Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

Abstract

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.

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“…3f–g and Supplementary Movie 4). This type of motion has been observed previously in in vitro microtubule gliding assays where immobilized microtubule motors propel partially immobilized microtubules13 and in vivo in Xenopus melanophores14 and epithelial cells1516. These results suggest that the subset of microtubules labelled by kinesin-1 undergoes constant undulations under the action of some localized force generators that, like kinesin-1, preferentially interact with this microtubule subset.…”

“…These observations are in line with previous studies, showing that bending of microtubules is caused by microtubule specific force generators141516. To illustrate our findings and aid future studies of intracellular transport and cytosol compartmentalization, we built a hypothetical ‘phase diagram' of the heterogeneous cell environment (Fig.…”

“…Further development of scalable tracers should allow precise mapping of those boundaries and a thorough description of the non-equilibrium mechanical environment of the cell22. Importantly, apart from the actin meshwork, other intracellular structures, such as intermediate filaments1423 and membrane organelles2124 are also likely to affect transport processes inside cells.…”

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells

“…Moreover, processes can be considered static in the time window of the experiments (Fig. 1A) as assessed by tracking the processes with a tracking routine that we have developed to track single filaments [40]. The algorithm shows the absence of significant motion of processes within the time window of the tracking experiments.…”

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“…The effective Lp of microtubules in PC3 cells was determined using the methodology described by Gittes et al, 37 for cells treated or not with hemin. Briefly, microtubule xy positions were recovered using a filament tracking routine, 38 a Fourier analysis was performed on the shape of the filaments and mode amplitudes were recorded. Finally, Lp* was obtained by fitting an inverse square law to the ensemble variance of these amplitudes as a function of the mode number.…”