Article

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

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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    • "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. "
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    • "Movement was quantified by tracking multiple points along each SF in the cell as a function of time (Fig. 1C). Points along the SFs were spaced ,5 mm apart, which is below the persistence length of F-actin (Arai et al., 1999; Brangwynne et al., 2007; van Mameren et al., 2009). If an SF moved or deformed, a displacement vector was mapped from one of the initial tracking points to a new point on the fibre at the next time step, normal to the initial SF position (Fig. 1C). "
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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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