Analysis of EGF receptor endocytosis dynamics based on semiquantitative processing of confocal immunofluorescent images of fixed cells

Abstract

Endocytosis of signaling receptors, EGF receptor in particular, starting at the plasma membrane and finishing in perinuclear lysosomes entails endosome multiple interactions with homotypic endosomes and vesicles of other origin (lysosomes, trans-Golgi network), which results in changes of endosome size. A distinctive feature of the endocytic pathway is endosome translocation from the cell periphery to the juxtanuclear region. Thus, endocytosis is a highly dynamic process developing in time and space. One of the most productive approaches to studying endocytosis regulation is light immunofluorescent microscopy, which allows determining the endocytosis dynamics at the level of single or several cells. Different effects that influence endocytic regulator components are inevitably reflected on the dynamics on endosome size and/or its translocation. This makes it possible to reveal both primary and secondary components of the regulatory machinery. However, visual determination of such effects is often subjective and does not allow statistically reliable data to be obtained. Comparison of different experiments, even in the case of the same series, also may be complicated. In this work, we use such parameters as apparent vesicle size (diameter, area, or volume) and vesicle number per cell to provide quantitative estimation of fusion efficacy. Moreover, we propose a coefficient reflecting vesicle clusterization in the perinuclear region as a measure of their translocation along microtubules toward the nucleus (D _clust). We present the application these parameters using EGF receptor endocytosis as an example.