ИСТИНА

Abstract—Microtubules (MTs) in fibroblast-like cells are thought to be organized in radial array and are mostly formed on centrosome-based microtubule-organizing center (MTOC). Contrary, previous studies showed that in 3T3 fibroblast low amount of MTs are connected with centrosome and are directly nucleated in cytoplasm (Vorobjev et. al., 2000). The default behavior of free MTs in the fibroblast interior is biased dynamic instability. MTs born at the centrosome show ‘dynamic instability’ type behavior with no boundary (Alieva et al., 2015). In the current study we investigated the subcellular organization of MTs in fibroblast-like 3T3 cells, during interphase. The present assay was performed on stably transfected 3T3-Swiss cell line expressing EB3-RFP (microtubule plus-end tracking protein). We provide a quantitative analysis of MT dynamic parameters in the cells (density of growing plus-ends, growth rate, length and duration of growth period). The highest density of growing MTs is presented near the leading edges. Centrosome as an active MTOC was absent in a half of the cell population, however these cells did not show any difference in MT growth pattern. In EB3-RFP transduced cells we defined centrosome as a dense cluster of comets in cell center (near the nucleus) with high values of integral brightness and a large number of short MT starting to grow from this area (18.0±7.0 growth events per minute). The brightness of centrosome was defined as a number of EB3-RFP comets started at centrosomal area and was around 30 EB3-RFP- comets and was measured as ratio between integrated density of MTOC and EB3-comets. In many cells centrosome might be inactive for prolonged periods of time and do not show any difference in MT growth pattern. To test whether centrosomal activity in cells could be changed by exogenous stimuli, we used MT modulators to disrupt or stabilize MT arrays chemically with nocodazole and paclitaxel and mechanically in the scratch assay and biased random walk. However it could be activated during recovery from depolymerization of MTs by nocodazole. Most of the observed cells had active bright centrosome MTOC at the first minutes after nocodazole withdrawal (28 cells out of 30), at the same time MTs started to grow in inner cytoplasm far from centrosomal area. Nucleation rate during the first hour after nocodazole treatment was lower than in untreated cells. We observed the opposite effect on cells after paclitaxel treatment. Centrosomal MTOC was inactivated in the most observed cells at the first minutes after inhibitor withdrawal (58 cells out of 60). The effect of the both inhibitors persist for more than 8 hours, while after 12 hours of inhibitor withdrawal the half of the cells demonstrate inactive centrosomal MTOC. Activation of centrosome restores its activity that is still minor related to the overall MT polymerization. As soon as destabilization of microtubule system by nocodazole activated the centrosome in non- migrating cells, we suggested that transition to the migration phase could also change centrosome activity and performed the scratch assay as a gold standard for quantitative evaluation of the cell migration. None of the cells that migrated to the wound demonstrated the switch of centrosome activity during 90 minutes of observation. As a next step, we observed cells in the biased random walking model to see if the transition to undirected movement can affect the activity of centrosome. We observed 30 motile stable-transfected with EB3-RFP 3T3 fibroblasts during 10 hours. Only 2 motile cells out of 30 had active centrosome but when they stopped locomotion, centrosome switched to inactive state. Our data confirm the crucial role of centrosome as MTOC in 3T3 fibroblasts and propose a model of non-centrosomal MTs as major players that create the cell asymmetry in the cells with mesenchymal type of motility. We suggest that increased density of dynamic MT near the active lamellum could be supported by microtubule-based MT nucleation.