Analysis of Microtubule Dynamics Heterogeneity in Cell Culture

Serikbaeva, Anara; Tvorogova, Anna; Kauanova, Sholpan; Vorobjev, Ivan A.

doi:10.1007/978-1-4939-7680-5_11

Cited by 4 publications

(5 citation statements)

References 77 publications

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“…We further analyzed the effect of nocodazole applied in nanomolar concentrations. In the range of concentrations, 30–50 nM, the velocity of MT growth estimated by manual tracking did not significantly change ( Serikbaeva et al, 2018 ). However, in some cases, smaller displacements of individual MT ends were evident ( Figure 9A ).…”

Section: Resultsmentioning

confidence: 99%

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

Urazbaev

Serikbaeva

Tvorogova

et al. 2021

Front. Mol. Biosci.

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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.

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Section: Resultsmentioning

confidence: 99%

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

Urazbaev

Serikbaeva

Tvorogova

et al. 2021

Front. Mol. Biosci.

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“…More detailed analysis shows that the minimal effective concentrations of all three drugs for inhibiting MT plusend growth are 10-30 nM for 3T3, HT1080, and A549 cells (Serikbaeva et al, 2018). Thus, we titrated all inhibitors in wound healing experiments in the range of concentrations from 1 nM to 1-3 µM.…”

Section: Effect Of Microtubule Inhibitors On Wound Closurementioning

confidence: 99%

The Frequent Sampling of Wound Scratch Assay Reveals the “Opportunity” Window for Quantitative Evaluation of Cell Motility-Impeding Drugs

Kauanova

Urazbayev

Vorobjev

2021

Front. Cell Dev. Biol.

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Wound healing assay performed with automated microscopy is widely used in drug testing, cancer cell analysis, and similar approaches. It is easy to perform, and the results are reproducible. However, it is usually used as a semi-quantitative approach because of inefficient image segmentation in transmitted light microscopy. Recently, several algorithms for wound healing quantification were suggested, but none of them was tested on a large dataset. In the current study, we develop a pipeline allowing to achieve correct segmentation of the wound edges in >95% of pictures and extended statistical data processing to eliminate errors of cell culture artifacts. Using this tool, we collected data on wound healing dynamics of 10 cell lines with 10 min time resolution. We determine that the overall kinetics of wound healing is non-linear; however, all cell lines demonstrate linear wound closure dynamics in a 6-h window between the fifth and 12th hours after scratching. We next analyzed microtubule-inhibiting drugs’, nocodazole, vinorelbine, and Taxol, action on the kinetics of wound healing in the drug concentration-dependent way. Within this time window, the measurements of velocity of the cell edge allow the detection of statistically significant data when changes did not exceed 10–15%. All cell lines show decrease in the wound healing velocity at millimolar concentrations of microtubule inhibitors. However, dose-dependent response was cell line specific and drug specific. Cell motility was completely inhibited (edge velocity decreased 100%), while in others, it decreased only slightly (not more than 50%). Nanomolar doses (10–100 nM) of microtubule inhibitors in some cases even elevated cell motility. We speculate that anti-microtubule drugs might have specific effects on cell motility not related to the inhibition of the dynamic instability of microtubules.

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“…The most common and most reliable way to extract microtubule life history data is to track the position of the microtubule end or to measure the length of a microtubule manually at every time point [74,75]. To account for changes in the curvature of the microtubule, a curve can be traced or fitted to the entire length of the microtubule [69].…”

Section: Data Analysis and Presentationmentioning

confidence: 99%

Measuring microtubule dynamics

Zwetsloot

Tut

Straube

2018

Essays in Biochemistry

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Microtubules are key players in cellular self-organization, acting as structural scaffolds, cellular highways, force generators and signalling platforms. Microtubules are polar filaments that undergo dynamic instability, i.e. transition between phases of growth and shrinkage. This allows microtubules to explore the inner space of the cell, generate pushing and pulling forces and remodel themselves into arrays with different geometry and function such as the mitotic spindle. To do this, eukaryotic cells employ an arsenal of regulatory proteins to control microtubule dynamics spatially and temporally. Plants and microorganisms have developed secondary metabolites that perturb microtubule dynamics, many of which are in active use as cancer chemotherapeutics and anti-inflammatory drugs. Here, we summarize the methods used to visualize microtubules and to measure the parameters of dynamic instability to study both microtubule regulatory proteins and the action of small molecules interfering with microtubule assembly and/or disassembly.

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Analysis of Microtubule Dynamics Heterogeneity in Cell Culture

Cited by 4 publications

References 77 publications

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

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

The Frequent Sampling of Wound Scratch Assay Reveals the “Opportunity” Window for Quantitative Evaluation of Cell Motility-Impeding Drugs

Measuring microtubule dynamics

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