Abstract
Microtubules in interphase fibroblast-like cells are thought to be organized in a radial array growing from a centrosome-based microtubule-organizing center (MTOC) to the cell edges. However, many morphogenetic processes require the asymmetry of the microtubules (MT) array. One of the possible mechanisms of this asymmetry could be the presence of non-centrosomal microtubules in different intracellular areas. To evaluate the role of centrosome-born and non-centrosomal microtubules in the organization of microtubule array in motile 3T3 fibroblasts, we have performed the high-throughput analysis of microtubule growth in different functional zones of the cell and distinguished three subpopulations of growing microtubules (centrosome-born, marginal and inner cytoplasmic).Centrosome as an active microtubule-organizing center was absent in half of the cell population. However, these cells do not show any difference in microtubule growth pattern. In cells with active centrosome, it was constantly forming short (ephemeral) MTs, and ∼15–20 MT per minute grow outwards for a distance >1 µm. Almost no persistent growth of microtubules was observed in these cells with the average growth length of 5–6 µm and duration of growth periods within 30 s.However, the number of growing ends increased towards cell margin, especially towards the active edges. We found the peripheral cytoplasmic foci of microtubule growth there. During recovery from nocodazole treatment microtubules started to grow around the centrosome in a normal way and independently in all the cell areas. Within 5 minutes microtubules continued to grow mainly near the cell edge. Thus, our data confirm the negligible role of centrosome as MTOC in 3T3 fibroblasts and propose a model of non-centrosomal microtubules as major players that create the cell asymmetry in the cells with a mesenchymal type of motility. We suggest that increased density of dynamic microtubules near the active lamellum could be supported by microtubule-based microtubule nucleation.
Highlights
Microtubules (MTs) are dynamic structurally polar cytoskeletal components that grow by addition of the GTP-loaded tubulin to their end
In our previous study we concluded that “in fibroblasts (1) the default behavior of free MTs in the cell interior is biased dynamic instability; (2) MTs born at the centrosome show ‘dynamic instability’ type behavior with no boundary; (3) that the extended radial array is formed predominantly by MTs not associated with the centrosome” [4]
The major pool of these short MT tracks in spread 3T3 cells grew in the cytoplasm or near the cell margin, whereas only a few of them started at the centrosome
Summary
Microtubules (MTs) are dynamic structurally polar cytoskeletal components that grow by addition of the GTP-loaded tubulin to their end. The centrosome is considered the major MT-nucleating center in the fibroblast-like cells [1]. Growing body of evidence shows that this is oversimplification [2] and in many types of animal cells centrosome is not working as MTOC through interphase [3]. In our previous study we concluded that “in fibroblasts (1) the default behavior of free MTs in the cell interior is biased dynamic instability (i.e., random walk of the plus ends with significant positive drift); (2) MTs born at the centrosome show ‘dynamic instability’ type behavior with no boundary; (3) that the extended radial array is formed predominantly by MTs not associated with the centrosome” [4]. We extended our analysis to describe MT array further and to determine what part of the MT array might be directly associated with the centrosome and what part is regulated by local properties of the cytoplasm
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