Microtubules and Actin Filaments Facilitate Fusions and Separations of Vesicules on the Endocytic Pathway of EGF-Receptor Complexes

Abstract

The effect of microtubules disassembly with nocodazole and depolymerization of the actin cytoskeleton with cytochalasin D, as well as their combined effect on endocytosis dynamics of complexes of epidermal growth factor and its receptor in HeLa cells, was studied. Using confocal microscopy, it was shown that in cells fixed at different intervals after endocytosis stimulation, there is a high degree of colocalization of the receptor with the marker of early endosomes EEA1, which correlates with enlargement of endosomes as a result of fusion at early stages, and translocation of endosomes to the juxtanuclear area (JNA) at later stages. Translocation towards the JNA depends on microtubules. Nocodazole does not interfere with the formation of endosomes, but blocks their fusions and translocations. At the same time, depolymerization of actin microfilaments, not affecting the process of EEA1-dependent fusions and transport of endosomes into the JNA, leads to enlargement of endosomes, preventing the separation of early endosomes and mature multivesicular endosomes. With the destruction of both types of cytoskeleton, the newly formed receptor-containing endocytic vesicles do not enlarge, do not mature, and do not move into the JNA. Live cell imaging of endosomes with EGF associated with quantum dots confirmed the data obtained on fixed cells and made it possible to analyze the nature of an endosome motility. It turned out that rare periods of a fast linear run are changed for prolonged chaotic oscillations within a small area, while the directions of linear runs can be different, and not only towards the MTOC. Our data suggest that the role of microtubules is not only to ensure the translocations of endosomes, but also to provide a platform for their fusion. This assumption can also explain the seemingly chaotic movements of endosomes, which make it possible to increase the probability of their contacts and, consequently, fusions.