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: 3.83). 08/2007; 93(1):346-59. DOI: 10.1529/biophysj.106.096966
Source: PubMed

ABSTRACT 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.

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    • "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. "
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    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
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    • "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). "
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    Molecular biology of the cell 12/2011; 22(24):4834-41. DOI:10.1091/mbc.E11-07-0611
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    • "Projectin 0.030 Lethocerus flight muscle [138] Mucins 0.036 Human ocular [139] Kettin 0.045 Recombinant fragments [138] DNA 0.053 <3000 bp [140] Intermediate filaments 1 In vitro polymerized human vimentin [77] Cofilactin 2.2 In vitro polymerized [92] Actin 9.0–17.7 In vitro polymerized with or without phalloidin [10] [12] [92] [141] Nanotubes 17–32 Single walled carbon nanotubes [142] Flagellar filaments 2.4–41.1 From bacteria [129] Thin filaments 44–121 See Table 1 Thick filaments 27–1742 From various species and muscle types [8] [9] [71] Microtubules 110–5200 In vitro polymerized with taxol or paclitaxel [12] [141] [143] contributes ∼20–50% to the total sarcomere compliance, indicating that the extension of actin plays a significant role in muscle contraction. "
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    BioMed Research International 06/2010; 2010:473423. DOI:10.1155/2010/473423
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