ИСТИНА

Short centrosomal microtubules (MTs) play a minor role in the organization of MT array in 3T3 fibroblasts. In contrast, the numerous MTs are nucleated in cytoplasm near the cell margin. Centrosome-independent MTs could contribute to the intracellular asymmetry of MT array. To understand the role of centrosomal and non-centrosomal MTs in the organization of MT array in 3T3 fibroblasts, we have performed detailed analysis of MT growth in different zones of the cells. The assay was performed on 3T3 Swiss cell line expressing EB3-RFP (MT plus-end tracking protein) co-transfected with centrin-eGFP (as a conventional centrosome marker). We analyzed the dynamic parameters and spatial distribution of MT plus-ends using time-lapse and z-stack movies of interphase cells. MTOC, visualized by clastering of EB3-RFP was colocalized with centrosome (centrin) in only half of cells. The average brightness of centrosome was 32.87 EB3-RFP comets and a large number of short MTs started to grow from this area (18.0±7.0 growth events/minute). Analysis of Z-stacks showed that MT cluster growing from centrosome and growing MTs at the cell margin are in different optical sections. Thus, most of the MTs started at the centrosome didn’t reach the cell margin. In order to explain high density of growing plus-ends at the cell edge, we found that there are special spots in cytoplasm characterized by a high density of growing plus-ends. Double-staining of tubulin and Golgi complex marker supported the theory of Golgi-independent formation of the cytoplasmic MTOC. We suggest that increased density of dynamic MTs near the active lamellum could be explained by MT-based MT nucleation. Our data confirm the negligible role of centrosome as MTOC in 3T3 fibroblasts and propose a model of non-centrosomal MTs as major players that create the asymmetry in the cells with a mesenchymal type of motility.