[INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only phospho-ERK2 could be detected after 60 min of endocytosis. In both cases, MAP-kinase activation dynamics was significantly different from the control. Our results suggest involvement of EGF-stimulated MAP-kinase pathway in cytoskeleton regulation. At the same time, they demonstrate that the two studied and widely used inhibitors are not equivalent with respect to not only the effect on MAP-kinase activity but also to such interdependent processes such as changes in cytoskeleton organization and signaling receptor' endocytosis.

[Analysis of vesicle subpopulations carrying early endosomal autoantigen EEA1].

Confocal immunofluorescent analysis of interphase HeLa cells has demonstrated that involved in regulation of homotypic fusions early endosomal autoantigene EEA1 is associated with vesicles represented by two populations differing in apparent size, localization and the level of bound EEA1. Before analysis the cells have been preincubated in serum-deprived medium for 12 h to minimize ligand-dependent endocytosis of serum growth factors. The first subpopulation is mainly represented by large vesicles strongly decorated with EEA1. These vesicles are localized presumably in juxtanuclear region. Microtubule depolimerization experiments have shown that this localization is maintained by tubulin cytoskeleton. The second subpopulation consists of numerous small vesicles slightly stained by EEA1 antibody and localized more peripherally. Double indirect immunofluorescent ananlysis of fixed cell images has revealed that juxtanuclear vesicles enriched in EEA1 are fully colocalized with key protein of early endosomes small GTPase Rab5, whereas about 50% of slightly decorated peripheral vesicles are Rab5-negative. It is found that the number of Rab5-positive vesicles per cell is higher than that of EEA1-positive vesicles. Thus, in serum-deprivated HeLa cells with low endocytic activity two subpopulations of EEA1-vesicles are revealed: the first one carries the both EEA1 at high level and Rab5 (EEA1+++/Rab5+), and the second subpopulation oconsists of weakly decorated EEA1-vesicles, that can be both Rab5-positive and -negative (EEA1+/Rab5- and EEA1+/Rab5+). Besides, there are vesicles carrying Rab5 only (EEA1-/Rab5+). The data obtained favor different functional role of all these subpopulations, which are associated with proteins widely considered as equivalent markers of early endosomes.

[Analysis of EGF receptor endocytosis dynamics based on semiquantative processing of confocal immunofluorescent images of fixed cells].

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.

[Acetylation of microtubules during endocytosis of epidermal growth factor receptor (c-ErbB1) in interphase HeLa cells].

Acetylation of microtubules (MT) during endocytosis of epidermal growth factor receptor, c-ErbB1, was studied by confocal immunofluorescence microscopy. It was found that stimulation of endocytosis of c-ErbB1 complexed with the epidermal growth factor (EGF), resulted in continuous raising of MT acetylation that reached its maximum at 60-90 min and then went down to the control level. Simultaneously, the receptor-containing endosomes grew in size and were redistributed into juxtranuclear region. Enlarged endosomes formed dense clusters around MTOC in 60-90 min. Another native c-ErbB1 ligand, transforming growth factor-alpha (TGF-alpha) and unlike EGF causing the receptor recycling, also initiated a wave of MT acetylation, but the effect was expressed more poorly. In this case, endosomes did not grow in size and did not form dense clusters near the MTOC. Cell treatment with deacetylase inhibitor trichostatin A (TSA) caused acetylation of the whole cellular MT population. Under these conditions, translocations of EGF-c-ErbB1-containing endosomes had the same pattern as in the cells untreated with the inhibitor, but the size of endosomes didn't increase during their redistribution into juxtranuclear region. Acetylation was especially pronounced in strongly bent MT regions positioned proximally to MTOC and forming there a dense meshwork whereas peripheral MT plus-ends were basically straight and not modified. We assume that MT acetylation is not so much crucial for preferential interaction with dynein or kinesin and, accordingly, for organization of endosome translocations in a certain direction. It is rather the result of stabilization of some MT pool which supports homotypic fusion of endosomes at early stage of their maturation.