛋白调节了分泌囊泡的胞内转运过程,但是GLUT4囊泡和分泌囊泡是否具有相同的胞内动态过程还未阐明。文章以3T3-L1纤维原细胞中的GSV和神经内分泌细胞PC12细胞中的分泌囊泡:致密核心大囊泡(large dense core vesicle,LDCV)为研究对象,使用消散场显微成像技术和单微粒跟踪技术直观观察了活体细胞内单个GSV和LDCV的三维运动轨迹。通过以适当方程拟合单个囊泡的均方位移曲线,发现两种囊泡都具有三种运动模式。定量分析显示作自由扩散运动和方向性扩散运动的GSV数量明显多于LDCV。对比GSV和LDCV的三维扩散系数,发现GSV的扩散系数中值为7.2×10-4μm2/s,而LDCV的扩散系数中值仅为1.94×10-4μm2/s。这一结果说明GSV的活动性远大于LDCV,提示GSV的胞内转运过程涉及不同的分子机制。

Dynamic difference between GLUT4 storage vesicles and secretory vesicles revealed by evanescent-field imaging and single particle tracking

Glucose transporter 4 (GLUT4) plays a crucial role in maintaining whole-body glucose homeostasis. It has been revealed that GLUT4 storage vesicles (GSV) and secretory vesicles in neuroendocrine cells contain similar proteins. Many studies have demonstrated that the intracellular transport of secretory vesicles is precisely mediated by these proteins. However, it is still unclear whether GSVs share similar dynamic features with secretory vesicles during intracellular trafficking. In this paper, GSV in 3T3-L1 fibroblasts and secretory large dense core vesicles (LDCVs) in neuroendocrine PC12 cells were taken as models for comparing GSV and LDCV mobility. Evanescent-field microscopy and Gaussian-fit-based single particle tracking were used to follow the three-dimensional trajectories of single GSVs and LDCVs. By fitting appropriate equations to particular mean squared displacement of single vesicles, three different motion modes were revealed for both GSVs and LDCVs. Quantitative analysis showed that the number of GSVs undergoing random diffusion and directed diffusion was much more than LDCVs. Furthermore, it was compared that three-dimensional diffusion coefficients for GSVs and LDCVs. The median diffusion coefficient of GSVs is 7.2×10-4 μm2/s and LDCVs 1.94×10-4 μm2/s. Although similar proteins have been found for both GSVs and secretory vesicles, our results reveal a significant difference between GSVs and LDCVs in their mobility, suggesting there are different molecular mechanism underlying the intracellular transport of GSVs.