Sims Peter A, Xie X Sunney
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Chemphyschem. 2009 Jul 13;10(9-10):1511-6. doi: 10.1002/cphc.200900113.
We report a label-free assay for simultaneous optical manipulation and tracking of endogenous lipid droplets as actively transported cargoes in a living mammalian cell with sub-millisecond time resolution. Using an EM-CCD camera as a highly sensitive quadrant detector, we can detect steps of dynein- and kinesin-driven cargoes under known force loads. We can distinguish single and multiple motor-driven cargoes and show that the stall forces for inward and outward transported cargoes are similar. By combining the stall force observable with the ability to detect individual steps, we can characterize kinesin- and dynein-driven active transport in different force regimes.
我们报告了一种无标记检测方法，可在活的哺乳动物细胞中以亚毫秒级时间分辨率同时对作为主动运输货物的内源性脂滴进行光学操纵和跟踪。使用电子倍增电荷耦合器件（EM-CCD）相机作为高灵敏度象限探测器，我们可以在已知力负载下检测动力蛋白和驱动蛋白驱动的货物的步移。我们能够区分单个和多个马达驱动的货物，并表明向内和向外运输货物的失速力相似。通过将可观测的失速力与检测单个步移的能力相结合，我们可以表征动力蛋白和驱动蛋白在不同力状态下驱动的主动运输。
