Compliance of actin filament networks measured by particle-tracking microrheology and diffusing wave spectroscopy

The fundamentals and best practices of multiple particle tracking microrheology are discussed, including methods for producing video microscopy data, analyzing data to obtain mean-squared displacements and displacement correlations, and, critically, the accuracy and errors (static and dynamic) associated with particle tracking. Applications presented include two-point microrheology, methods for characterizing heterogeneous material rheology, and shell models of local (non-continuum) heterogeneity. Particle tracking has a long history. The earliest descriptions of Brownian motion relied on precise observations, and later quantitative measurements, using light microscopy.

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A novel role for WAVE1 in controlling actin network growth rate and architecture

Molecular Biology of the Cell ◽

10.1091/mbc.e14-10-1477 ◽

2015 ◽

Vol 26 (3) ◽

pp. 495-505 ◽

Cited By ~ 9

Author(s):

Meredith O. Sweeney ◽

Agnieszka Collins ◽

Shae B. Padrick ◽

Bruce L. Goode

Keyword(s):

Actin Filament ◽

Specific Activity ◽

Inhibitory Effect ◽

Actin Network ◽

Inhibitory Effects ◽

Cellular Functions ◽

Network Growth ◽

Assembly Kinetics ◽

Filament Networks ◽

First Time

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

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Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin.

The Journal of Cell Biology ◽

10.1083/jcb.113.5.1057 ◽

1991 ◽

Vol 113 (5) ◽

pp. 1057-1067 ◽

Cited By ~ 149

Author(s):

M L Vitale ◽

A Rodríguez Del Castillo ◽

L Tchakarov ◽

J M Trifaró

Keyword(s):

Actin Filament ◽

Chromaffin Cells ◽

Chromaffin Cell ◽

Catecholamine Secretion ◽

Cortical Region ◽

Cortical Surface ◽

Filamentous Actin ◽

Cell Stimulation ◽

Rhodamine Phalloidin ◽

Filament Networks

Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca(2+)-dependent actin filament severing proteins, in the cortical surface of the chromaffin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K(+)-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-beta-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.

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Plate reader microrheology

10.1101/2020.07.10.197723 ◽

2020 ◽

Author(s):

Robert F. Hawkins ◽

Gregg A. Duncan

Keyword(s):

Particle Tracking ◽

Fluorescence Polarization ◽

Rotational Diffusion ◽

Polymeric Materials ◽

Soft Materials ◽

Clinical Samples ◽

Clinical Settings ◽

Particle Tracking Microrheology ◽

Plate Reader ◽

Potential Applications

AbstractIn this work, we report the development of a simplified microrheological method that can be used to rapidly study soft materials. This approach uses fluorescence polarization and a plate reader format to measure the rotational diffusion of nanoparticles within a sample of interest. We show that this measurement is sensitive to viscosity-dependent changes in polymeric soft materials and is correlated with particle tracking microrheology, a previously validated measure of microrheology. Using these fluorescence polarization-based measurements, we describe formalism that enables reasonable estimation of viscosity in polymeric materials after accounting for length-scale dependent effects of the polymer environment on the nanoparticle rotational diffusion. The use of a plate reader format allows this approach to be higher throughput, less technically challenging, and more widely accessible than standard macro- and microrheological methods, making it available to non-experts. This approach has potential applications in academic and industry settings where conventional rheological equipment may not be available, as well as in clinical settings to rapidly characterize human clinical samples.

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