Räägel Helin, Säälik Pille, Langel Ulo, Pooga Margus
Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
Methods Mol Biol. 2011;683:165-79. doi: 10.1007/978-1-60761-919-2_12.
The number of various cargo delivered into cells by CPPs demonstrates the effective transport abilities of these short-peptidic sequences. Over the years of research, the translocation process of CPP-cargo complexes has been mapped to being of mostly endocytic nature, however, there is still no consensus on which of the endocytic routes is prevalent and to which extent the interplay between different modes of endocytosis is taking place. The intracellular trafficking of CPPs attached to a cargo molecule is even less understood. Therefore, the internalization and the subsequent intracellular targeting of complexes need clarification in order to define cellular destinations and improve the targeting of the cargo molecule to specific cellular compartments depending on the cargo attached to the transporting vector. This chapter focuses on describing the methods for visualizing the CPP-protein complexes in relation to different endocytic markers, for example transferrin (marker for clathrin-mediated endocytosis) and cholera toxin (ambiguous marker for clathrin-, caveolin-, and flotillin-mediated, but also clathrin- and caveolin-independent endocytosis) to determine the role of the respective pathways during entry to cells, and to different intracellular targets, for instance the lysosomal organelles or the Golgi apparatus. Additionally, antibody staining of respective endocytic vesicles following the internalization of CPP-protein complexes will be discussed.
细胞穿透肽（CPPs）转运至细胞内的各种货物数量，证明了这些短肽序列具有有效的转运能力。经过多年研究，CPP-货物复合物的转位过程已被确定主要是内吞性质的，然而，对于哪种内吞途径占主导以及不同内吞模式之间的相互作用程度仍未达成共识。与货物分子相连的CPPs的细胞内运输情况则更不清楚。因此，为了确定细胞内的目的地，并根据与转运载体相连的货物，将货物分子更精准地靶向特定细胞区室，复合物的内化及其随后的细胞内靶向需要进一步阐明。本章重点描述了用于可视化与不同内吞标记物相关的CPP-蛋白质复合物的方法，例如转铁蛋白（网格蛋白介导的内吞作用的标记物）和霍乱毒素（网格蛋白、小窝蛋白和脂筏蛋白介导的内吞作用的模糊标记物，同时也是不依赖网格蛋白和小窝蛋白的内吞作用的标记物），以确定各自途径在进入细胞过程中的作用，以及在不同细胞内靶点（如溶酶体细胞器或高尔基体）中的作用。此外，还将讨论CPP-蛋白质复合物内化后对各自内吞小泡的抗体染色。
