ÀNALYSIS OF EGF RECEPTOR ENDOCYTOSIS DYNAMICS BASED ON SEMIQUANTATIVE PROCESSING OF CONFOCAL IMMUNOFLUORESCENT
IMAGES OF FIXED CELLS
M.V. Zlobina,1 M.V. Kharchenko,1 E S. Kornilova 1,2
1 Institute of Cytology, RAS, St.Petersburg and 2 St.Petersburg State University;
e-mail: aquimaria@gmail.com
Endocytosis of signaling receptors, and of 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. Besides that the characteristics of endocytic pathway
is endosomes' translocation from cell periphery to juxtanuclear region. Thus, endocytosis as highly dynamic process is developing in time and space. Obviously one of the most productive
approach is light immunofluorescent microscopy that allows to estimate endocytosis dynamics at the level of one or several cells. Different impacts influencing endocytic regulator
components are inevitably reflected on dynamics on 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 to get 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
(Dclust) and present these parameters application using EGF receptor endocytosis as an example.
Key words: EGF receptor, EEA1, endosomes, microtubules, fluorescent confocal microscopy, image processing
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