Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, and Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158.
Proc Natl Acad Sci U S A. 2013 Oct 1;110(40):16015-20. doi: 10.1073/pnas.1309676110. Epub 2013 Sep 16.
Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.
细胞严格调控细胞内细胞器的运输,但由于我们无法在尺寸小于衍射极限的情况下跟踪单个细胞器的分子组成,因此对这一过程的深入理解在技术上受到限制。在这里,我们开发了一种用于细胞内校准的超分辨率显微镜技术,该技术可以通过校正由于不成熟的荧光蛋白导致的低估和由于荧光团闪烁导致的高估,来测量单个细胞器的大小以及准确计数绝对分子数量。使用这种技术,我们对酵母内吞途径中单个小泡的大小以及它们所包含的可及的磷酸肌醇 3-磷酸结合位点的数量进行了表征。这种分析揭示了组成和大小的特征小泡成熟轨迹,其中包括随机和调节成分。该轨迹显示出一些细胞间的可变性,同一细胞内的细胞器之间的变化较小。这种方法还揭示了该轨迹中事件顺序的机制信息:与不同小泡成熟阶段的已知标志物的共定位分析表明,磷酸肌醇 3-磷酸的产生先于融合到更大的内体中。这种单个细胞器的分析方法可以潜在地应用于一系列小细胞器,以揭示它们的精确组成/结构关系、它们的调节动力学以及这些过程中的噪声。