Department of Emerging Infectious Diseases, Graduate School of Medicine, Tohoku University, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
Sensors (Basel). 2012;12(1):549-72. doi: 10.3390/s120100549. Epub 2012 Jan 5.
Specific protein domains known as protein transduction domains (PTDs) can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. It is, therefore, crucial to clarify the entry mechanisms and to identify the rate-limiting steps. Because of technical limitations for imaging PTD behavior on cells with conventional fluorescent-dyes, how PTDs enter the cells has been a topic of much debate. Utilizing quantum dots (QDs), we recently tracked the behavior of PTD that was derived from HIV-1 Tat (TatP) in living cells at the single-molecule level with 7-nm special precision. In this review article, we initially summarize the controversy on TatP entry mechanisms; thereafter, we will focus on our recent findings on single-TatP-QD tracking (SQT), to identify the major sequential steps of intracellular delivery in living cells and to discuss how SQT can easily provide direct information on TatP entry mechanisms. As a primer for SQT study, we also discuss the latest findings on single particle tracking of various molecules on the plasma membrane. Finally, we discuss the problems of QDs and the challenges for the future in utilizing currently available QD probes for SQT. In conclusion, direct identification of the rate-limiting steps of PTD entry with SQT should dramatically improve the methods for enhancing transduction efficiency.
已知特定的蛋白质结构域，即蛋白质转导结构域（PTDs），可以穿透细胞膜，并将蛋白质或生物活性物质递送到活细胞中。已经采用了各种方法来提高其转导效率。因此，阐明进入细胞的机制并确定限速步骤至关重要。由于传统荧光染料在对细胞内 PTD 行为进行成像方面的技术限制，PTD 如何进入细胞一直是一个备受争议的话题。利用量子点（QDs），我们最近以 7nm 的特殊精度，在单细胞水平上追踪了源自 HIV-1 Tat（TatP）的 PTD 在活细胞中的行为。在这篇综述文章中，我们首先总结了 TatP 进入机制的争议；此后，我们将重点介绍我们最近关于单 TatP-QD 追踪（SQT）的发现，以确定活细胞中细胞内递呈的主要顺序步骤，并讨论 SQT 如何轻松提供关于 TatP 进入机制的直接信息。作为 SQT 研究的入门，我们还讨论了最新关于各种分子在质膜上的单颗粒追踪的发现。最后，我们讨论了 QD 的问题以及未来利用当前可用的 QD 探针进行 SQT 的挑战。总之，利用 SQT 直接鉴定 PTD 进入的限速步骤，应该会极大地改进提高转导效率的方法。
