Bending Dynamics of Fluctuating Biopolymers Probed by Automated High-Resolution Filament Tracking

Harvard School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA.
Biophysical Journal (Impact Factor: 3.97). 08/2007; 93(1):346-59. DOI: 10.1529/biophysj.106.096966
Source: PubMed


Microscope images of fluctuating biopolymers contain a wealth of information about their underlying mechanics and dynamics. However, successful extraction of this information requires precise localization of filament position and shape from thousands of noisy images. Here, we present careful measurements of the bending dynamics of filamentous (F-)actin and microtubules at thermal equilibrium with high spatial and temporal resolution using a new, simple but robust, automated image analysis algorithm with subpixel accuracy. We find that slender actin filaments have a persistence length of approximately 17 microm, and display a q(-4)-dependent relaxation spectrum, as expected from viscous drag. Microtubules have a persistence length of several millimeters; interestingly, there is a small correlation between total microtubule length and rigidity, with shorter filaments appearing softer. However, we show that this correlation can arise, in principle, from intrinsic measurement noise that must be carefully considered. The dynamic behavior of the bending of microtubules also appears more complex than that of F-actin, reflecting their higher-order structure. These results emphasize both the power and limitations of light microscopy techniques for studying the mechanics and dynamics of biopolymers.

6 Reads
  • Source
    • "Whenever a MT plus end reached the cell margin it switched to shortening. We assumed that MTs inside the cell are flexible (Brangwynne et al., 2007), leading to fluctuations in the orientation of the growing MT tips (see Supplemental Experimental Procedures for details). At the beginning of the simulations, the initial length of all MTs (250 in total) was equal to zero and they were allowed to grow from the cell center. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/β1 and the components of the LL5β-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.
    Developmental Cell 10/2013; 27(2). DOI:10.1016/j.devcel.2013.09.010 · 9.71 Impact Factor
  • Source
    • "For instance, wavy sinusoidal and circular shapes are frequently observed when MTs are adsorbed to glass surfaces or confined between them. In this confined case the Fourier mode analysis of MT deformations systematically reveals that a few discrete modes have a larger amplitude than the fluctuations around it [26] [39] [40], cf. also [41] supplementary material. This is a strong hint towards the presence of some type of " frozen in " curvature -dynamically quenched on experimental timescales. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Microtubules have been in the focus of biophysical research for several decades. However, the confusing and mutually contradictory results regarding their elasticity and fluctuations have cast doubt on their present understanding. In this paper, we present the empirical evidence for the existence of discrete guanosine diphosphate (GDP)-tubulin fluctuations between a curved and a straight configuration at room temperature as well as for conformational tubulin cooperativity. Guided by a number of experimental findings, we build the case for a novel microtubule model, with the principal result that microtubules can spontaneously form micron-sized cooperative helical states with unique elastic and dynamic features. The polymorphic dynamics of the microtubule lattice resulting from the tubulin bistability quantitatively explains several experimental puzzles, including anomalous scaling of dynamic fluctuations of grafted microtubules, their apparent length-stiffness relation, and their remarkable curved-helical appearance in general. We point out that the multistability and cooperative switching of tubulin dimers could participate in important cellular processes, and could in particular lead to efficient mechanochemical signaling along single microtubules.
    Biophysics of Structure and Mechanism 12/2011; 41(2):217-39. DOI:10.1007/s00249-011-0778-0 · 2.22 Impact Factor
  • Source
    • "After ablation, confocal scans were collected using Zeiss LSM 510 4.2 software at 2–5 s per frame. The root-meansquare curvature was estimated from microtubule traces by fitting a one-dimensional Gaussian approximately orthogonally across the microtubule (Bicek et al., 2007, 2009; Brangwynne et al., 2007a). "
    [Show abstract] [Hide abstract]
    ABSTRACT: To determine forces on intracellular microtubules, we measured shape changes of individual microtubules following laser severing in bovine capillary endothelial cells. Surprisingly, regions near newly created minus ends increased in curvature following severing, whereas regions near new microtubule plus ends depolymerized without any observable change in shape. With dynein inhibited, regions near severed minus ends straightened rapidly following severing. These observations suggest that dynein exerts a pulling force on the microtubule that buckles the newly created minus end. Moreover, the lack of any observable straightening suggests that dynein prevents lateral motion of microtubules. To explain these results, we developed a model for intracellular microtubule mechanics that predicts the enhanced buckling at the minus end of a severed microtubule. Our results show that microtubule shapes reflect a dynamic force balance in which dynein motor and friction forces dominate elastic forces arising from bending moments. A centrosomal array of microtubules subjected to dynein pulling forces and resisted by dynein friction is predicted to center on the experimentally observed time scale, with or without the pushing forces derived from microtubule buckling at the cell periphery.
    Molecular biology of the cell 12/2011; 22(24):4834-41. DOI:10.1091/mbc.E11-07-0611 · 4.47 Impact Factor
Show more

Preview (2 Sources)

6 Reads
Available from