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

ArticleinBiophysical Journal 93(1):346-59 · August 2007with8 Reads
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.
    • "Therefore, the effective surface tension becomes σ ef f = σ + µ/2. Estimates of µ depend on the meshsize, however, for a meshsize of about 10 nm, µ ≈ 10 −3 N/m, which would bring the effective surface tension in the range for the pearling instability to set in [40]. "
    [Show abstract] [Hide abstract] ABSTRACT: During endocytosis, the cell membrane deforms to surround extracellular material and draw it into the cell. Experiments on clathrin-mediated endocytosis in yeast all agree that (i) actin polymerizes into a network of filaments exerting active forces on the membrane to deform it and (ii) the large scale membrane deformation is tubular in shape. Three competing ideas remain as to precisely how the actin filament network organizes itself to drive the deformation. To begin to address this issue, we use variational approaches and numerical simulations to analyze a meso-scale model of clathrin-mediated endocytosis in yeast. The meso-scale model breaks up the invagination process into three stages: (i) the initiation stage, where clathrin interacts with the membrane, (ii) the elongation stage, where the membrane is then pulled and/or squeezed via polymerizing actin filaments, followed by a (iii) final pinch-off stage. Our results suggest that the pinch-off mechanism is assisted by a pearling-like instability. In addition, we potentially rule out two of the three competing models for the organization of the actin filament network during the elongation stage. These two models could possibly be important in the pinch-off stage, however, where actin polymerization helps break off the vesicle. Implications and comparisons with earlier modeling of clathrin-mediated endocytosis in yeast is discussed.
    Full-text · Article · Oct 2013
    • "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.
    Full-text · Article · Oct 2013
    • "Particle tracking microrheology starts with the imaging experiments and the image treatment in a strict sense, however, obtaining particle trajectories from video microscopy is well described in the literature1617181920 including the statistical difficulties of the process212223. There are various implementations of particle tracking algorithms available in IDL (see the same website as for the rheology code) [11,24], Matlab [12,25], LabView [20,26] or C++ languages [27]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Particle tracking passive microrheology relates recorded trajectories of microbeads, embedded in soft samples, to the local mechanical properties of the sample. The method requires intensive numerical data processing and tools allowing control of the calculation errors. Results We report the development of a software package collecting functions and scripts written in Python for automated and manual data processing, to extract viscoelastic information about the sample using recorded particle trajectories. The resulting program package analyzes the fundamental diffusion characteristics of particle trajectories and calculates the frequency dependent complex shear modulus using methods published in the literature. In order to increase conversion accuracy, segmentwise, double step, range-adaptive fitting and dynamic sampling algorithms are introduced to interpolate the data in a splinelike manner. Conclusions The presented set of algorithms allows for flexible data processing for particle tracking microrheology. The package presents improved algorithms for mean square displacement estimation, controlling effects of frame loss during recording, and a novel numerical conversion method using segmentwise interpolation, decreasing the conversion error from about 100% to the order of 1%.
    Full-text · Article · Nov 2012
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