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 implies endosomes' multiple interactions with homotypic endosomes and vesicles of other origin (lysosomes, trans-Golgi network), which results in endosomal size changes. In addition, the characteristics of endocytic pathway is endosomes' translocation from cell periphery to juxtanuclear region. Thus, endocytosis as a highly dynamic process develops in time and space. Obviously one of the most productive approach is light immunofluorescent microscopy that allows of estimating endocytes dynamics at the level of one or several cells. Different impacts influencing endocytic regulator components are inevitably reflected on dynamics of endosome size and (or) its translocation. This provide possibility to uncover the both crucial and secondary components of regulatory machinery. However, visual estimation of such effects is often too subjective and does not allow getting statistically reliable data. Comparison of different experiments even in the case of the same series is also impeded. 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, as well as the coefficient reflecting vesicles' clasterization in perinuclear region as a measure of their translocation along microtubules toward nucleus (D(clust)) and present these parameters application using EGF receptor endocytosis as an example.