On the Relationship Between EB-3 Profiles and Microtubules Growth in Cultured Cells

Abstract

Microtubules are dynamic structures undergoing rapid growth and shrinkage in living cells and in vitro. The growth of microtubules in vitro was analyzed with subpixel precision (Maurer et al., Current Biology, 2014, 24 (4), 372–384); however, to what extent these results could be applied for microtubules growing in vivo remains largely unknown. Particularly, the question is whether microtubule growth velocity in cells could be sufficiently approximated by a Gaussian distribution or its variability requires a more sophisticated description? Addressing this question, we used time-lapse microscopy and mathematical modeling, and we analyzed EB-3 comets forming on microtubules of cultured cells with subpixel precision. Parameters of comets (shape, form, and velocity) were used as topological characteristics of 3D voxel objects. Using regression analysis, we determined the real positions of the microtubule tips in time-lapse sequences. By exponential decay fitting of the restored comet intensity profile, we found that in vivo EB-3 rapidly exchanges on growing microtubule ends with a decoration time ∼ 2 s. We next developed the model showing that the best correlation between comet length and microtubule end growth velocity is at time intervals close to the decoration time. In the cells, EB comet length positively correlates with microtubule growth velocity in preceding time intervals, while demonstrating no correlation in subsequent time intervals. Correlation between comet length and instantaneous growth velocity of microtubules remains under nocodazole treatment when mean values of both parameters decrease. Our data show that the growth of microtubules in living cells is well-approximated by a constant velocity with large stochastic fluctuations.

In Vitro Cell Culture Model for Osteoclast Activation during Estrogen Withdrawal

“…Furthermore, exposing human osteosarcoma cells to a E2 derivative (formed by the dimerization of E2) was shown to result in reduced MT dynamicity, with the speed of MT growth being slowed [ 84 ], similar to what we observed in the EB1-eGFP comets in the continuously E2-treated osteoclasts. Comet length of EB1 or its homologue EB3 was reported to positively correlate with the MT growth rate [ 85 , 86 , 87 ]. Consistent with our result of shorter EB1-eGFP comets and slower MT growth speed under E2 conditions, we speculate that E2 exposure would influence the recruitment of other MT +TIPs proteins that depend on EB1 to localize to the MT plus end such as CLIP-170 [ 87 , 88 ] and would alter MT interactions with the cell cortex [ 89 , 90 ].…”