Department of Cell Biology, Center of Anatomy, Hannover Medical School, Carl-Neuberg Str. 1, D-30625 Hannover, Germany.
Traffic. 2010 Sep;11(9):1129-40. doi: 10.1111/j.1600-0854.2010.01084.x. Epub 2010 Jun 2.
Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)-tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP-tagged LC variants. Cy3-labeled light chains (Cy3-LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1-2 min the Cy3-LC heavy chain complexes entered clathrin-coated structures, whereas uncomplexed Cy3-LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time-course of an endocytic cycle. To explore whether GFP-tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild-type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3- and Alexa 488-labeled LC behaved similar to wild-type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.
网格蛋白三腿复合物由三条重链和三条轻链（LCs）组成。绿色荧光蛋白（GFP）标记的 LCs 被广泛用于研究活细胞中网格蛋白的动力学，但它们是否能真实反映细胞中网格蛋白三腿复合物的行为尚未得到充分研究。作为一种替代方法，我们用 Alexa 488 或 Cy3 染料标记纯化的 LCs，并将其与 GFP 标记的 LC 变体进行比较。Cy3 标记的轻链（Cy3-LC）直接或与重链一起微注射到 HeLa 细胞中。在 1-2 分钟内，Cy3-LC 重链复合物进入网格蛋白包被结构，而未复合的 Cy3-LC 在 2 小时内没有进入。这些发现表明，在胞吞周期的过程中，LC 没有明显的交换。为了研究 GFP 标记的 LCs 是否具有功能性，我们对其进行了生化特性分析。与野生型 LCs 不同，连接 GFP 的重组 LCs 不能有效地抑制体外的网格蛋白组装，而 Cy3 和 Alexa 488 标记的 LC 在体外和体内与野生型 LCs 行为相似。因此，荧光标记的 LCs 是研究活细胞中网格蛋白复杂行为的一种有价值的工具。
